Method of generating lineage-restricted cells

Abstract

The invention relates to methods of generating lineage-restricted cells using forward programming.

Description

[0001] BIT-C-P3860PCT1

[0002] 1

[0003] METHOD OF GENERATING LINEAGE-RESTRICTED CELLS

[0004] FIELD OF THE INVENTION

[0005] The invention relates to methods of generating lineage-restricted cells, by overexpressing one or more polypeptides that have the activity of combinations of transcription factors and / or combinations of transcription factors themselves, i.e. through forward programming.

[0006] BACKGROUND OF THE INVENTION

[0007] Stem cell research holds great promise for research of human development, regenerative medicine, disease modelling, drug discovery, and cell transplantation. Moreover, stem cell- derived cells enable studying physiological and pathological responses of human cell populations that are not easily accessible. This often entails the study of genes (and other forms of regulatory mechanisms encoded in non-protein-coding RNAs - ncRNAs). Unfortunately, controllable transcription or expression of genetic information in human cells has been proven to be particularly difficult.

[0008] There is a real need for the ex vivo derivation of many highly desirable human cell types in a quantity and quality suitable for drug discovery and regenerative medicine purposes. Because directed differentiation of stem cells into desired cell types is often challenging, other approaches have emerged, including forward programming. This is a method of directly converting stem cells, including pluripotent stem cells, to lineage-restricted cells such as mature cell types, and has been recognised as a powerful strategy for the derivation of human cells. Forward programming generally involves the forced expression of polypeptides having the activity of key lineage transcription factors (or the transcription factors themselves) in order to convert the stem cell into lineage-restricted cells. Alternatively, lineage-restricted cells may be directly converted into a cell of a different lineage (i.e. without needing a pluripotent intermediary step). Such methods are known as transdifferentiation.

[0009] Generative pretrained models using artificial intelligence and learning from large-scale diverse genetic datasets have been developed and are readily available in the art. For example single cell Generative Pretrained Transformer (scGPT) is a model developed using a repository of over 33 million cells, as described in Cui et al., 2024, Nature Methods, 21 , pp1470-80. The scGPT codebase is publicly available at https: / / github.com / bowang-lab / scGPT. Another such model is scMulan, as described in Bian et al., doi: https: / / doi.org / 10.1101 / 2024.01.25.577152. The scMulan codebase is publicly available at https: / / github.com / SuperBianC / scMulan. These models provide a “signature” for each cell type based on what genes are upregulated and BIT-C-P3860PCT1

[0010] 2 which genes are downregulated. These signatures can be compared against the signatures of cells that have been forward programmed using a particular polypeptide having the activity of key lineage transcription factors (or the transcription factors themselves), or sets of polypeptides / transcription factors, and this allows for the screening and pairing of transcription factors to cell types.

[0011] There is a need in the art to provide methods for generating specific desired cell types suitable for use as potential therapeutic agents, in research and in tissue engineering.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to a method of generating lineage-restricted cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain those lineage-restricted cells.

[0014] The invention also relates to a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the one or more transcription factors are capable of generating (through forward programming or transdifferentiation) a cell into a desired lineage-restricted cell.

[0015] The particular resultant lineage-restricted cell types are set out below. The transcription factors suitable for generating the lineage-restricted cell types are set out in Table 1 below. The specific transcription factors suitable for generating each specific lineage restricted cell type is also set out below.

[0016] According to a further aspect of the invention, there is provided a cell obtainable by any one of the methods defined herein.

[0017] According to a further aspect of the invention, there is provided an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the transcription factor activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are capable of generating lineage-restricted cells.

[0018] According to a further aspect of the invention, there is provided a cell as defined herein, for use in therapy, in vitro diagnostics or drug screening. BIT-C-P3860PCT1

[0019] 3

[0020] According to a further aspect of the invention, there is provided a kit for generating a lineage- restricted cell comprising:

[0021] (i) a source cell and an agent that activates or increases the expression or amount of one or more transcription factors; and / or

[0022] (ii) one or more expression cassette(s) comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the one or more transcription factors are capable of generating those lineage- restricted cells.

[0023] According to a further aspect of the invention, there is provided a use of a kit as defined herein, for forward programming or transdifferentiating a cell into a lineage-restricted cell.

[0024] According to a further aspect of the invention, there is provided a method of drug screening comprising contacting a lineage-restricted cell generated using the method or a lineage- restricted cell as defined herein, with the drug and observing a change in the lineage-restricted cell induced by the drug.

[0025] According to a further aspect of the invention, there is provided a method of treating a subject having or at risk of a disease or disorder comprising administering to the subject a therapeutically effective amount of the lineage-restricted cells generated using the method or the lineage-restricted cells as defined herein.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1. Doxycycline induced cells (black) from large scale pooled screen cluster compared with no doxycycline controls (grey).

[0028] Figure 2. UMAP showing cells expressing ectoderm marker TUBB3 (black) within a large scale pooled screen.

[0029] Figure 3. UMAP showing cells expressing endoderm marker SOX17 (dark grey) within a large scale pooled screen.

[0030] Figure 4. UMAP showing cells expressing mesoderm marker KDR (dark grey) within a large scale pooled screen. BIT-C-P3860PCT1

[0031] 4

[0032] Figure 5. LIMAP showing ciliated cells (black) within the large scale pooled screen.

[0033] Figure 6. LIMAP showing ciliated cells expressing TF FOXJ1 (dark grey).

[0034] Figure 7. Heatmap showing gene expression of ciliated cells with induced programming by doxycycline compared to no doxycycline controls and other cell types made in the screen. X axis is sample ID and y axis is the marker expression of ciliated cells from CZ CellXGene. Z score shows the standardised expression of each gene across the samples.

[0035] Figure 8. LIMAP showing endothelial cells (black) within all cells from large scale pooled screen (other cells light grey).

[0036] Figure 9. UMAPs for endothelial cells showing cells expressing TFs (grey) with specific markers ERG, ETS1 and ETV2 broken (black).

[0037] Figure 10. LIMAP showing ciliated cells (black) within the large scale pooled screen expressing endogenous FOXJ1 using CRISPR activation (dark grey).

[0038] Figure 11. Heatmap showing gene expression of ciliated cells after bulk transcriptomic analysis of multiple replicates of monoclonal cell lines expressing induced exogenous FOXJ1. “NoDox” indicates an absence of doxycycline, so no exogenous gene expression, and “Dox” indicates a presence of doxycycline and exogenous gene expression. X axis is sample ID and y axis is the marker expression of ciliated cells from CZ CellXGene.

[0039] Figure 12. Heatmap showing endothelial cell marker gene expression after bulk transcriptomic analysis of multiple replicates of monoclonal cell lines. X axis is sample ID (“LIBT003” representing the exogenous expression of the ETV2 gene and “LIBT014” representing the exogenous expression of ETV2, ERG and ETS1 genes, “NoDox” indicating an absence of doxycycline, so no exogenous gene expression, and “Dox” indicating a presence of doxycycline and exogenous gene expression) and y axis indicates endothelial cell marker genes obtained from CZ CellXGene CellGuide.

[0040] Figure 13. Plots showing the enrichment p-values for all cell type marker gene sets among genes upregulated following both exogenous expression of the ETV2 gene (A) and exogenous expression of ETV2, ERG and ETS1 genes (B). Marker gene sets, obtained from the CZ BIT-C-P3860PCT1

[0041] 5

[0042] CELLxGENE CellGuide, are ordered from most to least significant (left to right). Endothelial gene sets are the highest scoring following exogenous TF expression in both cell lines. Enrichment was assessed using a hypergeometric test. P-values were adjusted using the Benjamini-Hochberg method.

[0043] Figure 14. Optical images showing cells after five days of exogenous expression of the ETV2 gene, induced using doxycycline (A) and equivalent cells without doxycycline (B).

[0044] Figure 15. Optical images showing cells after five days of exogenous expression of the ETV2, ERG and ETS1 genes, induced using doxycycline (A) and equivalent cells without doxycycline (B).

[0045] Figure 16. Heatmap showing cardiomyocyte gene expression after bulk transcriptomic analysis of multiple replicates of monoclonal cell lines. X axis is sample ID ((“LIBT056” representing the exogenous expression of the MYOD1 , MEF2D and ESRRG genes, “LIBT057” representing the exogenous expression of the MEF2D, MYF6 and ESRRB genes, “LIBT058” representing the exogenous expression of the MEF2D and ESRRB genes and “LIBT059” representing the exogenous expression of the MEF2C, MYF6 and ESRRG genes, “NoDox” indicating an absence of doxycycline, so no exogenous gene expression and “Dox” indicating a presence of doxycycline and exogenous gene expression) and y axis indicates ‘cardiac muscle cell’ marker genes obtained from CZ CellXGene CellGuide.

[0046] Figure 17. Optical images showing cells after five days of exogenous expression of the MEF2D, MYF6 and ESRRB genes (LIBT057 in Figure 16), induced using doxycycline (A) and equivalent cells without doxycycline (B).

[0047] Figure 18. Optical images showing cells after five days of exogenous expression of the MEF2C, MYF6 and ESRRG genes (LIBT059 in Figure 16), induced using doxycycline (A) and equivalent cells without doxycycline (B).

[0048] DETAILED DESCRIPTION

[0049] The present invention provides methods for producing lineage-restricted cells from source cells by expressing of one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of a select group of transcription factors which the present inventors have identified as inducing cell reprogramming into the lineage-restricted cells. BIT-C-P3860PCT1

[0050] 6

[0051] Definitions

[0052] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0053] References to “transcription factor” as used herein, refer to proteins that are involved in gene regulation in both prokaryotic and eukaryotic organisms. In one embodiment, transcription factors can have a positive effect on gene expression and, thus, may be referred to as an “activator” or a “transcriptional activation factor”. In another embodiment, a transcription factor can negatively affect gene expression and, thus, may be referred to as “repressors” or a “transcription repression factor”. Activators and repressors are generally used terms and their functions may be discerned by those skilled in the art.

[0054] The term “increasing the expression of” or “increasing the amount of” with respect to increasing an amount, level or expression of a transcription factor, refers to increasing the quantity of the transcription factor in a cell of interest (e.g., a source cell). In some embodiments, the amount of transcription factor is increased in a cell (e.g., via an expression cassette directing expression of a polynucleotide encoding one or more transcription factors) when the quantity of transcription factor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a control (e.g., a source cell without said expression cassette(s) or a control cell where the baseline expression is zero or negligible). In some of the embodiments, increasing the expression comprises “overexpressing” the transcription factor, i.e., increasing the expression of the transcription factor above the endogenous expression level of the transcription factor in the cell.

[0055] Methods of the invention may be used in a “cell population”, i.e., a collection of cells which may be differentiated into the desired cell type. Said cell population may comprise “source cells”, also referred to as “starting cells”, i.e., a cell type prior to differentiation into the desired cell type.

[0056] References herein to “pluripotent”’ refer to cells which have the potential to differentiate into all types of cell found in an organism. One form of pluripotent stem cell, known as induced pluripotent stem cells, are of particular interest to the present invention. “Induced pluripotent stem cells” (iPSCs) are cells that have been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. In 2006, it was shown that overexpression of four specific BIT-C-P3860PCT1

[0057] 7 transcription factors could convert adult cells into pluripotent stem cells. OCT-3 / 4 and certain members of the SOX gene family have been identified as potentially crucial transcriptional regulators involved in the induction process. Additional genes including certain members of the KLF family, the MYC family, NANOG, and LIN28, may increase the induction efficiency. Examples of the genes which may be used as reprogramming factors to generate iPSCs include OCT3 / 4, S0X2, S0X1, S0X3, S0X15, S0X17, KLF4, KLF2, C-MYC, N-MYC, L-MYC, NANOG, LIN28, F0X15, ERAS, ECAT15-2, TCL1, CTNNB1, LIN28B, SALLI4, ESRRB, TBX3 and GLIS1.

[0058] A “promoter” is a nucleotide sequence which is recognised by proteins involved in initiating and regulating transcription of a polynucleotide sequence. An “inducible promoter” is a nucleotide sequence where expression of a genetic sequence operably linked to the promoter is controlled by an analyte, co-factor, regulatory protein, etc. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional {e.g., controls and / or affects transcription or translation) segments of these regions.

[0059] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a genetic sequence is capable of effecting the expression of that sequence when the regulatory factors are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the genetic sequence and the promoter sequence can still be considered “operably linked” to the genetic sequence. Thus, the term “operably linked” is intended to encompass any spacing or orientation of the promoter element and the genetic sequence in the inducible cassette which allows for initiation of transcription of the inducible cassette upon recognition of the promoter element by a transcription complex.

[0060] The term “vector”, as used herein, is intended to refer to a nucleic acid molecule which is used as a vehicle to carry genetic material into a cell. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop or circle into which additional DNA segments may be ligated. Another type of vector is an infectious but non-pathogenic viral vector, wherein additional DNA segments may be ligated to certain viral genetic elements. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a BIT-C-P3860PCT1

[0061] 8 host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentiviral vectors, adenoviruses, Sendai viruses and adeno- associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. Another type of vector includes synthetic and in vitro transcribed RNA molecules, e.g., mRNA and stabilised RNA, to carry coding genetic information to the cells. This also includes synthetic-self- replicating RNA vectors.

[0062] References to “subject”, “patient” or “individual” refer to a subject, in particular a mammalian subject, to be treated. Mammalian subjects include humans, non-human primates, farm animals (such as cows), sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats or mice. In some embodiments, the subject is a human. In alternative embodiments, the subject is a non-human mammal, such as a mouse.

[0063] The term "sufficient amount" means an amount sufficient to produce a desired effect. The term "therapeutically effective amount" is an amount that is effective to ameliorate a symptom of a disease or disorder. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.

[0064] As used herein, the term “about” when used herein includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between” includes the values of the specified boundaries.

[0065] As used herein, the term “comprises”, and variations such as ‘comprises’ and ‘comprising’, means that the stated components are included, but not to the exclusion of any other component or group of components other than those listed. As used herein, the term “consists of” means that no further components are included other than those listed. As such, reference to transcription factor combinations that “do not consist of” a listed selection, are interpreted to exclude the listed combination only. BIT-C-P3860PCT1

[0066] 9

[0067] It will be understood that any method as described herein may have one or more, or all, steps performed in vitro, ex vivo or in vivo. The methods described herein do not modify the germ line genetic identity of a human being. Transcription factor activity

[0068] The method described herein may comprise increasing the expression (in particular, the protein expression) of a sufficient number of polypeptides having the activity of the transcription factors (or the transcription factors themselves) {e.g., as listed in Table 1 and variants and isoforms thereof) capable of causing forward programming or transdifferentiation of a cell population to lineage-restricted cells. In the context of the present invention, these factors may also be referred to as “reprogramming factors”. As described herein, the expression of an exogenous or endogenous (in particular an exogenous) transcription factor may be increased.

[0069] Table 1. Transcription factors for generation of lineage restricted cell types, including accession numbers (as accessed on 5 September 2025) BIT-C-P3860PCT1

[0070] 10 BIT-C-P3860PCT1

[0071] 11 BIT-C-P3860PCT1

[0072] 12 BIT-C-P3860PCT1

[0073] 13 BIT-C-P3860PCT1

[0074] 14 BIT-C-P3860PCT1

[0075] 15 BIT-C-P3860PCT1

[0076] 16 BIT-C-P3860PCT1

[0077] 17 BIT-C-P3860PCT1

[0078] 18 BIT-C-P3860PCT1

[0079] 19

[0080] In one embodiment, the method comprises expressing one or more polypeptides having the activity of two or more transcription factors, in particular three or more, four or more or five transcription factors and / or increasing the expression of two or more transcription factors, in particular three or more, four or more or five or more transcription factors.

[0081] In one embodiment, the method comprises expressing one or more polypeptides having the activity of less than eight transcription factors, in particular less than seven, less than six, less than five or less than four transcription factors and / or increasing the expression of less than eight transcription factors, in particular less than seven, less than six or less than five transcription factors.

[0082] In one embodiment, the method comprises expressing one or more polypeptides having the activity of between two and eight transcription factors, in particular between three and seven or between four and six transcription factors and / or increasing the expression of between two and eight transcription factors, in particular between three and seven or between four and six transcription factors.

[0083] Once the activity of a transcription factor is appreciated, the endogenous transcription machinery can be modulated using not only the transcription factors themselves, but also BIT-C-P3860PCT1

[0084] 20 polypeptides engineered to replicate the action of the transcription factor, such as synthetic transcription factors or artificial transcription factors. For example, CRISPR (clustered regularly interspaced palindromic repeats), TALE (transcriptional activator-like effector) or Zinc Finger technologies can be used to modulate the expression of endogenous cellular genes, to allow for faster and more efficient nuclear reprogramming under conditions amenable for clinical and commercial applications. This is set out in, for example, LIS2016 / 362705, incorporated herein. Alternatively, with the development of highly accurate protein structure prediction with artificial intelligence tools such as AlphaFold, it is now straightforward for polypeptides to be developed that have very similar structure and / or activity to a transcription factor of interest whilst at the same time having an amino acid sequence that has very little resemblance to that of the transcription factor of interest. For example, large language models trained on biological diversity have been used to develop proteins only around 70% identical to CRISPR-Cas proteins that occur in nature and yet with comparable or improved biological activity and specificity (Ruffolo et al. (2024) bioRxiv, doi: https: / / doi.org / 10.1101 / 2024.04.22.590591). Such polypeptides are covered within the scope of the invention.

[0085] In some embodiments of the present invention, a polypeptide (in particular a single polypeptide) is engineered to mimic the activity of more than one transcription factor of interest. In a further embodiment, polypeptides having the activity of one or more transcription factors is expressed, in combination with increasing the expression of another transcription factor.

[0086] In one embodiment, the method comprises expressing one or more polypeptides having the activity of one or more additional transcription factors, and / or increasing the expression of one or more additional transcription factors. The additional transcription factors may be one or more of the transcription factors listed in Table 1.

[0087] Methods of the invention encompass the use of variants of the transcription factors of interest (i.e., as described in Table 1). References to the transcription factors also encompasses species variants, isoforms, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure and / or function. Changes in the nucleic acid sequence of the transcription factor gene can result in conservative changes or substitutions in the amino acid sequence. Therefore, the invention includes polypeptides having conservative changes or substitutions. The invention includes sequences where conservative substitutions are made that do not alter the activity of the transcription factor protein of interest. BIT-C-P3860PCT1

[0088] 21

[0089] References to a “variant” when referring to a polypeptide could be an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the original, full-length polypeptide. When referring to a nucleic acid sequence, the term “variant” could be, for example, a nucleic acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the original, full-length nucleic acid sequence. The variant could be a fragment of full-length polypeptide, in particular a functional fragment of the polypeptide. The fragment may be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% as long as the full-length wild type polypeptide or a domain thereof having an activity of interest such as the ability to reprogram a source cell into a regulatory T cell. Variations known in the art to eliminate or substantially reduce the activity of the protein are preferably avoided. In some embodiments, the variant lacks an N- and / or C- terminal portion of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either terminus is lacking. In some embodiments, a functional variant or fragment has at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the full-length wild type polypeptide. One of skill in the art will be aware of, or will readily be able to ascertain, whether a particular polypeptide variant or fragment is functional using assays known in the art. For example, the ability of a variant of a transcription factor as listed in Table 1 to generate lineage-restricted cells can be assessed using the assays as described herein. In particular, the variant may be a biologically active variant. A "biologically active variant" includes any variant of a molecule having substantially, at least in part, the same functional and / or biological properties of said molecule, such as binding properties, and / or the same structural features, such as binding domain. It also refers to a molecule that exhibits the same functional features as the transcription factors disclosed herein.

[0090] In one embodiment, an isoform of the listed transcription factor is used. Many transcription factors have one or more isoforms which result, for example, from alternative splicing or from a shifted transcription initiation. Based on the different transcript variants (i.e. mRNA), different polypeptides are generated. It is possible that different transcript variants have different translation initiation sites.

[0091] Glial cells

[0092] The present invention provides a method of generating glial cells, such as oligodendrocyte precursor cells, oligodendrocytes or astrocytes, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain glial cells, wherein the transcription factors are selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , LMX1 B, OVOL1 and ASCL2, preferably BIT-C-P3860PCT1

[0093] 22 in combination with NEUROD1 , FOXJ1 or NEURODI and FOXJ1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 , FOXJ1 , ASCL2, LMX1 B and OVOL1.

[0094] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , LMX1 B, OVOL1 and ASCL2, preferably in combination with NEUROD1 , FOXJ1 or NEUROD1 and FOXJ1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 , FOXJ1. ASCL2, LMXI B and OVOL1.

[0095] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , LMX1 B, OVOL1 and ASCL2, preferably in combination with NEUROD1 , FOXJ1 or NEUROD1 and FOXJ1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 , FOXJ1 , ASCL2, LMX1 B and OVOL1 .

[0096] In one embodiment, the transcription factors are selected from the group consisting of: LMX1 A, SOX8, SOX9, SOX10, LMX1 B, OVOL1 and ASCL2, preferably in combination with one or more of NEUROD1 , FOXJ1 or SOX1 or wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 , FOXJ1 , ASCL2, LMX1 B and OVOL1 , wherein the transcription factors do not consist of SOX1 and NEUROD1 (this specific combination being particularly useful in generating photoreceptor cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 , FOXJ1. ASCL2, LMXI B and OVOL1. In one embodiment, the transcription factors are selected from the group consisting of: LMX1A, SOX8, SOX9 and SOX10, preferably in combination with one or more of NEUROD1 , FOXJ1 or SOX1 or wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 and FOXJ1 , wherein the transcription factors do not consist of SOX1 and NEUROD1 , or wherein the transcription factors are three or more BIT-C-P3860PCT1

[0097] 23 transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 and FOXJ1. These transcription factors have been found to be particularly useful in generating glial cells, such as oligodendrocyte precursor cells, oligodendrocytes or astrocytes.

[0098] In one embodiment, the transcription factors are selected from the group consisting of: LMX1 A, SOX9, SOX10, SOX8, NEUROD1 , FOXJ1 , SOX1 , OVOL1 , LMX1 B and ASCL2 in combination with one or more of HEY1 , ZBTB37 or REST. In one embodiment, the transcription factors are selected from the group consisting of: LMX1A, SOX9, SOX10, SOX8, NEUROD1 , FOXJ1 and SOX1 in combination with one or more of HEY1 , ZBTB37 and REST. These transcription factors have been found to be particularly useful in generating glial cells, such as oligodendrocyte precursor cells, oligodendrocytes, astrocytes or Schwann cells.

[0099] The present invention provides a method of generating glial cells, such as oligodendrocyte precursor cells, oligodendrocytes, astrocytes or Schwann cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain glial cells, wherein the transcription factors comprise ZBTB37, preferably in combination with one or more of LMX1A, SOX9, SOX10, SOX8, HEY1 , NEUROD1 , FOXJ1 , REST, SOX1 , OVOL1 , LMX1 B or ASCL2, or wherein the transcription factors comprise HEY1 and / or REST in combination with one or more of ZBTB37, LMX1A, SOX9, SOX10, SOX8, NEUROD1 , FOXJ1 , SOX1 , OVOL1 , LMX1 B or ASCL2.

[0100] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZBTB37, preferably in combination with one or more of LMX1A, SOX9, SOX10, SOX8, HEY1 , NEUROD1 , FOXJ1 , REST, SOX1 , OVOL1 , LMX1 B, or ASCL2 or wherein the transcription factors comprise HEY1 and / or REST in combination with one or more of ZBTB37, LMX1A, SOX9, SOX10, SOX8, NEUROD1 , FOXJ1 , SOX1 , OVOL1 , LMX1 B or ASCL2.

[0101] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ZBTB37, preferably in combination with one or more of LMX1A, SOX9, SOX10, BIT-C-P3860PCT1

[0102] 24

[0103] SOX8, HEY1 , NEUROD1 , FOXJ1 , REST, SOX1 , OVOL1 , LMX1 B or ASCL2, or wherein the transcription factors comprise HEY1 and / or REST in combination with one or more of ZBTB37, LMX1A, SOX9, SOX10, SOX8, NEUROD1 , FOXJ1 , SOX1 , OVOL1 , LMX1 B or ASCL2.

[0104] In one embodiment, the transcription factors comprise ZBTB37, preferably in combination with one or more of HEY1 , LMX1A, SOX9, SOX10, SOX8, SOX1 , NEUROD1 , REST or FOXJ1 , or the transcription factors comprise HEY1 and / or REST in combination with one or more of ZBTB37, LMX1A, SOX9, SOX10, SOX8, SOX1 , NEU ROD 1 or FOXJ1. These transcription factors have been found to be particularly useful in generating glial cells, such as oligodendrocyte precursor cells, oligodendrocytes, astrocytes or Schwann cells.

[0105] The transcription factors may be selected from the group consisting of: LMX1A, SOX9, SOX10, SOX1 , LMX1 B and OVOL1 preferably in combination with NEUROD1 , FOXJ1 or NEUROD1 and FOXJ1. In particular the transcription factors comprise LMX1A and SOX10. In a further embodiment, the transcription factors comprise LMX1A, SOX10, SOX1 and LMX1 B. In one embodiment, the transcription factors comprise SOX1 and SOX10 in combination with one or more of LMX1A, SOX9, NEUROD1 , FOXJ1 , LMX1 B and / or OVOL1. In one embodiment, the transcription factors comprise LMX1 B in combination with one or more of LMX1A, SOX9, SOX10, SOX1 , NEUROD1 , FOXJ1 and / or OVOL1. In one embodiment, the transcription factors may be selected from the group consisting of: LMX1A, SOX9 and SOX10. These transcription factors have been found to be particularly useful in generating oligodendrocyte precursor cells. In particular the transcription factors may comprise SOX9, SOX10 or both SOX9 and SOX10. These transcription factors have been found to be particularly useful in generating glial cells (particularly both SOX9 and SOX10) and oligodendrocytes.

[0106] The transcription factors may comprise SOX8 and / or ASCL2, preferably in combination with NEUROD1 , FOXJ1 or NEUROD1 and FOXJ1 or two or more transcription factors selected from the group consisting of: SOX8, NEUROD1 , FOXJ1 and ASCL2. In one embodiment, the transcription factors comprise SOX8, preferably in combination with NEUROD1 , FOXJ1 or both NEU ROD 1 and FOXJ1 or two or more transcription factors selected from the group consisting of: SOX8, NEUROD1 and FOXJ1. In one embodiment, the transcription factors are selected from the group consisting of: SOX8, NEUROD1 , FOXJ1 and ASCL2 in combination with one or more of ZBTB37, REST or SOX1 , wherein the transcription factors do not consist of NEUROD1 and SOX1 (this specific combination being particularly useful in generating photoreceptor cells). In one embodiment, the transcription factors are selected from the group consisting of: SOX8, NEUROD1 and FOXJ1 in combination with one or more of ZBTB37, BIT-C-P3860PCT1

[0107] 25

[0108] REST or SOX1 , wherein the transcription factors do not consist of NEUROD1 and SOX1. These transcription factors have been found to be particularly useful in generating astrocytes.

[0109] The transcription factors may comprise ZBTB37, preferably in combination with one or more of SOX8, REST, SOX1 , NEUROD1 , FOXJ1 or ASCL2, or may comprise REST in combination with one or more of ZBTB37, SOX8, SOX1 , NEUROD1 , FOXJ1 or ASCL2. In one embodiment, the transcription factors comprise ZBTB37, preferably in combination with one or more of SOX8, REST, SOX1 , NEUROD1 or FOXJ1 , or the transcription factors comprise REST in combination with one or more of ZBTB37, SOX8, SOX1 , NEUROD1 or FOXJ1 . These transcription factors have been found to be particularly useful in generating astrocytes.

[0110] The transcription factors may comprise HEY1 in combination with SOX8 and / or SOX9. These transcription factors have been found to be particularly useful in generating Schwann cells.

[0111] Retinal cells

[0112] The present invention provides a method of generating retinal cells, such as photoreceptor cells, retinal ganglion cells, amacrine cells or Muller cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain retinal cells, wherein the transcription factors are selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , ATOH1 , HOXB2, POLI4F1 and SOX2, preferably in combination with one or more of KLF4, ASCL1 , TCF3, LMX1A, SOX1 , SOX9 or SOXIO, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , SOX1 , SOX2, SOX9, SOX10, ATOH1 , HOXB2 POU4F1 , KLF4, ASCL1 , TCF3, HOXB2, LMX1A and NR2E1 , wherein the transcription factors do not consist of SOX9 and SOX10, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , SOX1 , SOX2, SOX9, SOX10, ATOH1 , HOXB2 POU4F1 , KLF4, ASCL1 , TCF3, HOXB2, LMX1A and NR2E1.

[0113] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: NEUROD1 , BIT-C-P3860PCT1

[0114] 26

[0115] NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , ATOH1 , HOXB2, POU4F1 and SOX2, preferably in combination with one or more of KLF4, ASCL1 , TCF3, LMX1A, SOX1 , SOX9 or SOX10, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , SOX1 , SOX2, SOX9, SOX10, ATOH1 , HOXB2 POU4F1 , KLF4, ASCL1 , TCF3, HOXB2, LMX1A and NR2E1 , wherein the transcription factors do not consist of SOX9 and SOX10 (this specific combination being particularly useful in generating oligodendrocytes), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , SOX1 , SOX2, SOX9, SOX10, ATOH1 , HOXB2 POU4F1 , KLF4, ASCL1 , TCF3, HOXB2, LMX1A and NR2E1.

[0116] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , ATOH1 , HOXB2, POLI4F1 and SOX2, preferably in combination with one or more of KLF4, ASCL1 , TCF3, LMX1A, SOX1 , SOX9 or SOX10, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , SOX1 , SOX2, SOX9, SOX10, ATOH1 , HOXB2 POU4F1 , KLF4, ASCL1 , TCF3, HOXB2, LMX1 A and NR2E1 , wherein the transcription factors do not consist of SOX9 and SOX10 (this specific combination being particularly useful in generating oligodendrocytes), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: NEUROD1 , NEUROD2, NEUROD4, ZBTB42, ZNF282, NR2E1 , SOX1 , SOX2, SOX9, SOX10, ATOH1 , HOXB2 POU4F1 , KLF4, ASCL1 , TCF3, HOXB2, LMX1A and NR2E1.

[0117] The transcription factors may be selected from the group consisting of: NEUROD1 , NEUROD2, ATOH1 , HOXB2 POU4F1 and SOX2, preferably in combination with SOX1. In one embodiment, the transcription factors comprise NEUROD1 in combination with one or more of NEUROD2, ATOH1 , HOXB2, SOX1 , POLI4F1 or SOX2. In one embodiment, the transcription factors are selected from the group consisting of: NEUROD1 , NEUROD2, ATOH1 , HOXB2, SOX1 , preferably in combination with POLI4F1 , SOX2 or both POLI4F1 and SOX2. In one embodiment, the transcription factors are selected from the group consisting of: NEUROD1 , NEUROD2, ATOH1 , HOXB2 and SOX1. These transcription factors have been found to be particularly useful in generating photoreceptor cells. The transcription factors may comprise ZNF282, preferably in combination with KLF4, ASCL1 or KLF4 and ASCL1 , or two or more BIT-C-P3860PCT1

[0118] 27 transcription factors selected from the group consisting of: ZNF282, ASCL1 and KLF4. In one embodiment the transcription factors comprise ASCL1 in combination with ZNF282, KLF4 or ZNF282 and KLF4. In one embodiment, the transcription factors comprise KLF4 in combination with ASCL1 , ZNF282 or both ASCL1 and ZNF282. In one embodiment, the transcription factors are selected from the group consisting of: ZNF282, KLF4 and ASCL1 in combination with one or more of NR2F1 , CDX2, HOXB2, MYT1 or SOX3, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and HOXB2, (iii) ZNF282 and CDX2, (iv) ZNF282 and HOXB2, (v) ASCL1 , CDX2 and HOXB2, (vi) ZNF282, CDX2 and HOXB2, (vii) ASCL1 , ZNF282 and CDX2, (viii) ASCL1 , ZNF282 and HOXB2 or (ix) ASCL1 , ZNF282, CDX2 and HOXB2 (these specific combinations being particularly useful in generating amacrine cells). In one embodiment, the transcription factors comprise CDX2, ASCL1 and KLF4. In one embodiment, the transcription factors comprise MYT1 in combination with ASCL1 , ZNF282 or both ASCL1 and ZNF282. These transcription factors have been found to be particularly useful in generating retinal ganglion cells.

[0119] The present invention provides a method of generating retinal ganglion cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain retinal ganglion cells, wherein the transcription factors comprise NR2F1 , preferably in combination with one or more of SOX3, ASCL1 , KLF4, ZNF282, CDX2, HOXB2 or MYT1 , or wherein the transcription factors are selected from the group consisting of: SOX3, CDX2, HOXB2 and MYT1 in combination with one or more of NR2F1 , ASCL1 , KLF4 or ZNF282, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and HOXB2, (iii) ZNF282 and CDX2, (iv) ZNF282 and HOXB2, (v) ASCL1 , CDX2 and HOXB2, (vi) ZNF282, CDX2 and HOXB2, (vii) ASCL1 , ZNF282 and CDX2, (viii) ASCL1 , ZNF282 and HOXB2 or (ix) ASCL1 , ZNF282, CDX2 and HOXB2 (these specific combinations being particularly useful in generating amacrine cells).

[0120] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise NR2F1 , preferably in combination with one or more of SOX3, ASCL1 , KLF4, ZNF282, CDX2, HOXB2 or MYT1 , or wherein the transcription factors are selected from the group consisting of: SOX3, CDX2, HOXB2 and MYT1 in combination with one or more of NR2F1 , ASCL1 , KLF4 or ZNF282, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and HOXB2, BIT-C-P3860PCT1

[0121] 28

[0122] (iii) ZNF282 and CDX2, (iv) ZNF282 and HOXB2, (v) ASCL1 , CDX2 and HOXB2, (vi) ZNF282, CDX2 and HOXB2, (vii) ASCL1 , ZNF282 and CDX2, (viii) ASCL1 , ZNF282 and HOXB2 or (ix) ASCL1 , ZNF282, CDX2 and HOXB2.

[0123] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise NR2F1 , preferably in combination with one or more of SOX3, ASCL1 , KLF4, ZNF282, CDX2, HOXB2 or MYT1 , or wherein the transcription factors are selected from the group consisting of: SOX3, CDX2, HOXB2 and MYT1 in combination with one or more of NR2F1 , ASCL1 , KLF4 or ZNF282, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and HOXB2, (iii) ZNF282 and CDX2, (iv) ZNF282 and HOXB2, (v) ASCL1 , CDX2 and HOXB2, (vi) ZNF282, CDX2 and HOXB2, (vii) ASCL1 , ZNF282 and CDX2, (viii) ASCL1 , ZNF282 and HOXB2 or (ix) ASCL1 , ZNF282, CDX2 and HOXB2.

[0124] In one embodiment, the transcription factors comprise KLF4, ASCL1 and CDX2. In one embodiment, the transcription factors comprise MYT1 in combination with ASCL1 , ZNF282 or both ASCL1 and ZNF282. These transcription factors have been found to be particularly useful in generating retinal ganglion cells.

[0125] The transcription factors may comprise NR2E1 , preferably in combination with one or more of ASCL1 , TCF3 or HOXB2, or two or more transcription factors selected from the group consisting of: NR2E1 , ASCL1 , TCF3 and HOXB2. In one embodiment, the transcription factors comprise ASCL1 , preferably in combination with one or more of HOXB2, TCF3 or NR2E1 . In one embodiment, the transcription factors comprise ASCL1 , preferably in combination with HOXB2, TCF3 or both HOXB2 and TCF3 (preferably TCF3), or two or more transcription factors selected from the group consisting of: ASCL1 , HOXB2 and TCF3. In one embodiment, the transcription factors are selected from the group consisting of: ASCL1 , HOXB2, TCF3 and NR2E1 in combination with one or more of FOXN2, ZNF263, ZNF282 or CDX2. In one embodiment, the transcription factors are selected from the group consisting of: ASCL1 , HOXB2 and TCF3 in combination with one or more of FOXN2, ZNF263, ZNF282 or CDX2. These transcription factors have been found to be particularly useful in generating amacrine cells (including glycinergic amacrine cells).

[0126] The present invention provides a method of generating amacrine cells, preferably glycinergic amacrine cells, comprising expressing one or more polypeptides having the activity BIT-C-P3860PCT1

[0127] 29 of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain amacrine cells, wherein the transcription factors comprise FOXN2 and / or ZNF263, preferably in combination with one or more transcription factors selected from the group consisting of: ZNF282, ASCL1 , CDX2, HOXB2, TCF3 and NR2E1 , or wherein the transcription factors comprise ZNF282 and / or CDX2 in combination with one or more transcription factors selected from the group consisting of: FOXN2, ZNF263, ASCL1 , HOXB2, TCF3 and NR2E1.

[0128] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise FOXN2 and / or ZNF263, preferably in combination with one or more of ZNF282, ASCL1 , CDX2, HOXB2, TCF3 or NR2E1 or wherein the transcription factors comprise ZNF282 and / or CDX2 in combination with one or more transcription factors selected from the group consisting of: FOXN2, ZNF263, ASCL1 , HOXB2, TCF3 and NR2E1.

[0129] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise FOXN2 and / or ZNF263, preferably in combination with one or more of ZNF282, ASCL1 , CDX2, HOXB2, TCF3 or NR2E1 orwherein the transcription factors comprise ZNF282 and / or CDX2 in combination with one or more transcription factors selected from the group consisting of: FOXN2, ZNF263, ASCL1 , HOXB2, TCF3 and NR2E1.

[0130] In one embodiment, the transcription factors comprise FOXN2 and / or ZNF263, preferably in combination with one or more transcription factors selected from the group consisting of: ZNF282, ASCL1 , CDX2, HOXB2 and TCF3, or wherein the transcription factors comprise ZNF282 and / or CDX2 in combination with one or more transcription factors selected from the group consisting of: FOXN2, ZNF263, ASCL1 , HOXB2 and TCF3. In one embodiment, the transcription factors comprise ZNF282 in combination with ASCL1 , HOXB2 or both ASCL1 and HOXB2. These transcription factors have been found to be particularly useful in generating amacrine cells, preferably glycinergic amacrine cells.

[0131] The transcription factors may comprise NEUROD4 and / or ZBTB42, preferably in combination with one or more of LMX1A, SOX9, SOX10, SOX2 or NR2E1 , or two or more transcription BIT-C-P3860PCT1

[0132] 30 factors selected from the group consisting of: NEUROD4, ZBTB42, LMX1A, SOX2, SOX9, SOX10 and NR2E1 , wherein the transcription factors do not consist of SOX9 and SOX10 (this specific combination being particularly useful in generating oligodendrocytes) or three or more transcription factors selected from the group consisting of: NEUROD4, ZBTB42, LMX1A, SOX2, SOX9, SOX10 and NR2E1. These transcription factors have been found to be particularly useful in generating Muller cells.

[0133] Kidney Cells

[0134] The present invention provides a method of generating kidney cells, such as podocytes, loop of Henle epithelial cells, collecting duct principal cells, interstitial fibroblasts, papillary tip cells, proximal tubule epithelial cells or parietal epithelial cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain kidney cells, wherein the transcription factors are selected from the group consisting of: FOXC2, FOXI3, FOXP1 , NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF7, CREBZF, CXXC4, MEF2B, NFIX, KLF2, HNF1A and TBX2, preferably in combination with one or more of RREB1 , KLF4, TFAP2E, FOXA2, MEF2D or GATA4, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: FOXA2, FOXC2, FOXI3, FOXP1 , TFAP2E, NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF4, KLF7, CREBZF, CXXC4, MEF2B, MEF2D, NFIX, KLF2, HNF1A, TBX2, RREB1 and GATA4, wherein the transcription factors do not consist of TFAP2E and FOXA2 (this specific combination being particularly useful in generating respiratory epithelial cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: FOXA2, FOXC2, FOXI3, FOXP1 , TFAP2E, NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF4, KLF7, CREBZF, CXXC4, MEF2B, MEF2D, NFIX, KLF2, HNF1A, TBX2, RREB1 and GATA4.

[0135] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: FOXC2, FOXI3, FOXP1 , NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF7, CREBZF, CXXC4, MEF2B, NFIX, KLF2, HNF1A and TBX2, preferably in combination with one or more of RREB1 , KLF4, TFAP2E, FOXA2, MEF2D or GATA4, or wherein the transcription factors are two or more transcription factors selected from BIT-C-P3860PCT1

[0136] 31 the group consisting of: FOXA2, FOXC2, FOXI3, FOXP1 , TFAP2E, NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF4, KLF7, CREBZF, CXXC4, MEF2B, MEF2D, NFIX, KLF2, HNF1A, TBX2, RREB1 and GATA4, wherein the transcription factors do not consist of TFAP2E and FOXA2 (this specific combination being particularly useful in generating respiratory epithelial cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: FOXA2, FOXC2, FOXI3, FOXP1 , TFAP2E, NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF4, KLF7, CREBZF, CXXC4, MEF2B, MEF2D, NFIX, KLF2, HNF1A, TBX2, RREB1 and GATA4.

[0137] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: FOXC2, FOXI3, FOXP1 , NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF7, CREBZF, CXXC4, MEF2B, NFIX, KLF2, HNF1 A and TBX2, preferably in combination with one or more of RREB1 , KLF4, TFAP2E, FOXA2, MEF2D or GATA4, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: FOXA2, FOXC2, FOXI3, FOXP1 , TFAP2E, NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF4, KLF7, CREBZF, CXXC4, MEF2B, MEF2D, NFIX, KLF2, HNF1A, TBX2, RREB1 and GATA4, wherein the transcription factors do not consist of TFAP2E and FOXA2 (this specific combination being particularly useful in generating respiratory epithelial cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: FOXA2, FOXC2, FOXI3, FOXP1 , TFAP2E, NKX2.6, BARHL2, NFIC, NR0B1 , POU6F2, SOX3, SOX15, CASZ1 , GRHL3, HES1 , BMP2, IRF1 , IRF5, KLF4, KLF7, CREBZF, CXXC4, MEF2B, MEF2D, NFIX, KLF2, HNF1A, TBX2, RREB1 and GATA4.

[0138] The transcription factors may be selected from the group consisting of: CREBZF, IRF5, BMP2, KLF7 and IRF1 , preferably in combination with GATA4. In one embodiment, the transcription factors comprise CREBZF, preferably in combination with one or more of GATA4, IRF5, IRF1 , BMP2 or KLF7, or two or more transcription factors selected from the group consisting of: CREBZF, GATA4, IRF5, I RF1 , BMP2 and KLF7. In one embodiment, the transcription factors comprise CREBZF, preferably in combination with GATA4, IRF5, or both GATA4 and IRF5, or wherein the transcription factors are two or more transcription factors selected from the group BIT-C-P3860PCT1

[0139] 32 consisting of: CREBZF, GATA4 and IRF5. These transcription factors have been found to be particularly useful in generating podocytes.

[0140] The transcription factors may be selected from the group consisting of: BARHL2, NR0B1 , POLI6F2, SOX15, NFIC and NKX2.6, preferably in combination with one or more of TFAP2E, HNF1A or IRF5, or two or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX15, HNF1A, NFIC, NKX2.6 and IRF5. In one embodiment, the transcription factors are selected from the group consisting of: BARHL2, NR0B1 , POLI6F2 and TFAP2E, preferably in combination with one or more of SOX15, NFIC, HNF1A, NKX2.6 or IRF5, or the transcription factors are two or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX15, HNF1A, NFIC, NKX2.6 and IRF5. In one embodiment, the transcription factors are selected from the group consisting of: BARHL2, NR0B1 , POLI6F2 and TFAP2E, preferably in combination with one or more of SOX15, NFIC, or HNF1A, or the transcription factors are two or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POLI6F2, SOX15, NFIC and HNF1A. In one embodiment, the transcription factors are selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX15, HNF1A, NFIC, NKX2.6 and IRF5 in combination with FAM200B, SOX9 or both FAM200B and SOX9. In one embodiment, the transcription factors are selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, NFIC, SOX15 and HNF1A in combination with FAM200B, SOX9 or both FAM200B and SOX9. These transcription factors have been found to be particularly useful in generating loop of Henle epithelial cells.

[0141] The present invention provides a method of generating loop of Henle epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain loop of Henle epithelial cells, wherein the transcription factors comprise FAM200B, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX9, SOX15, HNF1A, NFIC, NKX2.6 and IRF5, or wherein the transcription factors comprise SOX9 in combination with one or more transcription factors selected from the group consisting of: FAM200B, TFAP2E, BARHL2, NR0B1 , POU6F2, SOX15, HNF1A, NFIC, NKX2.6 and IRF5.

[0142] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides BIT-C-P3860PCT1

[0143] 33 having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise FAM200B, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX9, SOX15, HNF1A, NFIC, NKX2.6 and IRF5, or wherein the transcription factors comprise SOX9 in combination with one or more transcription factors selected from the group consisting of: FAM200B, TFAP2E, BARHL2, NR0B1 , POLI6F2, SOX15, HNF1A, NFIC, NKX2.6 and IRF5.

[0144] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise FAM200B, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX9, SOX15, HNF1A, NFIC, NKX2.6 and IRF5, or wherein the transcription factors comprise SOX9 in combination with one or more transcription factors selected from the group consisting of: FAM200B, TFAP2E, BARHL2, NR0B1 , POU6F2, SOX15, HNF1A, NFIC, NKX2.6 and IRF5.

[0145] In one embodiment, the transcription factors comprise FAM200B, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX9, SOX15, HNF1A and NFIC, or wherein the transcription factors comprise SOX9 in combination with one or more transcription factors selected from the group consisting of: FAM200B, TFAP2E, BARHL2, NR0B1 , POLI6F2, SOX15, HNF1A and NFIC. These transcription factors have been found to be particularly useful in generating loop of Henle epithelial cells.

[0146] The transcription factors may be selected from the group consisting of: CASZ1 , GRHL3 and HES1 , preferably in combination with one or more of I RF5, RREB1 , KLF4, FOXA2, orTFAP2E, or two or more transcription factors selected from the group consisting of: IRF5, RREB1 , CASZ1 , GRHL3, HES1 , KLF4, FOXA2 or TFAP2E, wherein the transcription factors do not consist of FOXA2 and TFAP2E (this specific combination being particularly useful in generating respiratory epithelial cells) or three or more transcription factors selected from the group consisting of: IRF5, RREB1 , CASZ1 , GRHL3, HES1 , KLF4, FOXA2 and TFAP2E. In one embodiment the transcription factors comprise HES1 in combination with one or more of IRF5, RREB1 , CASZ1 , GRHL3, KLF4, FOXA2 and / or TFAP2E. In one embodiment the transcription factors comprise IRF5 and RREB1. These transcription factors have been found to be particularly useful in generating collecting duct principal cells. BIT-C-P3860PCT1

[0147] 34

[0148] The transcription factors may be selected from the group consisting of: FOXC2, CXXC4, MEF2B, SOX3, TBX2, FOXI3 and NFIX, preferably in combination with MEF2D. In one embodiment, the transcription factors are selected from the group consisting of: CXXC4, FOXC2, MEF2B and TBX2, preferably in combination with one or more of MEF2D, SOX3, FOXI3 or NFIX or wherein the transcription factors are two or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D, SOX3, FOXI3 and NFIX. In one embodiment, the transcription factors are selected from the group consisting of: CXXC4, FOXC2, MEF2B and TBX2, preferably in combination with MEF2D, SOX3 or both MEF2D and SOX3, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3. In one embodiment, the transcription factors are selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3 in combination with HOXB9, ZNF570 or both HOXB9 and ZNF570. These transcription factors have been found to be particularly useful in generating interstitial fibroblasts.

[0149] The present invention provides a method of generating interstitial fibroblasts comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain interstitial fibroblasts, wherein the transcription factors comprise HOXB9 and / or ZNF570, preferably in combination with one or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D, SOX3, FOXI3 and NFIX.

[0150] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise HOXB9 and / or ZNF570, preferably in combination with one or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D, SOX3 FOXI3 and NFIX.

[0151] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise HOXB9 and / or ZNF570, preferably in combination with one or more transcription BIT-C-P3860PCT1

[0152] 35 factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D, SOX3, FOXI3 and NFIX.

[0153] In one embodiment, the transcription factors comprise HOXB9 and / or ZNF570, preferably in combination with one or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3. These transcription factors have been found to be particularly useful in generating interstitial fibroblasts.

[0154] The transcription factors may comprise KLF2. In one embodiment, the transcription factors comprise KLF2 in combination with one or more of KLF1 , TBX22, TFAP2B or GRHL3 (preferably KLF1), wherein the transcription factors do not consist of KLF2 and GRHL3 (this specific combination being particularly useful in generating type I pneumocytes). These transcription factors have been found to be particularly useful in generating papillary tip cells.

[0155] The present invention provides a method of generating papillary tip cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain papillary tip cells, wherein the transcription factors comprise TBX22, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2B, GRHL3, KLF1 and KLF2, or wherein the transcription factors are selected from the group consisting of: KLF1 , GRHL3, and TFAP2B in combination with KLF2, TBX22 or both TBX22 and KLF2, wherein the transcription factors do not consist of KLF2 and GRHL3 (this specific combination being particularly useful in generating type I pneumocytes).

[0156] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise TBX22, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2B, GRHL3, KLF1 and KLF2, or wherein the transcription factors are selected from the group consisting of: KLF1 , GRHL3, and TFAP2B in combination with KLF2, TBX22 or both KLF2 and TBX22, wherein the transcription factors do not consist of KLF2 and GRHL3.

[0157] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription BIT-C-P3860PCT1

[0158] 36 factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise TBX22, preferably in combination with one or more transcription factors selected from the group consisting of: TFAP2B, GRHL3, KLF1 and KLF2, or wherein the transcription factors are selected from the group consisting of: KLF1 , GRHL3 and TFAP2B in combination with KLF2, TBX22 or both KLF2 and TBX22, wherein the transcription factors do not consist of KLF2 and GRHL3.

[0159] The transcription factors may comprise HNF1A. In one embodiment, the transcription factors comprise HNF1A in combination with ANHX or both ANHX and HNF1 B. These transcription factors have been found to be particularly useful in generating proximal tubule epithelial cells.

[0160] The present invention provides a method of generating proximal tubule epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain proximal tubule epithelial cells, wherein the transcription factors comprise ANHX, preferably in combination with HNF1A, HNF1 B or both HNF1A and HNF1 B.

[0161] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ANHX, preferably in combination with HNF1A, HNF1 B or both HNF1A and HNF1 B.

[0162] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ANHX, preferably in combination with HNF1A, HNF1 B or both HNF1A and HNF1 B.

[0163] The transcription factors may comprise FOXP1. This transcription factor has been found to be particularly useful in generating parietal epithelial cells.

[0164] Lung cells

[0165] The present invention provides a method of generating lung cells, such as respiratory epithelial cells, basal epithelial cells of the tracheobronchial tree, pneumocytes, club cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors BIT-C-P3860PCT1

[0166] 37 and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain lung cells, wherein the transcription factors are selected from the group consisting of: PAX5, FOXA1 , FOXA2, WIZ, TFAP2B, EN1 , GBX1 , HOXD12, ZNF74, HEY1 and PITX2, preferably in combination with one or more of IRF5, MYC, KLF2, FOXA2, FOXJ1 , ZBTB42, TFAP2E, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, GFI1 B, RREB1 or BMP2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: PAX5, FOXA1 , FOXA2, FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2, wherein the transcription factors do not consist of FOXA2 and GFI1 B or CEBPB and GFI1 B, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: PAX5, FOXA1 , FOXA2, FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2 or variants thereof.

[0167] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: PAX5, FOXA1 , FOXA2, WIZ, TFAP2B, EN1 , GBX1 , HOXD12, ZNF74, HEY1 and PITX2, preferably in combination with one or more of IRF5, MYC, KLF2, FOXA2, FOXJ1 , ZBTB42, TFAP2E, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, GFI1 B, RREB1 or BMP2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: PAX5, FOXA1 , FOXA2, FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2, wherein the transcription factors do not consist of FOXA2 and GFI1 B (this specific combination being particularly useful in generating enteroendocrine cells) or CEBPB and GFI1 B (this specific combination being particularly useful in generating prostate epithelial cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: PAX5, FOXA1 , FOXA2, FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2.

[0168] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are BIT-C-P3860PCT1

[0169] 38 selected from the group consisting of: PAX5, FOXA1 , FOXA2, WIZ, TFAP2B, EN1 , GBX1 , HOXD12, ZNF74, HEY1 and PITX2, preferably in combination with one or more of IRF5, MYC, KLF2, FOXA2, FOXJ1 , ZBTB42, TFAP2E, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, GFI1 B, RREB1 or BMP2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: PAX5, FOXA1 , FOXA2, FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2, wherein the transcription factors do not consist of FOXA2 and GFI1 B (this specific combination being particularly useful in generating enteroendocrine cells) or CEBPB and GFI1 B (this specific combination being particularly useful in generating prostate epithelial cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: PAX5, FOXA1 , FOXA2, FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2.

[0170] In one embodiment, the transcription factors comprise FOXA2 in combination with one or more of PAX5, FOXA1 , FOXJ1 , GFI1 B, WIZ, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, ZNF74, ZBTB42, HEY1 , PITX2, IRF5, MYC, KLF2, CEBPB, HOXB2, NFIX, CREBZF, GRHL3, NFIC, RREB1 and BMP2, wherein the transcription factors do not consist of FOXA2 and GFI1 B (this specific combination being particularly useful in generating enteroendocrine cells).

[0171] The transcription factors may be selected from the group consisting of: FOXA2, TFAP2B, EN1 , GBX1 , WIZ and HOXD12, preferably in combination with one or more of IRF5, MYC, KLF2, FOXJ1 , ZBTB42 or TFAP2E, or two or more transcription factors selected from the group consisting of: FOXA2, TFAP2B, TFAP2E, EN1 , GBX1 , HOXD12, WIZ, IRF5, MYC, KLF2, FOXJ1 and ZBTB42. In one embodiment, the transcription factors comprise FOXA2, preferably in combination with one or more of GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 or ZBTB42. In one embodiment, the transcription factors are selected from the group consisting of: FOXA2, GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42 in combination with one or more of HEY2, PRDM6, ZBTB18, FOXA1. GFI1 , NFIX or SOXI . In one embodiment, the transcription factors comprise FOXA2 in combination with one or more of HEY2, PRDM6, ZBTB18, FOXA1 , GF11 , NFIX or SOX1. These transcription factors have been found to be particularly useful in generating respiratory epithelial cells, including epithelial cells of the lower respiratory tract. BIT-C-P3860PCT1

[0172] 39

[0173] The present invention provides a method of generating respiratory epithelial cells, preferably epithelial cells of the lower respiratory tract, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain respiratory epithelial cells, wherein the transcription factors are selected from the group consisting of: PRDM6, HEY2 and ZBTB18, preferably in combination with one or more transcription factors selected from the group consisting of: FOXA2, FOXA1 , GFI1 , NFIX, SOX1 , GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42, or wherein the transcription factors are selected from the group consisting of: FOXA1 , GF11 , NFIX and SOX1 in combination with one or more transcription factors selected from the group consisting of: PRDM6, HEY2, ZBTB18, FOXA2, GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42.

[0174] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: PRDM6, HEY2 and ZBTB18, preferably in combination with one or more transcription factors selected from the group consisting of: FOXA2, FOXA1 , GFI1 , NFIX, SOX1 , GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42, or wherein the transcription factors are selected from the group consisting of: FOXA1 , GF11 , NFIX and SOX1 in combination with one or more transcription factors selected from the group consisting of: PRDM6, HEY2, ZBTB18, FOXA2, GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42.

[0175] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: PRDM6, HEY2 and ZBTB18, preferably in combination with one or more transcription factors selected from the group consisting of: FOXA2, FOXA1 , GFI1 , NFIX, SOX1 , GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42, or wherein the transcription factors are selected from the group consisting of: FOXA1 , GFI1 , NFIX and SOX1 in combination with one or more transcription factors selected from the group consisting of: PRDM6, HEY2, ZBTB18, FOXA2, GBX1 , HOXD12, KLF2, WIZ, IRF5, MYC, TFAP2B, TFAP2E, EN1 , FOXJ1 and ZBTB42. BIT-C-P3860PCT1

[0176] 40

[0177] In one embodiment, the transcription factors are selected from the group consisting of: PRDM6, HEY2 and ZBTB18, preferably in combination with one or more transcription factors selected from the group consisting of: FOXA2, FOXA1 , GFI1 , NFIX and SOX1 , or the transcription factors are selected from the group consisting of: FOXA1 , GFI1 , NFIX and SOX1 in combination with one or more transcription factors selected from the group consisting of: PRDM6, HEY2, ZBTB18 and FOXA2. These transcription factors have been found to be particularly useful in generating respiratory epithelial cells, including epithelial cells of the lower respiratory tract.

[0178] In particular, the transcription factors may be selected from the group consisting of: GBX1 , WIZ and HOXD12, preferably in combination with one or more of IRF5, MYC or KLF2, or two or more transcription factors selected from the group consisting of: GBX1 , HOXD12, WIZ, IRF5, MYC and KLF2. In one embodiment, the transcription factors are selected from the group consisting of: GBX1 , WIZ, HOXD12 and KLF2, preferably in combination with IRF5, MYC or both IRF5 and MYC. These transcription factors have been found to be particularly useful in generating basal epithelial cells of the tracheobronchial tree.

[0179] The transcription factors may comprise PAX5, preferably in combination with one or more of GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, HOXB2 or CREBZF, or two or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 and HOXB2. In one embodiment, the transcription factors comprise GRHL3 and / or NFIC, preferably in combination with one or more of CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 or HOXB2, or two or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 and HOXB2, wherein the transcription factors do not consist of (i) CEBPB and KLF2 (this specific combination being particularly useful in generating club cells) or (ii) KLF2 and MYC (this specific combination being particularly useful in generating basal epithelial cells of the tracheobronchial tree), or three or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 and HOXB2. In one embodiment, the transcription factors comprise GRHL3 and / or NFIC, preferably in combination with one or more of CEBPB, MYC, KLF2 and NFIX, or two or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX, wherein the transcription factors do not consist of (i) CEBPB and KLF2 or (ii) KLF2 and MYC, or three or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX. In one embodiment, the transcription factors are selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX in combination with BIT-C-P3860PCT1

[0180] 41

[0181] ZNF441. These transcription factors have been found to be particularly useful in generating type 1 pneumocytes.

[0182] The present invention provides a method of generating type 1 pneumocytes comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain type 1 pneumocytes, wherein the transcription factors comprise ZNF441 , preferably in combination with one or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 and HOXB2.

[0183] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZNF441 , preferably in combination with one or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 and HOXB2.

[0184] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ZNF441 , preferably in combination with one or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2, NFIX, CREBZF, PAX5 and HOXB2.

[0185] In one embodiment, the transcription factors comprise ZNF441 , preferably in combination with one or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX. These transcription factors have been found to be particularly useful in generating type 1 pneumocytes.

[0186] The transcription factors may comprise HEY1 and / or PITX2. In one embodiment, the transcription factors comprise HEY1 and / or PITX2 in combination with one or more of SKOR2, SOX21 or CEBPB. These transcription factors have been found to be particularly useful in generating type 2 pneumocytes. BIT-C-P3860PCT1

[0187] 42

[0188] The present invention provides a method of generating type 2 pneumocytes comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain type 2 pneumocytes, wherein the transcription factors comprise SKOR2 and / or SOX21 , preferably in combination with one or more transcription factors selected from the group consisting of: CEBPB, HEY1 or PITX2.

[0189] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise SKOR2 and / or SOX21 , preferably in combination with one or more transcription factors selected from the group consisting of: CEBPB, HEY1 or PITX2.

[0190] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise SKOR2 and / or SOX21 , preferably in combination with one or more transcription factors selected from the group consisting of: CEBPB, HEY1 or PITX2.

[0191] In one embodiment, the transcription factors comprise SKOR2 and / or SOX21 , preferably in combination with CEBPB. These transcription factors have been found to be particularly useful in generating type 2 pneumocytes.

[0192] The transcription factors may comprise FOXA1 , preferably in combination with one or more of CEBPB, RREB1 , GFI1 B, KLF2 or FOXA2, or two or more transcription factors selected from the group consisting of: CEBPB, RREB1 , FOXA1 , FOXA2, GFI1 B and KLF2, wherein the transcription factors do not consist of CEBPB and GFI1 B (this specific combination being particularly useful in generating prostate epithelial cells), FOXA2 and GFI1 B (this specific combination being particularly useful in generating enteroendocrine cells), or KLF2 and CEBPB (this specific combination being particularly useful in generating type 1 pneumocytes), or three or more transcription factors selected from the group consisting of: FOXA1 , FOXA2, CEBPB, GFI1 B, KLF2 and RREB1. In one embodiment, the transcription factors comprise CEBPB and / or RREB1 , preferably in combination with one or more of GFI1 B, FOXA1 , GFI1 , KLF2 or FOXA2 (preferably GFI1 B), or FOXA1 in combination with one or more of CEBPB, RREB1 , GFI1 B, GF11 , KLF2 or FOXA2, wherein the transcription factors do not consist of (i) BIT-C-P3860PCT1

[0193] 43

[0194] FOXA1 and FOXA2, (ii) FOXA1 and GFI1 or (iii) FOXA1 , FOXA2 and GFI1 (these specific combinations being particularly useful in generating respiratory epithelial cells). In one embodiment, the transcription factors comprise CEBPB, preferably in combination with one or more of FOXA2, GFI1 or KLF2. In one embodiment, the transcription factors comprise RREB1 , preferably in combination with KLF2. In one embodiment, the transcription factors are selected from the group consisting of: CEBPB, GFI1 B, FOXA1 , GFI1 , KLF2, RREB1 and FOXA2 in combination with MAF. These transcription factors have been found to be particularly useful in generating club cells.

[0195] The present invention provides a method of generating club cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain club cells, wherein the transcription factors comprise MAF in combination with one or more of CEBPB, GFI1 B, FOXA1 , GFI1 , KLF2, RREB1 or FOXA2.

[0196] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise MAF in combination with one or more of CEBPB, GF11 B, FOXA1 , GF11 , KLF2, RREB1 or FOXA2.

[0197] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise MAF in combination with one or more of CEBPB, GFI1 B, FOXA1 , GFI1 , KLF2, RREB1 or FOXA2.

[0198] Muscle cells

[0199] The present invention provides a method of generating muscle cells, such as pericytes, cardiac muscle cells, skeletal muscle cells, skeletal satellite muscle cells, smooth muscle cells, enteric smooth muscle cells or bronchial smooth muscle cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain muscle cells, wherein the transcription factors are selected from the group consisting of: ESRRG, MEF2C, MEF2D, NKX2.3, MYF6, MYOD1 , ESRRB, PHOX2B, ATF6, BIT-C-P3860PCT1

[0200] 44

[0201] ZNF74, NKX2.1 and MEF2A, preferably in combination with SOX9, BMP2 or SOX9 and BMP2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 and ATF6.

[0202] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: ESRRG, MEF2C, MEF2D, NKX2.3, MYF6, MYOD1 , ESRRB, PHOX2B, ATF6, ZNF74, NKX2.1 and MEF2A, preferably in combination with SOX9, BMP2 or SOX9 and BMP2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 and ATF6.

[0203] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: ESRRG, MEF2C, MEF2D, NKX2.3, MYF6, MYOD1 , ESRRB, PHOX2B, ATF6, ZNF74, NKX2.1 and MEF2A, preferably in combination with SOX9, BMP2 or SOX9 and BMP2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 and ATF6.

[0204] In one embodiment, the transcription factors are selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6 and MYOD1 , preferably in combination with one or more of PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 and ATF6. In one embodiment, the transcription factors are selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6 and MYOD1 , preferably in combination with one or more of PHOX2B, SOX9 or ESRRB, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9 and ESRRB. In one embodiment, the transcription factors are selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB NKX2.1 , ZNF74, BIT-C-P3860PCT1

[0205] 45

[0206] MEF2A, BMP2 and ATF6 in combination with one or more of DPF1 , EN1 , FERD3L, FOXF2, HOXC4, KLF10, LEUTX, MESP2, MYOG, NR3C2, SMYD2, SOX15, ZNF429, ZNF619, NEUROD2 or TFEB, wherein the transcription factors do not consist of (i) ESRRB and TFEB (this specific combination being particularly useful in generating placental villous trophoblasts), (ii) NEUROD2 and PHOX2B (this specific combination being particularly useful in generating Purkinje cells) or (iii) SOX9 and SOX15 (this specific combination being particularly useful in generating loop of Henle epithelial cells). In one embodiment, the transcription factors are selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9 and ESRRB in combination with one or more of DPF1 , EN1 , FERD3L, FOXF2, HOXC4, KLF10, LEUTX, MESP2, MYOG, NR3C2, SMYD2, SOX15, ZNF429, ZNF619, NEUROD2 or TFEB, wherein the transcription factors do not consist of (i) ESRRB and TFEB, (ii) NEUROD2 and PHOX2B or (iii) SOX9 and SOX15. These transcription factors have been found to be particularly useful in generating muscle cells, such as cardiac muscle cells, skeletal muscle cells, skeletal satellite muscle cells, smooth muscle cells or enteric smooth muscle cells.

[0207] The present invention provides a method of generating muscle cells, such as cardiac muscle cells, skeletal muscle cells, skeletal satellite muscle cells, smooth muscle cells or enteric smooth muscle cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain muscle cells, wherein the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, and ZNF429, preferably in combination with one or more of MEF2C, MEF2D, ESRRG, NKX2.3, EN1 , SOX15, NEUROD2, TFEB, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6, or wherein the transcription factors are selected from the group consisting of: EN1 , SOX15, NEUROD1 and TFEB in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, ZNF429 MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6 wherein the transcription factors do not consist of (i) ESRRB and TFEB (this specific combination being particularly useful in generating placental villous trophoblasts), (ii) NEUROD2 and PHOX2B (this specific combination being particularly useful in generating Purkinje cells) or (iii) SOX9 and SOX15 (this specific combination being particularly useful in generating loop of Henle epithelial cells). BIT-C-P3860PCT1

[0208] 46

[0209] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, and ZNF429, preferably in combination with one or more of MEF2C, MEF2D, ESRRG, NKX2.3, EN1 , SOX15, NEUROD2, TFEB, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6, or wherein the transcription factors are selected from the group consisting of: EN1 , SOX15, NEUROD1 and TFEB in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, ZNF429 MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6 wherein the transcription factors do not consist of (i) ESRRB and TFEB, (ii) NEUROD2 and PHOX2B or (iii) SOX9 and SOX15.

[0210] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, and ZNF429, preferably in combination with one or more of MEF2C, MEF2D, ESRRG, NKX2.3, EN1 , SOX15, NEUROD2, TFEB, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6, or wherein the transcription factors are selected from the group consisting of: EN1 , SOX15, NEUROD1 and TFEB in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, ZNF429 MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9, ESRRB, NKX2.1 , ZNF74, MEF2A, BMP2 or ATF6 wherein the transcription factors do not consist of (i) ESRRB and TFEB, (ii) NEUROD2 and PHOX2B or (iii) SOX9 and SOX15.

[0211] In one embodiment, the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, and ZNF429, preferably in combination with one or more of MEF2C, MEF2D, ESRRG, NKX2.3, EN1 , SOX15, NEUROD2, TFEB, MYF6, MYOD1 , PHOX2B, SOX9 or ESRRB, or wherein the transcription factors are selected from the group consisting of: EN1 , SOX15, NEUROD1 and TFEB in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, FOXF2, HOXC4, FERD3L, KLF10, MESP2, SMYD2, ZNF429 MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9 or ESRRB, wherein the transcription factors do not consist BIT-C-P3860PCT1

[0212] 47 of (i) ESRRB and TFEB, (ii) NEUROD2 and PHOX2B or (iii) SOX9 and SOX15. These transcription factors have been found to be particularly useful in generating muscle cells, such as cardiac muscle cells, skeletal muscle cells, skeletal satellite muscle cells, smooth muscle cells or enteric smooth muscle cells.

[0213] The transcription factors may comprise ATF6. This transcription factor has been found to be particularly useful in generating pericytes.

[0214] The transcription factors may be selected from the group consisting of: ESRRG, MEF2C, MYF6, ESRRB and MEF2A, preferably in combination with MEF2D, MYOD1 or MEF2D and MYOD1 , or two or more transcription factors selected from the group consisting of: ESRRG, MEF2C, MEF2D, MYF6, ESRRB MYOD1 and MEF2A. In one embodiment, the transcription factors are selected from the group consisting of: ESRRG, MEF2C, MEF2D, MYF6 and MYOD1 , preferably in combination with ESRRB, MEF2A or both ESRRB and MEF2A or the transcription factors are two or transcription factors selected from the group consisting of: ESRRG, MEF2C, MEF2D, MYF6, MYOD1 , ESRRB and MEF2A. In one embodiment, the transcription factors are selected from the group consisting of: ESRRG, MEF2C, MEF2D, MYF6 and MYOD1 , preferably in combination with ESRRB. In one embodiment, the transcription factors comprise MYF6 in combination with one or more of MEF2C, ESRRG, MEF2D, MYOD1 , ESRRB or MEF2A. In one embodiment, the transcription factors may comprise (i) MEF2C, ESRRG and ESRRB; (ii) MEF2D, MYOD1 and MYF6; (iii) MEF2C, MEF2D, ESRRG, MYOD1 MYF6 and ESRRB, (iv) MEF2D and ESRRB, preferably in combination with MYF6, (v) MYF6, ESRRG and MEF2C or (vi) ESRRG, MEF2D and MYOD1. These transcription factors have been found to be particularly useful in generating cardiac muscle cells.

[0215] The transcription factors may comprise MYOD1 and / or NKX2.3, preferably in combination with one or more of SOX9, MYF6 or both SOX9 and MYF6, or two or more transcription factors selected from the group consisting of: NKX2.3, MYOD1 , SOX9 and MYF6. In one embodiment, the transcription factors comprise NKX2.3, preferably in combination with one or more of MYOD1 , SOX9 or MYF6, or wherein the transcription factors are two of more transcription factors selected from the group consisting of: NKX2.3, MYOD1 , SOX9 and MYF6, wherein the transcription factors do not consist of MYOD1 and MYF6 (this specific combination being particularly useful in generating cardiac muscle cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: NKX2.3, MYOD1 , SOX9 and MYF6. In one embodiment, the transcription factors comprise MYOD1 and MYF6 BIT-C-P3860PCT1

[0216] 48 in combination with NKX2.3, SOX9 or both NKX2.3 and SOX9. In one embodiment, the transcription factors are selected from the group consisting of: NKX2.3, MYOD1 , SOX9 and MYF6 in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, EN1 , NEUROD2 or TFEB. In one embodiment, the transcription factors comprise MYOD1 and / or MYF6 in combination with one or more of MYOG, NR3C2 or EN1. These transcription factors have been found to be particularly useful in generating skeletal muscle cells.

[0217] In particular the transcription factors may comprise NKX2.3, preferably in combination with one or more of MYOD1 , SOX9 or MYOD1 and SOX9, or two or more transcription factors selected from the group consisting of: NKX2.3, MYOD1 and SOX9. In one embodiment the transcription factors comprise NKX2.3, MYOD1 and SOX9. In one embodiment, the transcription factors are selected from the group consisting of: NKX2.3, MYOD1 and SOX9 in combination with one or more of DPFI , LEUTX, ZNF619, NEUROD2 or TFEB. In one embodiment, the transcription factors comprise MYOD1 and / or SOX9 in combination with TFEB. These transcription factors have been found to be particularly useful in generating skeletal satellite muscle cells.

[0218] The present invention provides a method of generating skeletal muscle cells, such as skeletal satellite muscle cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain skeletal muscle cells, wherein the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LEUTX and ZNF619, preferably in combination with one or more of EN1 , NEUROD2, TFEB, NKX2.3, MYOD1 , MYF6 and SOX9, or wherein the transcription factors are selected from the group consisting of: EN1 , NEUROD2 and TFEB, in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, NKX2.3, MYOD1 , MYF6 and SOX9.

[0219] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LEUTX and ZNF619, preferably in combination with one or more of EN1 , NEUROD2, TFEB, NKX2.3, MYOD1 , MYF6 or SOX9, or wherein the transcription factors are selected from the group consisting of: EN1 , NEUROD2 and TFEB, in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, NKX2.3, MYOD1 , MYF6 or SOX9. BIT-C-P3860PCT1

[0220] 49

[0221] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: MYOG, NR3C2, DPF1 , LELITX and ZNF619, preferably in combination with one or more of EN1 , NEUROD2, TFEB, NKX2.3, MYOD1 , MYF6 or SOX9, or wherein the transcription factors are selected from the group consisting of: EN1 , NEUROD2 and TFEB, in combination with one or more of MYOG, NR3C2, DPF1 , LELITX, ZNF619, NKX2.3, MYOD1 , MYF6 or SOX9.

[0222] In one embodiment, the transcription factors are selected from the group consisting of: MYOG and NR3C2, preferably in combination with one or more of EN1 , MYOD1 and MYF6.

[0223] In one embodiment, the transcription factors are selected from the group consisting of: DPF1 , LELITX and ZNF619, preferably in combination with one or more of NEUROD2, TFEB, NKX2.3, MYOD1 or SOX9, or the transcription factors comprise NEUROD2 and / or TFEB in combination with one or more of DPF1 , LELITX, ZNF619, NKX2.3, MYOD1 or SOX9. In one embodiment, the transcription factors comprise MYOD1 and / or SOX9 in combination with TFEB. These transcription factors have been found to be particularly useful in generating skeletal satellite muscle cells.

[0224] The transcription factors may be selected from the group consisting of: MEF2D, PHOX2B, NKX2.1 and ZNF74, preferably in combination with BMP2. In one embodiment, the transcription factors comprise MEF2D and PHOX2B. In one embodiment, the transcription factors are selected from the group consisting of: MEF2D, PHOX2B, NKX2.1 , ZNF74 and BMP2 in combination with one or more of FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2, SOX15 or ZNF429. In one embodiment, the transcription factors comprise MEF2D and / or PHOX2B in combination with one or more of FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2, SOX15 or ZNF429. In one embodiment, the transcription factors comprise PHOX2B in combination with FOXF2, HOXC4 or both FOXF2 and HOXC4. These transcription factors have been found to be particularly useful in generating smooth muscle cells.

[0225] In particular the transcription factors may comprise MEF2D. In one embodiment, the transcription factors comprise MEF2D in combination with one or more of FERD3L, KLF10, MESP2, SMYD2, SOX15 or ZNF429 (preferably MESP2). This transcription factor has been found to be particularly useful in generating enteric smooth muscle cells. BIT-C-P3860PCT1

[0226] 50

[0227] The present invention provides a method of generating smooth muscle cells, such as enteric smooth muscle cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain smooth muscle cells, wherein the transcription factors are selected from the group consisting of: FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2 and ZNF429, preferably in combination with one or more of SOX15, MEF2D, PHOX2B, NKX2.1. ZNF74 or BMP2.

[0228] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2 and ZNF429, preferably in combination with one or more of SOX15, MEF2D, PHOX2B, NKX2.1 , ZNF74 or BMP2.

[0229] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2 and ZNF429, preferably in combination with one or more of SOX15, MEF2D, PHOX2B, NKX2.1 , ZNF74 or BMP2.

[0230] In one embodiment, the transcription factors are selected from the group consisting of: FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2 and ZNF429, preferably in combination with one or more of SOX15, MEF2D or PHOX2B, or the transcription factors comprise SOX15 in combination with one or more of: FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2, ZNF429, MEF2D or PHOX2B. These transcription factors have been found to be particularly useful in generating smooth muscle cells. In one embodiment, the transcription factors are selected from the group consisting of FOXF2 and HOXC4, preferably in combination with PHOX2B, MEF2D or NKX2.1 (preferably PHOX2B). In one embodiment, the transcription factors are selected from the group consisting of: FERD3L, KLF10, MESP2, SMYD2 and ZNF429, preferably in combination with SOX15, MEF2D or both SOX15 and MEF2D, or the transcription factors comprise SOX15 in combination with one or more of FERD3L, KLF10, MESP2, SMYD2, ZNF429 or MEF2D. In one embodiment the transcription factors comprise MESP2 and MEF2D. These transcription factors have been found to be particularly useful in generating enteric smooth muscle cells. BIT-C-P3860PCT1

[0231] 51

[0232] Alternatively the transcription factors may comprise ZNF74, preferably in combination with BMP2. These transcription factors have been found to be particularly useful in generating bronchial smooth muscle cells.

[0233] Intestinal epithelial cells

[0234] The present invention provides a method of generating intestinal epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain intestinal epithelial cells, wherein the transcription factors are selected from the group consisting of: HMX1 , RREB1 and GATA5, preferably in combination with ESRRA, HNF1A or both ESRRA and HNF1A.

[0235] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: HMX1 , RREB1 and GATA5, preferably in combination with ESRRA, HNF1A or both ESRRA and HNF1A.

[0236] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: HMX1 , RREB1 and GATA5, preferably in combination with ESRRA, HNF1A or both ESRRA and HNF1A.

[0237] In one embodiment, the transcription factors comprise HMX1 and / or GATA5, preferably in combination with one or more of RREB1 , ESRRA or HNF1A. In one embodiment the transcription factors comprise HMX1 and / or GATA5 in combination with one or more of RREB1 , ESRRA, HNF1A, FOXQ1, GATA4, GATA6 or KLF2. In one embodiment the transcription factors comprise FOXQ1 and / or KLF2 in combination with one or more of HMX1 , GATA5, RREB1 , ESRRA, HNF1A, GATA4 or GATA6, wherein the transcription factor do not consist of RREB1 and KLF2 (this specific combination being particularly useful in generating club cells). These transcription factors have been found to be particularly useful in generating intestinal epithelial cells. BIT-C-P3860PCT1

[0238] 52

[0239] The present invention provides a method of generating intestinal epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain intestinal epithelial cells, wherein the transcription factors comprise GATA6 in combination with one or more of HMX1 , GATA5, RREB1 , ESRRA, HNF1A, FOXQ1 , GATA4 or KLF2, wherein the transcription factors do not consist of (i) GATA6 and ESRRA, (ii) GATA6 and GATA4, (iii) GATA6 and HNF1 A, (iv) GATA6 and RREB1 , (v) GATA6, ESRRA and GATA4, (vi) GATA6, ESRRA and HNF1A, (vii) GATA6, ESRRA and RREB1 , (viii) GATA6, GATA4 and HNF1A, (ix) GATA6, GATA4 and RREB1 , (x) GATA6, HNF1A and RREB1 , (xi) GATA6, ESRRA, GATA4 and HNF1A, (xii) GATA6, ESRRA, GATA4 and RREB1 , (xii) GATA6, ESRRA, HNF1A and RREB1 , (xiii) GATA6, GATA4, HNF1A and RREB1 or (xiv) GATA6, ESRRA, GATA4, HNF1A and RREB1 (these specific combinations being particularly useful in generating hepatocytes).

[0240] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise GATA6 in combination with one or more of HMX1 , GATA5, RREB1 , ESRRA, HNF1A, FOXQ1 , GATA4 or KLF2, wherein the transcription factors do not consist of (i) GATA6 and ESRRA, (ii) GATA6 and GATA4, (iii) GATA6 and HNF1A, (iv) GATA6 and RREB1 , (v) GATA6, ESRRA and GATA4, (vi) GATA6, ESRRA and HNF1A, (vii) GATA6, ESRRA and RREB1 , (viii) GATA6, GATA4 and HNF1A, (ix) GATA6, GATA4 and RREB1 , (x) GATA6, HNF1A and RREB1 , (xi) GATA6, ESRRA, GATA4 and HNF1A, (xii) GATA6, ESRRA, GATA4 and RREB1 , (xii) GATA6, ESRRA, HNF1A and RREB1 , (xiii) GATA6, GATA4, HNF1A and RREB1 or (xiv) GATA6, ESRRA, GATA4, HNF1A and RREB1.

[0241] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise GATA6 in combination with one or more of: HMX1 , GATA5, RREB1 , ESRRA, HNF1 A, FOXQ1 , GATA4 or KLF2, wherein the transcription factors do not consist of (i) GATA6 and ESRRA, (ii) GATA6 and GATA4, (iii) GATA6 and HNF1A, (iv) GATA6 and RREB1 , (v) GATA6, ESRRA and GATA4, (vi) GATA6, ESRRA and HNF1A, (vii) GATA6, ESRRA and RREB1 , (viii) GATA6, GATA4 and HNF1A, (ix) GATA6, GATA4 and RREB1 , (x) GATA6, HNF1A and RREB1 , (xi) GATA6, ESRRA, GATA4 and HNF1A, (xii) GATA6, ESRRA, GATA4 BIT-C-P3860PCT1

[0242] 53 and RREB1 , (xii) GATA6, ESRRA, HNF1A and RREB1 , (xiii) GATA6, GATA4, HNF1A and RREB1 or (xiv) GATA6, ESRRA, GATA4, HNF1A and RREB1.

[0243] T cells

[0244] The present invention provides a method of generating T cells, such as regulatory T cell or gamma delta T cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain T cells, wherein the transcription factors are selected from the group consisting of: FOXC1 , FOX11 , FOXP3, GLIS1 , GLIS3, TCF3, SOX7, BCL11 B and TBR1 , preferably in combination with one or more of TFEB, GFI1 , SOX8 or FOXC2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: FOXC1 , FOXC2, FOXI1 , FOXP3, GLIS1 , GLIS3, TCF3, SOX7, BCL11 B, TBR1 , TFEB, GFI1 and SOX8.

[0245] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: FOXC1 , FOXI1 , FOXP3, GLIS1 , GLIS3, TCF3, SOX7, BCL11 B and TBR1 , preferably in combination with one or more of TFEB, GF11 , SOX8 or FOXC2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: FOXC1 , FOXC2, FOXI1 , FOXP3, GLIS1 , GLIS3, TCF3, SOX7, BCL11 B, TBR1 , TFEB, GFI1 and SOX8.

[0246] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: FOXC1 , FOXI1 , FOXP3, GLIS1 , GLIS3, TCF3, SOX7, BCL11 B and TBR1 , preferably in combination with one or more of TFEB, GFI1 , SOX8 or FOXC2, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: FOXC1 , FOXC2, FOXI1 , FOXP3, GLIS1 , GLIS3, TCF3, SOX7, BCL11 B, TBR1 , TFEB, GFI1 and SOX8.

[0247] The transcription factors may be selected from the group consisting of: FOXP3, GLIS1 , GLIS3, TCF3 and SOX7, preferably in combination with TFEB, SOX8 or TFEB and SOX8, or two or more transcription factors selected from the group consisting of: FOXP3, GLIS1 , TCF3, GLIS3, TFEB SOX7 and SOX8. In one embodiment, the transcription factors comprise FOXP3 in BIT-C-P3860PCT1

[0248] 54 combination with one or more of GLIS1 , TCF3, GLIS3, TFEB SOX7 and / or SOX8. In one embodiment, the transcription factors comprise (i) GLIS1 , TCF3 and FOXP3, or (ii) GLIS1 , TCF3, SOX8, SOX7, TFEB and GLIS3. These transcription factors have been found to be particularly useful in generating regulatory T cells.

[0249] The transcription factors may be selected from the group consisting of: FOXC1 , FOXI1 , BCL11 B, and TBR1 , preferably in combination with FOXC2, GFI1 or FOXC2 and GFI1 , or two or more transcription factors selected from the group consisting of: FOXC1 , FOXC2, FOXI1 , BCL11 B, TBR1 and GFI1. In particular these transcription factors comprise FOXC1 , FOXC2, FOXI1 , GFI1 , BCL11 B and TBR1. These transcription factors have been found to be particularly useful in generating gamma delta T cells.

[0250] Endocrine cells

[0251] The present invention provides a method of generating endocrine cells, such as enteroendocrine cells or neural endocrine cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endocrine cells, wherein the transcription factors are selected from the group consisting of: ASCL1 , PTF1A, FOXA2 and GF11 , preferably in combination with KLF2, HMX1 or KLF and HMX1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ASCL1 , PTF1A, GF11 , FOXA2, KLF2 and HMX1.

[0252] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: ASCL1 , PTF1A, FOXA2 and GFI1 , preferably in combination with KLF2, HMX1 or KLF and HMX1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 and HMX1.

[0253] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: ASCL1 , PTF1A, FOXA2 and GFI1 , preferably in combination with KLF2, HMX1 or KLF and HMX1 , or wherein the transcription factors are two BIT-C-P3860PCT1

[0254] 55 or more transcription factors selected from the group consisting of: ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 and HMX1.

[0255] The transcription factors may be selected from the group consisting of: PTF1A, FOXA2 and GFI1 , preferably in combination with one of more of ASCL1 , KLF2 or HMX1 , or two or more transcription factors selected from the group consisting of: ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 and HMX1. In one embodiment, the transcription factors comprise ASCL1 in combination with one or more of FOXA2, GFI1 or PTF1A. In particular the transcription factors comprise ASCL1 and GFI1. In one embodiment, the transcription factors are selected from the group consisting of: ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 and HMX1 , in combination with one or more of GLISI , NKX3.2, RFX6, CDX2, KLF1 , KLF4, TCF3 or GFI1B, wherein the transcription factors do not consist of (i) ASCL1 and CDX2 (this specific combination being particularly useful in generating retinal ganglion cells), (ii) ASCL1 and KLF4 (this specific combination being particularly useful in generating retinal ganglion cells), (iii) ASCL1 , CDX2 and KLF4 (this specific combination being particularly useful in generating retinal ganglion cells), (iv) ASCL1 and TCF3 (this specific combination being particularly useful in generating amacrine cells), (v) FOXA2 and KLF4 (this specific combination being particularly useful in generating collecting duct principal cells), (vi) GFI1 and KLF4 (this specific combination being particularly useful in generating luminal epithelial cells of the mammary gland), (vii) KLF1 and FOXA2 (this specific combination being particularly useful in generating prostate epithelial cells), (viii) KLF1 and GFI1 (this specific combination being particularly useful in generating prostate epithelial cells), (ix) KLF1 and PTF1A (this specific combination being particularly useful in generating prostate epithelial cells), (x) KLF1 , FOXA2 and GFI1 (this specific combination being particularly useful in generating prostate epithelial cells), (xi) KLF1 , FOXA2 and PTF1A (this specific combination being particularly useful in generating prostate epithelial cells), (xii) KLF1 , GFI1 and PTF1A (this specific combination being particularly useful in generating prostate epithelial cells) or (xiii) KLF1 , FOXA2, GFI1 and PTF1A (this specific combination being particularly useful in generating prostate epithelial cells). In one embodiment, the transcription factors are selected from the group consisting of: ASCL1 , PTF1A, GFI1 and FOXA2 in combination with one or more of GLISI , NKX3.2, RFX6, CDX2, KLF1 , KLF4, TCF3 or GFI1B, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and KLF4, (iii) ASCL1 , CDX2 and KLF4, (iv) ASCL1 and TCF3, (v) FOXA2 and KLF4, (vi) GFI1 and KLF4, (vii) KLF1 and FOXA2, (viii) KLF1 and GFI1 , (ix) KLF1 and PTF1A, (x) KLF1 , FOXA2 and GFI1 , (xi) KLF1 , FOXA2 and PTF1A, (xii) KLF1 , GFI1 and PTF1A or (xiii) KLF1 , FOXA2, GFI1 and PTF1A. These transcription factors have been found to be particularly useful in generating enteroendocrine cells. BIT-C-P3860PCT1

[0256] 56

[0257] The present invention provides a method of generating enteroendocrine cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain enteroendocrine cells, wherein the transcription factors comprise NKX3.2 and / or RFX6, preferably in combination with one or more of ASCL1 , PTF1A, GFI1 , FOXA2, GLIS1 , CDX2, KLF1 , KLF4, TCF3, GFI1 B, KLF2 or HMX1 , or wherein the transcription factors are selected from the group consisting of: CDX2, KLF1 , KLF4, TCF3, GFI1 B and GLIS1 in combination with one or more of NKX3.2, RFX6, ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 and HMX1 , wherein the transcription factors do not consist of (i) ASCL1 and CDX2 (this combination being particularly useful for retinal ganglion cells), (ii) ASCL1 and KLF4 (this combination being particularly useful for retinal ganglion cells), (iii) ASCL1 , CDX2 and KLF4 (this combination being particularly useful for retinal ganglion cells), (iv) ASCL1 and TCF3 (this combination being particularly useful for amacrine cells), (v) FOXA2 and KLF4 (this combination being particularly useful for collecting duct principal cells), (vi) GFI1 and KLF4 (this combination being particularly useful for luminal epithelial cells of the mammary gland), (vii) KLF1 and FOXA2 (this combination being particularly useful for prostate epithelial cells), (viii) KLF1 and GFI1 (this combination being particularly useful for prostate epithelial cells), (ix) KLF1 and PTF1A (this combination being particularly useful for prostate epithelial cells), (x) KLF1 , FOXA2 and GFI1 (this combination being particularly useful for prostate epithelial cells), (xi) KLF1 , FOXA2 and PTF1A (this combination being particularly useful for prostate epithelial cells), (xii) KLF1 , GFI1 and PTF1A (this combination being particularly useful for prostate epithelial cells) or (xiii) KLF1 , FOXA2, GFI1 and PTF1A (this combination being particularly useful for prostate epithelial cells).

[0258] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise NKX3.2 and / or RFX6, preferably in combination with one or more of ASCL1 , PTF1A, GF11 , FOXA2, GLIS1 , CDX2, KLF1 , KLF4, TCF3, GFI1 B, KLF2 or HMX1 , or wherein the transcription factors are selected from the group consisting of: CDX2, KLF 1 , KLF4, TCF3, GF11 B and GLIS1 in combination with one or more of NKX3.2, RFX6, ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 or HMX1 , wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and KLF4, (iii) ASCL1 , CDX2 and KLF4, (iv) ASCL1 and TCF3, (v) FOXA2 and KLF4, (vi) GFI1 and KLF4, (vii) KLF1 BIT-C-P3860PCT1

[0259] 57 and FOXA2, (viii) KLF1 and GFH , (ix) KLF1 and PTF1A, (x) KLF1 , FOXA2 and GFH , (xi) KLF1 , FOXA2 and PTF1A, (xii) KLF1 , GFI1 and PTF1A or (xiii) KLF1 , FOXA2, GFI1 and PTF1A.

[0260] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise NKX3.2 and / or RFX6, preferably in combination with one or more of ASCL1 , PTF1A, GFI1 , FOXA2, GLIS1 , CDX2, KLF1 , KLF4, TCF3, GFI1 B, KLF2 and HMX1 , or wherein the transcription factors are selected from the group consisting of: CDX2, KLF1 , KLF4, TCF3, GFI1 B and GLIS1 in combination with one or more of NKX3.2, RFX6, ASCL1 , PTF1A, GFI1 , FOXA2, KLF2 or HMX1 , wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and KLF4, (iii) ASCL1 , CDX2 and KLF4, (iv) ASCL1 and TCF3, (v) FOXA2 and KLF4, (vi) GFI1 and KLF4, (vii) KLF1 and FOXA2, (viii) KLF1 and GFI1 , (ix) KLF1 and PTF1A, (x) KLF1 , FOXA2 and GFI1 , (xi) KLF1 , FOXA2 and PTF1A, (xii) KLF1 , GFI1 and PTF1A or (xiii) KLF1 , FOXA2, GFI1 and PTF1A.

[0261] In one embodiment, the transcription factors comprise NKX3.2 and / or RFX6, preferably in combination with one or more transcription factors selected from the group consisting of: ASCL1 , PTF1A, GFI1 , FOXA2, GLIS1 , CDX2, KLF1 , KLF4, TCF3 and GFI1 B, or wherein the transcription factors are selected from the group consisting of: CDX2, KLF1 , KLF4, TCF3, GFI1 B and GLIS1 in combination with one or more of NKX3.2, RFX6, ASCL1 , PTF1A, GFI1 or FOXA2, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and KLF4, (iii) ASCL1 , CDX2 and KLF4, (iv) ASCL1 and TCF3, (v) FOXA2 and KLF4, (vi) GFI1 and KLF4, (vii) KLF1 and FOXA2, (viii) KLF1 and GFI1 , (ix) KLF1 and PTF1A, (x) KLF1 , FOXA2 and GFI1 , (xi) KLF1 , FOXA2 and PTF1A, (xii) KLF1 , GFI1 and PTF1A or (xiii) KLF1 , FOXA2, GFI1 and PTF1A. These transcription factors have been found to be particularly useful in generating enteroendocrine cells.

[0262] The transcription factors may comprise ASCL1. In one embodiment, the transcription factors comprise ASCL1 in combination with GLI2 or both GLI2 and RFX6. These transcription factors have been found to be particularly useful in generating neural endocrine cells such as lung neural endocrine cells.

[0263] The present invention provides a method of generating neural endocrine cells, preferably lung neural endocrine cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more BIT-C-P3860PCT1

[0264] 58 transcription factors in a cell population and culturing the cell population to obtain neural endocrine cells, wherein the transcription factors comprise GLI2, preferably in combination with ASCL1 , RFX6 or both ASCL1 and RFX6.

[0265] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise GLI2, preferably in combination with ASCL1 , RFX6 or both ASCL1 and RFX6.

[0266] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, preferably in combination with ASCL1 , RFX6 or both ASCL1 and RFX6.

[0267] Ciliated Cells

[0268] The present invention provides a method of generating ciliated cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain ciliated cells, wherein the transcription factors comprise FOXJ1.

[0269] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise FOXJ1.

[0270] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise FOXJ1.

[0271] In one embodiment, the transcription factors comprise FOXJ1 , preferably in combination with FOXQ1 . These transcription factors have been found to be particularly useful in generating ciliated cells, including multicilitated cells. BIT-C-P3860PCT1

[0272] 59

[0273] The present invention provides a method of generating ciliated cells, including multiciliated cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain ciliated cells, wherein the transcription factors comprise FOXQ1 , preferably in combination with FOXJ1.

[0274] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise FOXQ1 , preferably in combination with FOXJ1 .

[0275] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise FOXQ1 , preferably in combination with FOXJ1.

[0276] Neutrophils

[0277] The present invention provides a method of generating neutrophils comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain neutrophils, wherein the transcription factors comprise CEBPB.

[0278] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise CEBPB.

[0279] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise CEBPB.

[0280] In one embodiment, the transcription factors comprise CEBPB in combination with POLI4F3. These transcription factors have been found to be particularly useful in generating neutrophils. BIT-C-P3860PCT1

[0281] 60

[0282] The present invention provides a method of generating neutrophils, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain neutrophils, wherein the transcription factors comprise POLI4F3, preferably in combination with CEBPB.

[0283] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise POLI4F3, preferably in combination with CEBPB.

[0284] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise POLI4F3, preferably in combination with CEBPB.

[0285] Endocardial cells

[0286] The present invention provides a method of generating endocardial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endocardial cells, wherein the transcription factors are selected from the group consisting of: ETS1 , TBXT, ETV4 and TAL1.

[0287] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: ETS1 , TBXT, ETV4 and TALI .

[0288] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: ETS1 , TBXT, ETV4 and TAL1. BIT-C-P3860PCT1

[0289] 61

[0290] In one embodiment, the transcription factors comprise TAL1 in combination with one or more of ETS1 , TBXT and / or ETV4. In one embodiment the transcription factors comprising ETS1 and ETV4 in combination with TBXT, TAL1 or TBXT and TAL1. In one embodiment, the transcription factors comprise TBXT, preferably in combination with one or more of ETS1 , ETV4 or TAL1. In one embodiment, the transcription factors are selected from the group consisting of: TBXT, ETS1 , ETV4 and TAL1 in combination with one or more of PATZ1 , ZNF84, ZSCAN9 or ETV2, wherein the transcription factors do not consist of (i) ETV2 and ETS1 , (ii) ETV2 and ETV4, (iii) ETV2 and TAL1 , (iv) ETV2, ETS1 and ETV4, (v) ETV2, ETS1 and TAL1 , (vi) ETV2, ETV4 and TAL1 , or (vii) ETV2, ETS1 , ETV4 and TAL1 (these specific combinations being particularly useful in generating endothelial cells), These transcription factors have been found to be particularly useful in generating endocardial cells.

[0291] The present invention provides a method of generating endocardial cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endocardial cells, wherein the transcription factors are selected from the group consisting of: PATZ1 , ZNF84 and ZSCAN9, preferably in combination with one or more of TBXT, ETS1 , ETV2, ETV4 or TAL1 , or wherein the transcription factors comprise ETV2 and TBXT.

[0292] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of PATZ1 , ZNF84 and ZSCAN9, preferably in combination with one or more of TBXT, ETS1 , ETV2, ETV4 or TALI , or wherein the transcription factors comprise ETV2 and TBXT.

[0293] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: PATZ1 , ZNF84 and ZSCAN9, preferably in combination with one or more of TBXT, ETS1 , ETV2, ETV4 or TAL1 , or wherein the transcription factors comprise ETV2 and TBXT. BIT-C-P3860PCT1

[0294] 62

[0295] Endothelial cells

[0296] The present invention provides a method of generating endothelial cells, such as endothelial cells of the lymph vessel, sinusoidal endothelial cells and endothelial cells of the pericentral hepatic sinusoid, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endothelial cells, wherein the transcription factors are selected from the group consisting of: ARID5A, ETS2, SNAI1, ETV2, HNF1B, MSX1, ERG, FLI1, ZNF467, GSX1, MSX2 and NFATC1, preferably in combination with one or more of ETS1, ETV4, TAL1, POLI4F1 or FOXJ1, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1, ZNF467, TAL1, HNF1B, GSX1, MSX2 NFATC1, FOXJ1 or POU4F1.

[0297] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: ARID5A, ETS2, SNAI1, ETV2, HNF1B, MSX1, ERG, FLI1, ZNF467, GSX1, MSX2 and NFATC1, preferably in combination with one or more of ETS1, ETV4, TAL1, POU4F1 or FOXJ1, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1, ZNF467, TAL1, HNF1B, GSX1, MSX2, NFATC1, FOXJ1 or POU4F1.

[0298] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: ARID5A, ETS2, SNAI1, ETV2, HNF1B, MSX1, ERG, FLI1, ZNF467, GSX1, MSX2 and NFATC1, preferably in combination with one or more of ETS1, ETV4, TAL1, POU4F1 or FOXJ1, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1, ZNF467, TAL1, HNF1B, GSX1, MSX2, NFATC1, FOXJ1 or POU4F1.

[0299] In one embodiment, the transcription factors are selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1 and ZNF467, preferably in combination with one or more of HNF1B, TAL1, GSX1, MSX2, NFATC1, POU4F1 or FOXJ1, BIT-C-P3860PCT1

[0300] 63 or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1, ZNF467, TAL1, HNF1B, GSX1, MSX2, NFATC1, FOXJ1 and POU4F1. In one embodiment, the transcription factors are selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1 and ZNF467, preferably in combination with HNF1B, TAL1 or both HNF1 B and TAL, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS2, SNAI1, ETV2, MSX1, ERG, FLI1, ZNF467, ETV4, ETS1, HNF1B and TAL1. In one embodiment, the transcription factors are selected from the group consisting of ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG and ZNF467, preferably in combination with TAL1. In one embodiment, the transcription factors are selected from the group consisting of: ETS1, ETV2 and ETV4, preferably in combination with TAL1. In one embodiment, the transcription factors are selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1, ZNF467, HNF1B, TAL1, GSX1, MSX2, NFATC1, FOXJ1 and POU4F1, in combination with ZNF711, TBX6 or both ZNF711 and TBX6. In one embodiment, the transcription factors are selected from the group consisting of: ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, FLI1,ZNF467, HNF1B and TAL1 , in combination ZNF711 , TBX6 or both ZNF711 and TBX6. In one embodiment, the transcription factors are selected from the group consisting of: ETS1, ETS2, SNAI1, ETV2, ETV4, MSX1, ERG, ZNF467 and TAL1, in combination ZNF711, TBX6 or both ZNF711 and TBX6. These transcription factors have been found to be particularly useful in generating endothelial cells, such as endothelial cells of the lymph vessel, sinusoidal endothelial cells and endothelial cells of the pericentral hepatic sinusoid.

[0301] The present invention provides a method of generating endothelial cells, such as endothelial cells of the lymph vessel, sinusoidal endothelial cells and endothelial cells of the pericentral hepatic sinusoid, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endothelial cells, wherein the transcription factors comprise ZNF711, preferably in combination with one or more of ARID5A, ERG, ETS1, ETS2, ETV2, ETV4, FLI1, FOXJ1, GSX1, HNF1B, MSX1, MSX2, NFATC1, POU4F1, SNAI1, TAL1, TBX6 orZNF467, orwhereinthe transcription factors comprise TBX6 in combination with one or more of ZNF711, ARID5A, ERG, ETS1, ETS2, ETV2, ETV4, FLI1, FOXJ1, GSX1, HNF1B, MSX1, MSX2, NFATC1, POU4F1, SNAI1, TAL1 orZNF467. BIT-C-P3860PCT1

[0302] 64

[0303] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZNF711, preferably in combination with one or more of ARID5A, ERG, ETS1, ETS2, ETV2, ETV4, FLI1, FOXJ1, GSX1, HNF1B, MSX1, MSX2, NFATC1, POU4F1, SNAI1, TAL1 , TBX6 or ZNF467 or wherein the transcription factors comprise TBX6 in combination with one or more of ZNF711, ARID5A, ERG, ETS1, ETS2, ETV2, ETV4, FLI1, FOXJ1, GSX1, HNF1B, MSX1, MSX2, NFATC1, POU4F1, SNAI1, TAL1 orZNF467.

[0304] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ZNF711, preferably in combination with one or more of ARID5A, ERG, ETS1, ETS2, ETV2, ETV4, FLI1, FOXJ1, GSX1, HNF1B, MSX1, MSX2, NFATC1, POU4F1, SNAI1, TAL1, TBX6 or ZNF467 or wherein the transcription factors comprise TBX6 in combination with one or more of ZNF711, ARID5A, ERG, ETS1, ETS2, ETV2, ETV4, FLI1, FOXJ1.GSX1, HNF1B, MSX1, MSX2, NFATC1, POU4F1, SNAI1, TAL1 orZNF467.

[0305] In one embodiment, the transcription factors comprise NFATC1 in combination with one or more of ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, TAL1, HNF1B, GSX1, MSX1, MSX2, ERG, FLI1, ZNF467, FOXJ1 and / or POU4F1. In one embodiment, the transcription factors comprise ARID5A in combination with one or more of ETS1, ETS2, SNAI1, ETV2, ETV4, TAL1, HNF1B, NFATC1, GSX1, MSX1, MSX2, ERG, FLI1, ZNF467, FOXJ1 and / or POU4F1. In one embodiment, the transcription factors comprise ETV2 and ERG in combination with one or moreof ARID5A, ETS1, ETS2, SNAI1, ETV4, TAL1, HNF1B, NFATC1, GSX1, MSX1, MSX2, FLI1, ZNF467, FOXJ1 and / or POU4F1. In one embodiment, the transcription factors comprise ETS1 or ETS2 in combination with one or more of ARID5A, SNAI1, ETV2, ETV4, TAL1, HNF1B, NFATC1, GSX1, MSX1, MSX2, ERG, FLI1, ZNF467, FOXJ1 and / or POU4F1. In one embodiment, the transcription factors comprise TAL1 and MSX1 in combination with ARID5A, ETS1, ETS2, SNAI1, ETV2, ETV4, HNF1B, NFATC1, GSX1, MSX2, ERG, FLI1, ZNF467, FOXJ1 and / or POU4F1. In particular, the transcription factors may comprise (i) ETV2, ERG and ETS1; or (ii) ETV2, ERG, ETS1, ETV4, ETS2 and TAL1; or (iii) ETS1, ETV2, ETV4 and TAL1. BIT-C-P3860PCT1

[0306] 65

[0307] In one embodiment, the transcription factors comprise ZNF711 , preferably in combination with one or more of ARID5A, ERG, ETS1 , ETS2, ETV2, ETV4, FLI1 , HNF1 B, MSX1 , SNAI1 , TAL1 , TBX6 or ZNF467. In one embodiment, the transcription factors comprise TBX6 in combination with one or more of ZNF711 , ARID5A, ERG, ETS1 , ETS2, ETV2, ETV4, FLI1 , HNF1 B, MSX1 , SNAI1 , TAL1 or ZNF467. In one embodiment, the transcription factors comprise ZNF711 , preferably in combination with one or more of ERG, ETS1 , ETS2, ETV2, ETV4, MSX1 , SNA11 , TAL1 , TBX6 or ZNF467. In one embodiment, the transcription factors comprise TBX6 in combination with one or more of ZNF711 , ERG, ETS1 , ETS2, ETV2, ETV4, MSX1 , SNAI1 , TAL1 or ZNF467. In one embodiment, the transcription factors comprise ZNF711 , preferably in combination with one or more of ERG, ETS1 and / or ETV2. In one embodiment, the transcription factors comprise TBX6 in combination with one or more of ERG, ETS1 and / or ETV2. These transcription factors have been found to be particularly useful in generating endothelial cells, such as endothelial cells of the lymph vessel, sinusoidal endothelial cells and endothelial cells of the pericentral hepatic sinusoid.

[0308] The transcription factors may be selected from the group consisting of: NFATC1 , ETV2 and FLI1 , preferably in combination with ETV4, POU4F1 or ETV4 and POU4F1 , or two or more transcription factors selected from the group consisting of: NFATC1 , ETV2, ETV4, FLI1 and POU4F1. In one embodiment, the transcription factors are selected from the group consisting of: ETV2, ETV4 and FLI1 , preferably in combination with NFATC1 , POU4F1 or both NFATC1 and POU4F1. In one embodiment, the transcription factors are selected from the group consisting of: ETV2, ETV4, FLI1 , NFATC1 and POU4F1 in combination with ERG. In one embodiment, the transcription factors are selected from the group consisting of: ETV2, ETV4 and FLI1 in combination with ERG. These transcription factors have been found to be particularly useful in generating endothelial cells of the lymph vessel.

[0309] In one embodiment, the transcription factors comprise ERG, preferably in combination with one or more of FLI1 , ETV2, ETV4, NFATC1 or POU4F1. In one embodiment, the transcription factors comprise ERG, preferably in combination with one or more of FLI1 , ETV2 or ETV4. These transcription factors have been found to be particularly useful in generating endothelial cells of the lymph vessel.

[0310] The transcription factors may be selected from the group consisting of: ARID5A, HNF1 B, ETS2 and SNAI1 , preferably in combination with ETS1. In one embodiment, the transcription factors are selected from the group consisting of: ARID5A, ETS1 , ETS2 and SNAI1 , preferably in combination with HNF1 B. In one embodiment, the transcription factors are selected from BIT-C-P3860PCT1

[0311] 66

[0312] ETS1 , ETS2 and / or SNAI1. These transcription factors have been found to be particularly useful in generating sinusoidal endothelial cells. In particular the transcription factors comprise ARID5A and / or HNF1 B. In one embodiment, the transcription factors comprise ARID5A, preferably in combination with HNF1 B. These transcription factors have been found to be particularly useful in generating endothelial cells of the pericentral hepatic sinusoid.

[0313] In one embodiment, the endothelial cell generated is selected from the group consisting of: endothelial cells of the lymph vessel, sinusoidal endothelial cells, endothelial cells of the pericentral hepatic sinusoid, alveolar capillary type 1 endothelial cells, endothelial stalk cells, capillary endothelial cells such as kidney capillary endothelial cells, lung microvascular endothelial cells, pulmonary capillary endothelial cells, cerebral cortex endothelial cells, lung endothelial cells and squamous endothelial cells.

[0314] Neurons

[0315] The present invention provides a method of generating neurons, such as excitatory neurons, glutamatergic neurons, inhibitory neurons, GABAergic neurons, enteric neurons or peripheral nervous system neurons, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain neurons, wherein the transcription factors are selected from the group consisting of: PAX2, ZNF704, LBX1 , NEUROG1 , NEUROG2, MYT1 , MIXL1 and INSM1 , preferably in combination with one or more of SOX1 , NEUROD1 , NEUROD2, NEUROD4, HOXB2, ASCL1 , NR2E1 , SOX8, SOX10, ATOH1 and PHOX2B, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1 , NEUROD1 , NEUROD2, NEUROD4, NEUROG1 , NEUROG2, MYT1 , MIXL1 , INSM1 , HOXB2, ASCL1 , NR2E1 , SOX1 , SOX8, SOX10, ATOH1 and PHOX2B, wherein the transcription factors do not consist of NEUROD1 and NEUROD2 (this specific combination being particularly useful in generating photoreceptor cells) or HOXB2 and ASCL1 (this specific combination being particularly useful in generating amacrine cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1 , NEUROD1 , NEUROD2, NEUROD4, NEUROG1 , NEUROG2, MYT1 , MIXL1 , INSM1 , HOXB2, ASCL1 , NR2E1 , SOX1 , SOX8, SOX10, ATOH1 and PHOX2B.

[0316] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, BIT-C-P3860PCT1

[0317] 67 wherein the transcription factors are selected from the group consisting of: PAX2, ZNF704, LBX1, NEUROG1, NEUROG2, MYT1, MIXL1 and INSM1, preferably in combination with one or more of SOX1, NEUROD1, NEUROD2, NEUROD4, HOXB2, ASCL1, NR2E1, SOX8, SOX10, ATOH1 and PHOX2B, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1, NEUROD1, NEUROD2, NEUROD4, NEUROG1, NEUROG2, MYT1, MIXL1, INSM1, HOXB2, ASCL1, NR2E1, SOX1, SOX8, SOX10, ATOH1 and PHOX2B, wherein the transcription factors do not consist of NEUROD1 and NEUROD2 (this specific combination being particularly useful in generating photoreceptor cells) or HOXB2 and ASCL1 (this specific combination being particularly useful in generating amacrine cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1, NEUROD1, NEUROD2, NEUROD4, NEUROG1, NEUROG2, MYT1, MIXL1, INSM1, HOXB2, ASCL1, NR2E1, SOX1, SOX8, SOX10, ATOH1 and PHOX2B.

[0318] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: PAX2, ZNF704, LBX1, NEUROG1, NEUROG2, MYT1, MIXL1 and INSM1, preferably in combination with one or more of SOX1, NEUROD1, NEUROD2, NEUROD4, HOXB2, ASCL1, NR2E1, SOX8, SOX10, ATOH1 and PHOX2B, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1, NEUROD1, NEUROD2, NEUROD4, NEUROG1, NEUROG2, MYT1, MIXL1, INSM1, HOXB2, ASCL1, NR2E1, SOX1, SOX8, SOX10, ATOH1 and PHOX2B, wherein the transcription factors do not consist of NEUROD1 and NEUROD2 (this specific combination being particularly useful in generating photoreceptor cells) or HOXB2 and ASCL1 (this specific combination being particularly useful in generating amacrine cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1, NEUROD1, NEUROD2, NEUROD4, NEUROG1, NEUROG2, MYT1, MIXL1, INSM1, HOXB2, ASCL1, NR2E1, SOX1, SOX8, SOX10, ATOH1 and PHOX2B.

[0319] In one embodiment, the transcription factors comprise ATOH1 in combination with one or more of PAX2, ZNF704, LBX1, NEUROD1, NEUROD2, NEUROD4, NEUROG1, NEUROG2, MYT1, MIXL1, INSM1, HOXB2, ASCL1, NR2E1, SOX1, SOX8, SOX10 and / or PHOX2B. In one embodiment, the transcription factors comprise INSM1, NEUROD1 and PHOX2B in BIT-C-P3860PCT1

[0320] 68 combination with one or more of PAX2, ZNF704, LBX1 , NEUROD2, NEUROD4, NEUROG1 , NEUROG2, MYT1 , MIXL1 , HOXB2, ASCL1 , NR2E1 , SOX1 , SOX8, SOX10 and / or ATOH1.

[0321] The transcription factors may comprise MYT1 , preferably in combination with one or more of ASCL1 , SOX8, SOX10 or HOXB2, or two or more transcription factors selected from the group consisting of: MYT1 , ASCL1 , HOXB2, SOX8 and SOX10, wherein the transcription factors do not consist of ASCL1 and HOX2B (this specific combination being particularly useful in generating amacrine cells), or three or more transcription factors selected from the group consisting of: MYT1 , ASCL1 , HOXB2, SOX8 and SOX10. These transcription factors have been found to be particularly useful in generating excitatory neurons.

[0322] In particular, the transcription factors may comprise SOX8 and SOX10. In one embodiment, the transcription factors comprise SOX10 and / or SOX8 in combination with one or more transcription factors selected from the group consisting of: ZNF131 , GFI1 , GLIS3 and NEUROG1. These transcription factors have been found to be particularly useful in generating glutamatergic neurons.

[0323] The present invention provides a method of generating glutamatergic neurons, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain glutamatergic neurons, wherein the transcription factors comprise ZNF131 , preferably in combination with one or more of GF11 , GLIS3, NEUROG1 , SOX10 or SOX8, or wherein the transcription factors comprise GFI1 and / or GLIS3 in combination with one or more of NEUROG1 , ZNF131 , SOX10 or SOX8.

[0324] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZNF131 , preferably in combination with one or more of GFI1 , GLIS3, NEUROG1 , SOX10 or SOX8, or wherein the transcription factors comprise GFI1 and / or GLIS3 in combination with one or more of NEUROG1 , ZNF131 , SOX10 or SOX8.

[0325] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors BIT-C-P3860PCT1

[0326] 69 comprise ZNF131 , preferably in combination with one or more of GF11 , GLIS3, NEUROG1 , SOX10 or SOX8, or wherein the transcription factors comprise GFI1 and / or GLIS3 in combination with one or more of NEUROG1 , ZNF131 , SOX10 or SOX8.

[0327] In one embodiment, the transcription factors comprise ZNF131 , preferably in combination with one or more of GFI1 , GLIS3, NEUROG1 or SOX10, or wherein the transcription factors comprise GFI1 and / or GLIS3 and in combination with one or more of NEUROG1 , ZNF131 or SOX10. These transcription factors have been found to be particularly useful in generating glutamatergic neurons.

[0328] The transcription factors may be selected from the group consisting of: PAX2, ZNF704, LBX1 , NEUROG1 and NEUROG2, preferably in combination with one or more of NEUROD1 , NEUROD2, HOXB2, MYT1 , ASCL1 or NR2E1 , or two or more transcription factors selected from the group consisting: of PAX2, ZNF704, LBX1 , NEUROG1 , NEUROG2, NEUROD1 , NEUROD2, HOXB2, MYT1 , ASCL1 and NR2E1 , wherein the transcription factors do not consist of NEUROD1 and NEUROD2 (this specific combination being particularly useful in generating photoreceptor cells), HOXB2 and ASCL1 (this specific combination being particularly useful in generating amacrine cells) or HOXB2 and MYT 1 (this specific combination being particularly useful in generating excitatory neurons), or three or more transcription factors selected from the group consisting of: PAX2, ZNF704, LBX1 , NEUROG1 , NEUROG2, NEUROD1 , NEUROD2, HOXB2, MYT1 , ASCL1 and NR2E1. These transcription factors have been found to be particularly useful in generating inhibitory neurons.

[0329] In particular, the transcription factors may selected from the group consisting of: LBX1 , NEUROG1 and NEUROG2, preferably in combination with one or more of NR2E1 , NEUROD1 , NEUROD2, HOXB2 or MYT1 , or two or more transcription factors selected from the group consisting of: LBX1 , NR2E1 , NEUROD1 , NEUROD2, NEUROG1 , NEUROG2, HOXB2 and MYT1 , wherein the transcription factors do not consist of NEUROD1 and NEUROD2 (this specific combination being particularly useful in generating photoreceptor cells) or HOXB2 and MYT1 (this specific combination being particularly useful in generating excitatory neurons), or three or more transcription factors selected from the group consisting of: LBX1 , NR2E1 , NEUROD1 , NEUROD2, NEUROG1 , NEUROG2, HOXB2 and MYT1. In one embodiment, the transcription factors comprise LBX1 and / or NR2E1 , preferably in combination with one or more of NEUROG1 , NEUROG2, NEUROD1 , NEUROD2, HOXB2 or MYT1 . In one embodiment, the transcription factors comprise LBX1 and / or NR2E1 , preferably in combination with one or more of NEUROG3, NEUROG1 , NEUROG2, NEUROD1 , NEUROD2, HOXB2 or MYT1. These BIT-C-P3860PCT1

[0330] 70 transcription factors have been found to be particularly useful in generating GABAergic neurons.

[0331] The present invention provides a method of generating GABAergic neurons, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain GABAergic neurons, wherein the transcription factors comprise NEUROG3, in combination with one or more of LBX1 , NEUROG1 , NEUROG2, NR2E1 , NEUROD1 , NEUROD2, HOXB2 or MYT1 , wherein the transcription factors do not consist of (i) NEUROG3 and MYT 1 , (ii) NEUROG3 and NEUROD1 , (iii) NEUROG3 and NEUROD2, (iv) NEUROG3 and NEUROG2, (v) NEUROG3, MYT1 and NEUROD1 , (vi) NEUROG3, MYT1 and NEUROD2, (vii) NEUROG3, MYT1 and NEUROG2, (viii) NEUROG3, NEUROD1 and NEUROD2, (ix) NEUROG3, NEUROD1 and NEUROG2, (x) NEUROG3, NEUROD2 and NEUROG2, (xi) NEUROG3, MYT1 , NEUROD1 and NEUROD2, (xii) NEUROG3, MYT1 , NEUROD1 and NEUROG2, (xiii) NEUROG3, MYT1 , NEUROD2 and NEUROG2, (xiv) NEUROG3, NEUROD1 , NEUROD2 and NEUROG2, or (xv) NEUROG3, MYT1 , NEUROD1 , NEUROD2 and NEUROG2 (these specific combinations being particularly useful in generating peripheral nervous system neurons).

[0332] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise NEUROG3 in combination with one or more of LBX1 , NEUROG1 , NEUROG2, NR2E1 , NEUROD1 , NEUROD2, HOXB2 or MYT1 , wherein the transcription factors do not consist of (i) NEUROG3 and MYT1 , (ii) NEUROG3 and NEUROD1 , (iii) NEUROG3 and NEUROD2, (iv) NEUROG3 and NEUROG2, (v) NEUROG3, MYT1 and NEUROD1 , (vi) NEUROG3, MYT1 and NEUROD2, (vii) NEUROG3, MYT1 and NEUROG2, (viii) NEUROG3, NEUROD1 and NEUROD2, (ix) NEUROG3, NEUROD1 and NEUROG2, (x) NEUROG3, NEUROD2 and NEUROG2, (xi) NEUROG3, MYT1 , NEUROD1 and NEUROD2, (xii) NEUROG3, MYT1 , NEUROD1 and NEUROG2, (xiii) NEUROG3, MYT1 , NEUROD2 and NEUROG2, (xiv) NEUROG3, NEUROD1 , NEUROD2 and NEUROG2, or (xv) NEUROG3, MYT1 , NEUROD1 , NEUROD2 and NEUROG2.

[0333] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors BIT-C-P3860PCT1

[0334] 71 comprise NEUROG3 in combination with one or more of LBX1 , NEUROG1 , NEUROG2, NR2E1 , NEUROD1 , NEUROD2, HOXB2 or MYT1 , wherein the transcription factors do not consist of (i) NEUROG3 and MYT1 , (ii) NEUROG3 and NEUROD1 , (iii) NEUROG3 and NEUROD2, (iv) NEUROG3 and NEUROG2, (v) NEUROG3, MYT1 and NEUROD1 , (vi) NEUROG3, MYT1 and NEUROD2, (vii) NEUROG3, MYT1 and NEUROG2, (viii) NEUROG3, NEUROD1 and NEUROD2, (ix) NEUROG3, NEUROD1 and NEUROG2, (x) NEUROG3, NEUROD2 and NEUROG2, (xi) NEUROG3, MYT1 , NEUROD1 and NEUROD2, (xii) NEUROG3, MYT1 , NEUROD1 and NEUROG2, (xiii) NEUROG3, MYT1 , NEUROD2 and NEUROG2, (xiv) NEUROG3, NEUROD1 , NEUROD2 and NEUROG2, or (xv) NEUROG3, MYT1 , NEUROD1 , NEUROD2 and NEUROG2.

[0335] The transcription factors may comprise MIXL1 , preferably in combination with SOX1 , NEUROD4 or both SOX1 and NEUROD4, or two or more transcription factors selected from the group consisting of: MIXL1 , SOX1 and NEUROD4. In one embodiment, the transcription factors comprise NEUROD4, preferably in combination with MIXL1 , SOX1 or both MIXL1 and SOX1. These transcription factors have been found to be particularly useful in generating enteric neurons.

[0336] The transcription factors may comprise INSM1 , preferably in combination with one or more of NEUROD1 , ATOH1 , MYT1 , PHOX2B, NEUROD4 or MIXL1 , or two or more transcription factors selected from the group consisting of: INSM1 , NEUROD1 , ATOH1 , MYT1 , PHOX2B, NEUROD4 and MIXL1. In one embodiment, the transcription factors comprise INSM1 , preferably in combination with one or more of NEUROD1 , ATOH1 or MYT1. In one embodiment, the transcription factors are selected from the group consisting of: INSM1 , NEUROD1 , ATOH1 , MYT1 , NEUROD4, PHOX2B and MIXL1 in combination with one or more of AHCTF1 , DLX1 , DLX3, PRDM4, PURG, ZBTB7A, ASCL1 , MEF2C, NEUROG1 , NEUROG2, NEUROG3, ZNF282 or ZNF746, wherein the transcription factors do not consist of (i) MYT1 and ASCL1 (this specific combination being particularly useful in generating retinal ganglion cells), (ii) MYT1 and ZNF282 (this specific combination being particularly useful in generating retinal ganglion cells), (iii) MYT1 , ASCL1 and ZNF282 (this specific combination being particularly useful in generating retinal ganglion cells), (iv) ATOH1 and NEUROG1 (this specific combination being particularly useful in generating Purkinje cells), (v) ATOH1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (vi) MYT1 and NEUROG1 (this specific combination being particularly useful in generating Purkinje cells), (vii) MYT1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (viii) NEUROD1 and NEUROG1 (this specific combination being particularly BIT-C-P3860PCT1

[0337] 72 useful in generating Purkinje cells), (ix) NEUROD1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (x) ATOH1 , MYT1 and NEUROG1 (this specific combination being particularly useful in generating Purkinje cells), (xi) ATOH1 , MYT1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xii) ATOH1 , NEUROD1 and NEUROG1 (this specific combination being particularly useful in generating Purkinje cells), (xiii) ATOH1 , NEUROD1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xiv) MYT1 , NEUROD1 and NEUROG1 (this specific combination being particularly useful in generating Purkinje cells), (xv) MYT1 , NEUROD1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xvi) ATOH1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xvii) MYT1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xviii) NEUROD1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xix) ATOH1 , MYT1 , NEUROD1 and NEUROG1 (this specific combination being particularly useful in generating Purkinje cells), (xx) ATOH1 , MYT1 , NEUROD1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xxi) ATOH1 , MYT1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xxii) ATOH1 , NEUROD1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), (xxiii) MYT1 , NEUROD1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells), or (xxiv) ATOH1 , MYT1 , NEUROD1 , NEUROG1 and NEUROG2 (this specific combination being particularly useful in generating Purkinje cells). In one embodiment, the transcription factors are selected from the group consisting of: INSM1 , NEUROD1 , ATOH1 and MYT1 in combination with one or more of AHCTF1 , DLX1 , DLX3, PRDM4, PURG, ZBTB7A, ASCL1 , MEF2C, NEUROG1 , NEUROG2, NEUROG3, ZNF282 or ZNF746, wherein the transcription factors do not consist of (i) MYT1 and ASCL1 , (ii) MYT1 and ZNF282, (iii) MYT1 , ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1 , (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1 , (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1 , (ix) NEU ROD 1 and NEUROG2, (x) ATOH1 , MYT1 and NEUROG1 , (xi) ATOH1 , MYT1 and NEUROG2, (xii) ATOH1 , NEUROD1 and NEUROG1 , (xiii) ATOH1 , NEUROD1 and NEUROG2, (xiv) MYT1 , NEUROD1 and NEUROG1 , (xv) MYT1 , NEUROD1 and NEUROG2, (xvi) ATOH1 , NEUROG1 and NEUROG2, (xvii) MYT1 , NEUROG1 and NEUROG2, (xviii) NEUROD1 , NEUROG1 and NEUROG2, (xix) ATOH1 , MYT1 , NEUROD1 and NEUROG1 , (xx) ATOH1 , MYT1 , NEUROD1 and NEUROG2, (xxi) ATOH1 , MYT1 , NEUROG1 and NEUROG2, (xxii) ATOH1 , NEUROD1 , NEUROG1 and NEUROG2, (xxiii) MYT1 , NEUROD1 , NEUROG1 and NEUROG2, or (xxiv) ATOH1 , MYT1 , NEUROD1 , NEUROG1 and NEUROG2. These BIT-C-P3860PCT1

[0338] 73 transcription factors have been found to be particularly useful in generating peripheral nervous system neurons.

[0339] The present invention provides a method of generating peripheral nervous system neurons, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain peripheral nervous system neurons, wherein the transcription factors are selected from the group consisting of: AHCTF1, DLX1, DLX3, PRDM4, PLIRG and ZBTB7A, preferably in combination with one or more of INSM1, NEUROG3, NEUROD1, ATOH1, MYT1, ASCL1, MEF2C, NEUROG1, NEUROG2, ZNF282, ZNF746, NEUROD4, PHOX2B or MIXL1 , orwherein the transcription factors are selected from the group consisting of: ASCL1, MEF2C, NEUROG1, NEUROG2, NEUROG3, ZNF282 and ZNF746 in combination with one or more of AHCTF1, DLX1, DLX3, PRDM4, PURG, ZBTB7A, INSM1, NEUROD1, ATOH1, MYT1, NEUROD4, PHOX2B or MIXL1 , wherein the transcription factors do not consist of (i) MYT1 and ASCL1, (ii) MYT1 and ZNF282, (iii) MYT1 , ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1, (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1, (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1, (ix) NEUROD1 and NEUROG2, (x) ATOH1, MYT1 and NEUROG1, (xi) ATOH1, MYT1 and NEUROG2, (xii) ATOH1, NEUROD1 and NEUROG1, (xiii) ATOH1, NEUROD1 and NEUROG2, (xiv) MYT1, NEUROD1 and NEUROG1, (xv) MYT1, NEU ROD 1 and NEUROG2, (xvi) ATOH1, NEU ROG 1 and NEUROG2, (xvii) MYT1, NEUROG1 and NEUROG2, (xviii) NEUROD1, NEUROG1 and NEUROG2, (xix) ATOH1, MYT1, NEUROD1 and NEUROG1, (xx) ATOH1, MYT1, NEUROD1 and NEUROG2, (xxi) ATOH1, MYT1, NEUROG1 and NEUROG2, (xxii) ATOH1, NEUROD1, NEUROG1 and NEUROG2, (xxiii) MYT1, NEUROD1, NEUROG1 and NEUROG2, or (xxiv) ATOH1, MYT1, NEUROD1, NEUROG1 and NEUROG2.

[0340] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: AHCTF1, DLX1, DLX3, PRDM4, PURG and ZBTB7A, preferably in combination with one or more of INSM1, NEUROG3, NEUROD1, ATOH1, MYT1, ASCL1, MEF2C, NEUROG1, NEUROG2, ZNF282, ZNF746, NEUROD4, PHOX2B or MIXL1, orwherein the transcription factors are selected from the group consisting of: ASCL1, MEF2C, NEUROG1, NEUROG2, NEUROG3, ZNF282 and ZNF746 in combination with one or more of AHCTF1 , DLX1 , DLX3, PRDM4, PURG, ZBTB7A, INSM1, NEUROD1, ATOH1, MYT1, NEUROD4, PHOX2B or BIT-C-P3860PCT1

[0341] 74

[0342] MIXL1 , wherein the transcription factors do not consist of (i) MYT 1 and ASCL1 , (ii) MYT 1 and ZNF282, (iii) MYT1, ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1, (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1, (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1, (ix) NEU ROD 1 and NEUROG2, (x) ATOH1, MYT1 and NEUROG1, (xi) ATOH1, MYT1 and NEUROG2, (xii) ATOH1, NEUROD1 and NEUROG1, (xiii) ATOH1, NEUROD1 and NEUROG2, (xiv) MYT1, NEUROD1 and NEUROG1, (xv) MYT1, NEUROD1 and NEUROG2, (xvi) ATOH1, NEUROG1 and NEUROG2, (xvii) MYT1, NEUROG1 and NEUROG2, (xviii) NEUROD1, NEUROG1 and NEUROG2, (xix) ATOH1, MYT1, NEUROD1 and NEUROG1, (xx) ATOH1, MYT1, NEUROD1 and NEUROG2, (xxi) ATOH1, MYT1, NEUROG1 and NEUROG2, (xxii) ATOH1, NEUROD1, NEUROG1 and NEUROG2, (xxiii) MYT1, NEUROD1, NEUROG1 and NEUROG2, or (xxiv) ATOH1, MYT1, NEUROD1, NEUROG1 and NEUROG2.

[0343] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: AHCTF1, DLX1, DLX3, PRDM4, PURG and ZBTB7A, preferably in combination with one or more of INSM1, NEUROG3, NEUROD1, ATOH1, MYT1, ASCL1, MEF2C, NEUROG1, NEUROG2, ZNF282, ZNF746, NEUROD4, PHOX2B or MIXL1, or wherein the transcription factors are selected from the group consisting of: ASCL1 , MEF2C, NEUROG1 , NEUROG2, NEUROG3, ZNF282 and ZNF746 in combination with one or more of AHCTF1, DLX1, DLX3, PRDM4, PURG, ZBTB7A, INSM1, NEUROD1, ATOH1, MYT1, NEUROD4, PHOX2B or MIXL1, wherein the transcription factors do not consist of (i) MYT1 and ASCL1, (ii) MYT1 and ZNF282, (iii) MYT1, ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1, (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1, (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1, (ix) NEUROD1 and NEUROG2, (x) ATOH1, MYT1 and NEUROG1, (xi) ATOH1, MYT1 and NEUROG2, (xii) ATOH1, NEUROD1 and NEUROG1, (xiii) ATOH1, NEUROD1 and NEUROG2, (xiv) MYT1, NEUROD1 and NEUROG1, (xv) MYT1, NEUROD1 and NEUROG2, (xvi) ATOH1, NEUROG1 and NEUROG2, (xvii) MYT1, NEUROG1 and NEUROG2, (xviii) NEUROD1, NEUROG1 and NEUROG2, (xix) ATOH1, MYT1, NEUROD1 and NEUROG1, (xx) ATOH1, MYT1, NEUROD1 and NEUROG2, (xxi) ATOH1, MYT1, NEUROG1 and NEUROG2, (xxii) ATOH1, NEUROD1, NEUROG1 and NEUROG2, (xxiii) MYT1, NEUROD1, NEUROG1 and NEUROG2, or (xxiv) ATOH1, MYT1, NEUROD1, NEUROG1 and NEUROG2.

[0344] In one embodiment, the transcription factors are selected from the group consisting of: AHCTF1, DLX1, DLX3, PRDM4, PURG and ZBTB7A, preferably in combination with one or BIT-C-P3860PCT1

[0345] 75 more of INSM1 , NEUROG3, NEUROD1 , ATOH1 , MYT1 , ASCL1 , MEF2C, NEUROG1 , NEUROG2, ZNF282 or ZNF746, or wherein the transcription factors are selected from the group consisting of: ASCL1 , MEF2C, NEUROG1 , NEUROG2, NEUROG3, ZNF282 and ZNF746 in combination with one or more of AHCTF1 , DLX1 , DLX3, PRDM4, PURG, ZBTB7A, INSM1 , NEUROD1 , ATOH1 or MYT1 , wherein the transcription factors do not consist of (i) MYT1 and ASCL1 , (ii) MYT1 and ZNF282, (iii) MYT1 , ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1 , (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1 , (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1 , (ix) NEUROD1 and NEUROG2, (x) ATOH1 , MYT1 and NEUROG1 , (xi) ATOH1 , MYT1 and NEUROG2, (xii) ATOH1 , NEUROD1 and NEUROG1 , (xiii) ATOH1 , NEUROD1 and NEUROG2, (xiv) MYT1 , NEUROD1 and NEUROG1 , (xv) MYT1 , NEUROD1 and NEUROG2, (xvi) ATOH1 , NEUROG1 and NEUROG2, (xvii) MYT1 , NEUROG1 and NEUROG2, (xviii) NEUROD1 , NEUROG1 and NEUROG2, (xix) ATOH1 , MYT1 , NEUROD1 and NEUROG1 , (xx) ATOH1 , MYT1 , NEUROD1 and NEUROG2, (xxi) ATOH1 , MYT1 , NEUROG1 and NEUROG2, (xxii) ATOH1 , NEUROD1 , NEUROG1 and NEUROG2, (xxiii) MYT1 , NEUROD1 , NEUROG1 and NEUROG2, or (xxiv) ATOH1 , MYT1 , NEUROD1 , NEUROG1 and NEUROG2. These transcription factors have been found to be particularly useful in generating peripheral nervous system neurons.

[0346] Mesothelial cells

[0347] The present invention provides a method of generating mesothelial cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain mesothelial cells, wherein the transcription factors are selected from the group consisting of: ZNF646, ZNF668, NR5A2, NANOG, NANOGP8, KLF1 , KLF17, NACC2 and TIGD5.

[0348] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: ZNF646, ZNF668, NR5A2, NANOG, NANOGP8, KLF1 , KLF17, NACC2 and TIGD5.

[0349] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are BIT-C-P3860PCT1

[0350] 76 selected from the group consisting of: ZNF646, ZNF668, NR5A2, NANOG, NANOGP8, KLF1 , KLF17, NACC2 and TIGD5.

[0351] Hepatoblasts

[0352] The present invention provides a method of generating hepatoblasts, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain hepatoblasts, wherein the transcription factors comprise ESRRA.

[0353] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ESRRA.

[0354] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ESRRA.

[0355] Trophoblasts

[0356] The present invention provides a method of generating trophoblasts, such as extravillous trophoblasts or trophoblast giant cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain trophoblasts, wherein the transcription factors comprise GATA4, preferably in combination with one or more of TFEB, LMX1A, ZNF282, CEBPB, KLF4 or OVOLI , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1 A, ZNF282, CEBPB, KLF4 and OVOL1.

[0357] The present invention provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise GATA4, preferably in combination with one or more of TFEB, LMX1A, ZNF282, CEBPB, KLF4 or OVOL1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4 and OVOL1. BIT-C-P3860PCT1

[0358] 77

[0359] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise GATA4, preferably in combination with one or more of TFEB, LMX1A, ZNF282, CEBPB, KLF4 or OVOL1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4 and OVOL1 .

[0360] In one embodiment, the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4, wherein the transcription factors do not consist of (i) CEBPB and KLF4 (this specific combination being particularly useful in generating goblet cells) or (ii) KLF4 and ZNF282 (this specific combination being particularly useful in generating retinal ganglion cells), or three or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4. In one embodiment, the transcription factors are selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4 and OVOL1 in combination with one or more of ZNF804A, ZSCAN30, DMRT1 , ESRRA, KLF7 or ESRRB, wherein the transcription factors do not consist of ESRRA and GATA4 (this specific combination being particularly useful in generating hepatocytes). In one embodiment, the transcription factors are selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4 in combination with one or more of ZNF804A, ZSCAN30, DMRT1 , ESRRA, KLF7 or ESRRB, wherein the transcription factors do not consist of ESRRA and GATA4. These transcription factors have been found to be particularly useful in generating trophoblasts, such as extravillous trophoblasts, trophoblast giant cells or placental villous trophoblasts.

[0361] The present invention provides a method of generating trophoblasts, such as extravillous trophoblasts, trophoblast giant cells or placental villous trophoblasts, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain trophoblasts, wherein the transcription factors comprise ZNF804A and / or ZSCAN30, preferably in combination with one or more of DMRT1 , TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4, ESRRA, KLF7, ESRRB or OVOLI , or wherein the transcription factors are selected from the group consisting of: DMRT1 , ESRRA, ESRRB and KLF7 in combination with one or more of ZNF804A, ZSCAN30, TFEB, GATA4, LMX1A, BIT-C-P3860PCT1

[0362] 78

[0363] ZNF282, CEBPB, KLF4 or OVOL1 , wherein the transcription factors do not consist of ESRRA and GATA4.

[0364] The present invention provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZNF804A and / or ZSCAN30, preferably in combination with one or more of DMRT1 , TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4, ESRRA, KLF7, ESRRB or OVOL1 , or wherein the transcription factors are selected from the group consisting of: DMRT1 , ESRRA, ESRRB and KLF7 in combination with one or more of ZNF804A, ZSCAN30, TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4 or OVOL1 , wherein the transcription factors do not consist of ESRRA and GATA4.

[0365] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ZNF804A and / or ZSCAN30, preferably in combination with one or more of DMRT 1 , TFEB, GATA4, LMX1A, ZNF282, CEBPB, KLF4, ESRRA, KLF7, ESRRB or OVOL1 , or wherein the transcription factors are selected from the group consisting of: DMRT1 , ESRRA, ESRRB and KLF7 in combination with one or more of ZNF804A, ZSCAN30, TFEB, GATA4, LMX1 A, ZNF282, CEBPB, KLF4 or OVOL1 , wherein the transcription factors do not consist of ESRRA and GATA4.

[0366] In one embodiment, the transcription factors comprise ZNF804A and / or ZSCAN30, preferably in combination with one or more of DMRT 1 , TFEB, GATA4, LMX1 A, ZNF282, CEBPB, KLF4, ESRRA, KLF7 or ESRRB, or wherein the transcription factors are selected from the group consisting of: DMRT1 , ESRRA, ESRRB and KLF7 in combination with one or more of ZNF804A, ZSCAN30, TFEB, GATA4, LMX1A, ZNF282, CEBPB or KLF4, wherein the transcription factors do not consist of ESRRA and GATA4. These transcription factors have been found to be particularly useful in generating trophoblasts, such as extravillous trophoblasts, trophoblast giant cells or placental villous trophoblasts.

[0367] The transcription factors may comprise GATA4, preferably in combination with one or more of LMX1 A, ZNF282, TFEB or OVOL1 , or two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282 and OVOL1. In one embodiment, the transcription factors are two or more transcription factors selected from the group consisting BIT-C-P3860PCT1

[0368] 79 of: TFEB, GATA4, LMX1A and ZNF282. In one embodiment, the transcription factors are selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282 and OVOL1 in combination with one or more of ZNF804A, ZSCAN30, DMRT 1 , ESRRA or KLF7, wherein the transcription factors do not consist of ESRRA and GATA4 (this specific combination being particularly useful in generating hepatocytes). In one embodiment, the transcription factors are one or more selected from the group consisting of: TFEB, GATA4, LMX1A and ZNF282 in combination with one or more of ZNF804A, ZSCAN30, DMRT 1 , ESRRA or KLF7, wherein the transcription factors do not consist of ESRRA and GATA4. These transcription factors have been found to be particularly useful in generating extravillous trophoblasts.

[0369] In one embodiment, the transcription factors comprise ZNF804A and / or ZSCAN30, preferably in combination with one or more of DMRT1 , TFEB, GATA4, LMX1A, ZNF282, ESRRA, KLF7 or OVOL1 , or wherein the transcription factors are selected from the group consisting of: DMRT1 , ESRRA and KLF7 in combination with one or more of ZNF804A, ZSCAN30, TFEB, GATA4, LMX1A, ZNF282 or OVOL1 , wherein the transcription factors do not consist of ESRRA and GATA4 (this specific combination being particularly useful in generating hepatocytes). In one embodiment, the transcription factors comprise ZNF804A and / or ZSCAN30, preferably in combination with one or more of DMRT1 , TFEB, GATA4, LMX1A, ZNF282, ESRRA or KLF7, or the transcription factors are selected from the group consisting of: DMRT1 , ESRRA and KLF7 in combination with one or more of ZNF804A, ZSCAN30, TFEB, GATA4, LMX1A or ZNF282, wherein the transcription factors do not consist of ESRRA and GATA4. These transcription factors have been found to be particularly useful in generating extravillous trophoblasts.

[0370] The transcription factors may comprise TFEB in combination with KLF4, CEBPB, or both CEBPB and KLF4. In one embodiment, the transcription factors comprise TFEB in combination with KLF4. These transcription factors have been found to be particularly useful in generating trophoblast giant cells.

[0371] In one embodiment, the transcription factors comprise ESRRB in combination with KLF4, TFEB or both KLF and TFEB. These transcription factors have been found to be particularly useful in generating placental villous trophoblasts.

[0372] Erythroblasts

[0373] The present invention provides method of generating erythroblasts, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing BIT-C-P3860PCT1

[0374] 80 the expression of one or more transcription factors in a cell population and culturing the cell population to obtain erythroblasts, wherein the transcription factors comprise MYC.

[0375] The present invention also provides cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise MYC.

[0376] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise MYC.

[0377] Melanocytes

[0378] The present invention provides a method of generating melanocytes comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain melanocytes, wherein the transcription factors comprise TFEB and / or SOHLH1 , preferably in combination with one or more of ZNF282, SOX8 or SOX9, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, SOHLH1 , ZNF282, SOX8 and SOX9.

[0379] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise TFEB and / or SOHLH1 , preferably in combination with one or more of ZNF282, SOX8 or SOX9, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, SOHLH1 , ZNF282, SOX8 and SOX9.

[0380] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise TFEB and / or SOHLH 1 , preferably in combination with one or more of ZNF282, SOX8 or SOX9, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, SOHLH1 , ZNF282, SOX8 and SOX9. BIT-C-P3860PCT1

[0381] 81

[0382] The transcription factors may comprise TFEB and SOX9.

[0383] Transit amplifying cells

[0384] The present invention provides a method of generating transit amplifying cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain transit amplifying cells, wherein the transcription factors comprise IRF5 in combination with KLF2, RREB1 or both KLF2 and RREB1.

[0385] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise IRF5 in combination with KLF2, RREB1 or both KLF2 and RREB1.

[0386] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise IRF5 in combination with KLF2, RREB1 or both KLF2 and RREB1.

[0387] In one embodiment, the transcription factors may comprise KLF2 and RREB1 , preferably in combination with IRF5. In one embodiment, the transcription factors are one or more selected from the group consisting of: KLF2, RREB1 and IRF5 in combination with one or more of FOXP3, ZBTB7B or MYC, wherein the transcription factors do not consist of (i) MYC and I RF5, (ii) MYC and KLF2, or (iii) MYC, IRF5 and KLF2 (these specific combinations being particularly useful in generating basal epithelial cells of the tracheobronchial tree). These transcription factors have been found to be particularly useful in generating transit amplifying cells.

[0388] The present invention provides a method of generating transit amplifying cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain transit amplifying cells, wherein the transcription factors comprise ZBTB7B, preferably in combination with one or more of FOXP3, MYC, RREB1 , IRF5 or KLF2, orwherein the transcription factors comprise FOXP3 and / or MYC BIT-C-P3860PCT1

[0389] 82 in combination with one or more of ZBTB7B, RREB1 , IRF5 or KLF2, wherein the transcription factors do not consist of (i) MYC and IRF5, (ii) MYC and KLF2, or (iii) MYC, IRF5 and KLF2.

[0390] The present invention provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZBTB7B, preferably in combination with one or more of FOXP3, MYC, RREB1 , IRF5 or KLF2, or wherein the transcription factors comprise FOXP3 and / or MYC in combination with one or more of ZBTB7B, RREB1 , IRF5 or KLF2, wherein the transcription factors do not consist of (i) MYC and IRF5, (ii) MYC and KLF2, or (iii) MYC, IRF5 and KLF2.

[0391] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ZBTB7B, preferably in combination with one or more of FOXP3, MYC, RREB1 , IRF5 or KLF2, or wherein the transcription factors comprise FOXP3 and / or MYC in combination with one or more of ZBTB7B, RREB1 , IRF5 or KLF2, wherein the transcription factors do not consist of (i) MYC and IRF5, (ii) MYC and KLF2, or (iii) MYC, IRF5 and KLF2.

[0392] Prostate epithelial cells

[0393] The present invention provides a method of generating prostate epithelial cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain prostate epithelial cells, wherein the transcription factors are selected from the group consisting of: RORA, TP73 and ZBTB6, preferably in combination with CEBPB, GFI1 or both CEBPB and GFI1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: CEBPB, GF11 , RORA, TP73 and ZBTB6.

[0394] The present invention provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: RORA, TP73 and ZBTB6, preferably in combination with CEBPB, GFI1 or both CEBPB and GFI1 , or wherein the BIT-C-P3860PCT1

[0395] 83 transcription factors are two or more transcription factors selected from the group consisting of: CEBPB, GFI1 , RORA, TP73 and ZBTB6.

[0396] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: RORA, TP73 and ZBTB6, preferably in combination with CEBPB, GFI1 or both CEBPB and GF11 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: CEBPB, GFI1 , RORA, TP73 and ZBTB6.

[0397] In one embodiment, the transcription factors are selected from the group consisting of: CEBPB, GFI1 , RORA, TP73 and ZBTB6 in combination with one or more of MAF, IRF7, SPDEF, ZNF552, ZNF614, FOXA2, KLF1 , KLF2, MYOD1 , PTF1A or ESRRB, wherein the transcription factors do not consist of (i) CEBPB and FOXA2 (this specific combination being particularly useful in generating club cells), (ii) CEBPB and KLF2 (this specific combination being particularly useful in generating club cells), (iii) GFI1 and FOXA2 (this specific combination being particularly useful in generating club cells), (iv) GFI1 and KLF2 (this specific combination being particularly useful in generating club cells), (v) CEBPB, GFI1 and FOXA2 (this specific combination being particularly useful in generating club cells), (vi) CEBPB, GFI1 and KLF2 (this specific combination being particularly useful in generating club cells), (vii) CEBPB, FOXA2 and KLF2 (this specific combination being particularly useful in generating club cells), (viii) GF11 , FOXA2 and KLF2 (this specific combination being particularly useful in generating club cells), (ix) CEBPB, GF11 , FOXA2 and KLF2 (this specific combination being particularly useful in generating club cells), (x) CEBPB and ESRRB (this specific combination being particularly useful in generating trophoblasts). In one embodiment, the transcription comprise CEBPB and / or GFI1 in combination with one or more of MAF, IRF7, SPDEF, ZNF552, ZNF614, FOXA2, KLF1 , KLF2, MYOD1 , PTF1A or ESRRB, wherein the transcription factors do not consist of (i) CEBPB and FOXA2, (ii) CEBPB and KLF2, (iii) GFI1 and FOXA2, (iv) GFI1 and KLF2, (v) CEBPB, GFI1 and FOXA2, (vi) CEBPB, GFI1 and KLF2, (vii) CEBPB, FOXA2 and KLF2, (viii) GFI1 , FOXA2 and KLF2, (ix) CEBPB, GFI1 , FOXA2 and KLF2, (x) CEBPB and PTF1A and (xi) CEBPB and ESRRB. In one embodiment, the transcription factors comprise CEBPB and KLF1. These transcription factors have been found to be particularly useful in generating prostate epithelial cells. BIT-C-P3860PCT1

[0398] 84

[0399] The present invention provides a method of generating prostate epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain prostate epithelial cells, wherein the transcription factors are selected from the group consisting of: MAF, SPDEF, IRF7, ZNF552 and ZNF614 preferably in combination with one or more of CEBPB, GFI1 , FOXA2, KLF1 , KLF2, MYOD1 , PTF1A, ESRRB, RORA, TP73 or ZBTB6, or wherein the transcription factors are selected from the group consisting of: FOXA2, KLF1 , KLF2, MYOD1 , PTF1A and ESRRB in combination with one or more of MAF, SPDEF, IRF7, ZNF552, ZNF614, CEBPB, GFI1 , RORA, TP73 or ZBTB6, wherein the transcription factors do not consist of (i) CEBPB and FOXA2 (this specific combination being particularly useful in generating club cells), (ii) CEBPB and KLF2 (this specific combination being particularly useful in generating club cells), (iii) GFI1 and FOXA2 (this specific combination being particularly useful in generating club cells), (iv) GFI1 and KLF2 (this specific combination being particularly useful in generating club cells), (v) CEBPB, GFI1 and FOXA2 (this specific combination being particularly useful in generating club cells), (vi) CEBPB, GFI1 and KLF2 (this specific combination being particularly useful in generating club cells), (vii) CEBPB, FOXA2 and KLF2 (this specific combination being particularly useful in generating club cells), (viii) GFI1 , FOXA2 and KLF2 (this specific combination being particularly useful in generating club cells), (ix) CEBPB, GF11 , FOXA2 and KLF2 (this specific combination being particularly useful in generating club cells), (x) CEBPB and ESRRB (this specific combination being particularly useful in generating trophoblasts).

[0400] The present invention provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: MAF, SPDEF, IRF7, ZNF552 and ZNF614 preferably in combination with one or more of CEBPB, GF11 , FOXA2, KLF1 , KLF2, MYOD1 , PTF1A, ESRRB, RORA, TP73 or ZBTB6, or wherein the transcription factors are selected from the group consisting of: FOXA2, KLF1 , KLF2, MYOD1 , PTF1A and ESRRB in combination with one or more of MAF, SPDEF, IRF7, ZNF552, ZNF614, CEBPB, GF11 , RORA, TP73 or ZBTB6, wherein the transcription factors do not consist of (i) CEBPB and FOXA2, (ii) CEBPB and KLF2, (iii) GFI1 and FOXA2, (iv) GFI1 and KLF2, (v) CEBPB, GFI1 and FOXA2, (vi) CEBPB, GFI1 and KLF2, (vii) CEBPB, FOXA2 and KLF2, (viii) GFI1 , FOXA2 and KLF2, (ix) CEBPB, GFI1 , FOXA2 and KLF2, (x) CEBPB and PTF1A and (xi) CEBPB and ESRRB. BIT-C-P3860PCT1

[0401] 85

[0402] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: MAF, SPDEF, IRF7, ZNF552 and ZNF614 preferably in combination with one or more of CEBPB, GFI1 , FOXA2, KLF1 , KLF2, MYOD1 , PTF1A, ESRRB, RORA, TP73 or ZBTB6, or wherein the transcription factors are selected from the group consisting of: FOXA2, KLF1 , KLF2, MYOD1 , PTF1A and ESRRB in combination with one or more of MAF, SPDEF, IRF7, ZNF552, ZNF614, CEBPB, GFI1 , RORA, TP73 or ZBTB6, wherein the transcription factors do not consist of (i) CEBPB and FOXA2, (ii) CEBPB and KLF2, (iii) GFI1 and FOXA2, (iv) GFI1 and KLF2, (v) CEBPB, GFI1 and FOXA2, (vi) CEBPB, GFI1 and KLF2, (vii) CEBPB, FOXA2 and KLF2, (viii) GFI1 , FOXA2 and KLF2, (ix) CEBPB, GFI1 , FOXA2 and KLF2, (x) CEBPB and PTF1A and (xi) CEBPB and ESRRB.

[0403] In one embodiment, the transcription factors are selected from the group consisting of: MAF, SPDEF, IRF7, ZNF552 and ZNF614 preferably in combination with one or more of CEBPB, GFI1 , FOXA2, KLF1 , KLF2, MYOD1 , PTF1A or ESRRB, or wherein the transcription factors are selected from the group consisting of: FOXA2, KLF1 , KLF2, MYOD1 , PTF1A and ESRRB in combination with one or more of MAF, SPDEF, IRF7, ZNF552, ZNF614, CEBPB or GFI1 , wherein the transcription factors do not consist of (i) CEBPB and FOXA2, (ii) CEBPB and KLF2, (iii) GFI1 and FOXA2, (iv) GFI1 and KLF2, (v) CEBPB, GFI1 and FOXA2, (vi) CEBPB, GFI1 and KLF2, (vii) CEBPB, FOXA2 and KLF2, (viii) GFI1 , FOXA2 and KLF2, (ix) CEBPB, GFI1 , FOXA2 and KLF2, (x) CEBPB and PTF1A and (xi) CEBPB and ESRRB. In one embodiment, the transcription factors comprise CEBPB and KLF1. These transcription factors have been found to be particularly useful in generating prostate epithelial cells,

[0404] Basement membrane fibroblasts

[0405] The present invention provides a method of generating basement membrane fibroblasts comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain basement membrane fibroblasts, wherein the transcription factors comprise RHOXF2B or RHOXF2B and PTF1A.

[0406] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise RHOXF2B or RHOXF2B and PTF1A. BIT-C-P3860PCT1

[0407] 86

[0408] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise RHOXF2B or RHOXF2B and PTF1A.

[0409] Mammary gland cells

[0410] The present invention provides a method of generating mammary gland cells, such as luminal epithelial cells of the mammary gland or myoepithelial cells of the mammary gland, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain mammary gland cells, wherein the transcription factors are selected from the group consisting of: KLF16, PLIRA, KLF4 and GFI1 B, preferably in combination with WIZ.

[0411] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: KLF16, PLIRA, KLF4, and GFI1 B, preferably in combination with WIZ.

[0412] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: KLF16, PLIRA, KLF4 and GFI1 B, preferably in combination with WIZ.

[0413] In one embodiment, the transcription factors comprise KLF16 and / or PLIRA, preferably in combination with one or more of KLF4, GFI1 B and WIZ, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ, wherein the transcription factors do not consist of KLF4 and WIZ (this specific combination being particularly useful in generating corneal epithelial cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ. In one embodiment, the transcription factors are selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2 orTEAD3. These transcription BIT-C-P3860PCT1

[0414] 87 factors have been found to be particularly useful in generating mammary gland cells, such as luminal epithelial cells of the mammary gland or myoepithelial cells of the mammary gland.

[0415] The present invention provides a method of generating mammary gland cells, such as luminal epithelial cells of the mammary gland or myoepithelial cells of the mammary gland, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain mammary gland cells, wherein the transcription factors are selected from the group consisting of: HAND1 , KLF11 , LHX8 and OVOL2, preferably in combination with one or more of TEAD3, KLF16, PLIRA, KLF4, GFI1 B or WIZ, or wherein the transcription factors comprise TEAD3 in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2, KLF16, PURA, KLF4, GFU B or WIZ.

[0416] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: HAND1 , KLF11 , LHX8 and OVOL2, preferably in combination with one or more of TEAD3, KLF16, PURA, KLF4, GFI1 B or WIZ, or wherein the transcription factors comprise TEAD3 in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2, KLF16, PURA, KLF4, GFI1 B or WIZ.

[0417] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: HAND1 , KLF11 , LHX8 and OVOL2, preferably in combination with one or more of TEAD3, KLF16, PURA, KLF4, GFU B or WIZ, or wherein the transcription factors comprise TEAD3 in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2, KLF16, PURA, KLF4, GFI1 B or WIZ.

[0418] The transcription factors may comprise KLF4 and / or GFI1 B. In one embodiment, the transcription factors comprise KLF4 and GFI1 B. These transcription factors have been found to be particularly useful in generating luminal epithelial cells of the mammary gland.

[0419] The transcription factors may comprise KLF16 and / or PURA, preferably in combination with KLF4, WIZ or KLF4 and WIZ, or two or more transcription factors selected from the group BIT-C-P3860PCT1

[0420] 88 consisting of: KLF16, PLIRA, KLF4 and WIZ. In one embodiment, the transcription factors comprise KLF16 and / or PLIRA, preferably in combination with WIZ. In one embodiment, the transcription factors are selected from the group consisting of: KLF16, PLIRA, WIZ and KLF4 in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2 or TEAD3. In one embodiment, the transcription factors are selected from the group consisting of: KLF16, PLIRA and WIZ in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2 or TEAD3. These transcription factors have been found to be particularly useful in generating myoepithelial cells of the mammary gland.

[0421] In one embodiment, the transcription factors are selected from the group consisting of: HAND1 , KLF11 , LHX8 and OVOL2, preferably in combination with one or more of TEAD3, KLF16, PLIRA, WIZ or KLF4, or wherein the transcription factors comprise TEAD3 in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2, KLF16, PURA, WIZ or KLF4. In one embodiment, the transcription factors are selected from the group consisting of: HAND1 , KLF11 , LHX8 and OVOL2, preferably in combination with one or more of TEAD3, KLF16, PURA or WIZ, or wherein the transcription factors comprise TEAD3 in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2, KLF16, PURA or WIZ. These transcription factors have been found to be particularly useful in generating myoepithelial cells of the mammary gland.

[0422] Mesenchymal stem cells

[0423] The present invention provides a method of generating mesenchymal stem cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain mesenchymal stem cells, wherein the transcription factors comprise EOMES and / or TCF4.

[0424] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise EOMES and / or TCF4.

[0425] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise EOMES and / or TCF4. BIT-C-P3860PCT1

[0426] 89

[0427] The present invention provides a method of generating mesenchymal stem cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain mesenchymal stem cells, wherein the transcription factors comprise TBX6, preferably in combination with EOMES, TCF4 or both EOMES and TCF4.

[0428] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise TBX6, preferably in combination with EOMES, TCF4 or both EOMES and TCF4.

[0429] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise TBX6, preferably in combination with EOMES, TCF4 or both EOMES and TCF4.

[0430] Goblet cells

[0431] The present invention provides a method of generating goblet cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain goblet cells, wherein the transcription factors comprise ZNF853 and / or REST, preferably in combination with KLF4, CEBPB or both KLF4 and CEBPB, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST, wherein the transcription factors do not consist of CEBPB and KLF4 (this specific combination being particularly useful in generating trophoblast giant cells), or wherein the transcription factors are three or more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST.

[0432] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ZNF853 and / or REST, preferably in combination with KLF4, CEBPB or both KLF4 and CEBPB, or wherein the transcription factors are two or BIT-C-P3860PCT1

[0433] 90 more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST, wherein the transcription factors do not consist of CEBPB and KLF4, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST.

[0434] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise ZNF853 and / or REST, preferably in combination with KLF4, CEBPB or both KLF4 and CEBPB, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST, wherein the transcription factors do not consist of CEBPB and KLF4, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST.

[0435] In one embodiment, the transcription factors comprise ZNF853 and / or KLF4, preferably in combination with CEBPB, REST or both CEBPB and REST, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: ZNF853, KLF4, CEBPB and REST. In one embodiment, the transcription factors are selected from the group consisting of: ZNF853, KLF4, CEBPB and REST in combination with one or more of DBX1 , KLF17, ZNF503 and RLINX1. These transcription factors have been found to be particularly useful in generating goblet cells.

[0436] The present invention provides a method of generating goblet cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain goblet cells, wherein the transcription factors are selected from the group consisting of: DBX1 and ZNF503, preferably in combination with one or more of RUNX1 , KLF17, ZNF853, KLF4, CEBPB or REST, or comprise RUNX1 and / or KLF17 in combination with one or more of DBX1 , ZNF503, ZNF853, KLF4, CEBPB or REST.

[0437] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: DBX1 and ZNF503, preferably in combination with one or more of RLINX1 , KLF17, ZNF853, BIT-C-P3860PCT1

[0438] 91

[0439] KLF4, CEBPB or REST, or comprise RLINX1 and / or KLF17 in combination with one or more of DBX1 , ZNF503, ZNF853, KLF4, CEBPB or REST.

[0440] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors are selected from the group consisting of: DBX1 and / or ZNF503, preferably in combination with one or more of RUNX1 , KLF17, ZNF853, KLF4, CEBPB or REST, or comprise RUNX1 and / or KLF17 in combination with one or more of DBX1 , ZNF503, ZNF853, KLF4, CEBPB or REST.

[0441] Migratory enteric neural crest cells

[0442] The present invention provides a method of generating migratory enteric neural crest cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain migratory enteric neural crest cells, wherein the transcription factors comprise SOX18, preferably in combination with LMX1 B, SOX1 or LMX1 B and SOX1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: SOX18, LMX1 B and SOX1.

[0443] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise SOX18, preferably in combination with LMX1 B, SOX1 or LMX1 B and SOX1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: SOX18, LMX1 B and SOX1.

[0444] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors SOX18, preferably in combination with LMX1 B, SOX1 or LMX1 B and SOX1 , or wherein the transcription factors are two or more transcription factors selected from the group consisting of: SOX18, LMX1 B and SOX1. BIT-C-P3860PCT1

[0445] 92

[0446] Neuronal brush cells

[0447] The present invention provides a method of generating neuronal brush cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain neuronal brush cells, wherein the transcription factors comprise NEUROD1 , NEUROD2, ATOH1 and NEUROG1.

[0448] The present invention also provides a cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise NEUROD1 , NEUROD2, ATOH1 and NEUROG1.

[0449] The present invention also provides an expression cassette comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding the transcription factors themselves, wherein the transcription factors comprise NEUROD1 , NEUROD2, ATOH1 and NEUROG1.

[0450] Cell Types

[0451] The method may be used on any cell type, including stem cells. In the case of stem cells, the generation of lineage-restricted cells using the method may be referred to as “cellular reprogramming”, “forward reprogramming”, “direct programming” or “direct differentiation”, i.e., the pluripotent stem cell is differentiated into a lineage-restricted cell.

[0452] Sources of cells suitable for methods of the invention may include any cells that are different from the desired lineage-restricted cells, for example, pluripotent stem cells. For example, the stem cells may be induced pluripotent stem cells, embryonic stem cells or pluripotent stem cells derived by nuclear transfer or cell fusion. It may be preferred that the embryonic stem cell is derived without destruction of the embryo, particularly where the cells are human. In some embodiments, the stem cells are not derived from human or animal embryos, i.e., the invention does not extend to any methods which involve the destruction of human or animal embryos. The stem cells may also include multipotent stem cells, oligopotent stem cells, or unipotent stem cells. The stem cells may also include fetal stem cells or adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, skin stem cells. In certain aspects, the stem cells may be isolated from umbilical, placenta, amniotic fluid, chorionic villi, blastocysts, bone marrow, adipose tissue, brain, peripheral blood, cord blood, menstrual blood, blood vessels, skeletal muscle, skin and liver. BIT-C-P3860PCT1

[0453] 93

[0454] In one embodiment, the cell population is of human origin. The source cell may be of human origin.

[0455] In one embodiment, the cell population is of animal origin. The source cell e.g., a stem cell, may be of animal origin. In certain aspects, the cell is preferably one from a livestock animal. Livestock animals include, for example, pigs, cows, horses, buffalo, bison, goats, sheep, deer, reindeer, donkeys, bantengs, yaks, chickens, ducks and turkeys.

[0456] In one embodiment, the cell population comprises stem cells, e.g., induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), haematopoietic stem cells, mesenchymal stem cells or neuronal stem cells. In a further embodiment, the cell population comprises pluripotent stem cells, e.g., iPSCs or ESCs.

[0457] In one embodiment, the source cell is a stem cell, e.g., an iPSC, an ESC, a haematopoietic stem cell, a mesenchymal stem cell or a neuronal stem cell. In a further embodiment, the source cell is a pluripotent stem cell, e.g., an iPSC or an ESC. In some embodiments, the source cell is an iPSC.

[0458] Methods of preparing induced pluripotent stem cells are also known in the art. Induction of iPSCs typically require the expression of or exposure to at least one member from Sox family and at least one member from Oct family. Sox and Oct are thought to be central to the transcriptional regulatory hierarchy that specifies ES cell identity. For example, Sox may be Sox-1 , Sox-2, Sox-3, Sox- 15, or Sox-18; Oct may be Oct-4. Additional factors may increase the reprogramming efficiency, like Nanog, Lin28, Klf4, or c-Myc; specific sets of reprogramming factors may be a set comprising Sox-2, Oct-4, Nanog and, optionally, Lin-28; or comprising Sox-2, Oct4, Klf and, optionally, c-Myc. In one method, iPSC may be generated by transfecting cells with transcription factors Oct4, Sox2, c-Myc and Klf4 using viral transduction. In an alternative method, iPSC may be generated by transfecting cells with RNA encoding transcription factors inducing the development of stem cell characteristics, such as transcription factors selected from Oct4, Sox2, c-Myc and Klf4.

[0459] In one embodiment, the cell population comprises somatic cells, e.g., differentiated cells such as fibroblasts. The source cell may therefore be a somatic cell and the method may be a transdifferentiation method (i.e. the conversion of one cell type to another). Alternatively, the BIT-C-P3860PCT1

[0460] 94 method may be used to convert a related somatic cell (e.g. a T lymphocyte other than a regulatory T cell to a regulatory T cell).

[0461] In one embodiment, the lineage-restricted cells are human lineage-restricted cells.

[0462] In one embodiment, the induced pluripotent stem cells are derived from somatic or germ cells of the patient. Such use of autologous cells would remove the need for matching cells to a recipient. Alternatively, commercially available iPSC may be used, such as those available from WICELL (WiCell Research Institute, Inc, Wisconsin, US). Alternatively, the cells may be a tissue-specific stem cell which may also be autologous or donated.

[0463] Delivery of transcription factors

[0464] It will be understood that methods for expressing polypeptides having transcription factor activity and / or increasing the expression of the transcription factors in the cells to be programmed into lineage-restricted cells may include any method known in the art, for example, by induction of expression of one or more expression cassettes previously introduced into the cells, or by introduction of nucleic acids (such as DNA or RNA), polypeptides, or small molecules to the cells to stimulate expression of the endogenous or exogenous transcription factors. Increasing the expression of certain endogenous but transcriptionally repressed genes may also reverse the silencing or inhibitory effect on the expression of these genes by regulating the upstream transcription factor expression or epigenetic modulation. Therefore, methods of the invention may involve culturing the cell population under conditions to artificially increase the expression level of one or more of the transcription factors described herein.

[0465] In one embodiment, the expression of the polypeptides having transcription factor activity and / or the transcription factors is increased by contacting the cell population with the polypeptides and / or the transcription factors (i.e., the proteins encoding the transcription factors). Delivery of the transcription factors may occur using direct electroporation of transcription factor proteins to the cells.

[0466] In an alternative embodiment, the expression of the transcription factors is increased by introducing a promoter (e.g. a strong promoter) ahead of an endogenous gene encoding the transcription factor(s).

[0467] In a further alternative embodiment, the expression of the polypeptides having transcription factor activity and / or the transcription factors is increased by contacting the cell population with BIT-C-P3860PCT1

[0468] 95 one or more agents that activate or increase the expression amount of the (exogenous or endogenous) transcription factors. In the case of polypeptides having transcription factor activity or exogenous transcription factors, the agents may still be used after the genes for the polypeptides and / or transcription factors have been inserted into the cell.

[0469] In one embodiment, the agent is selected from the group consisting of: a nucleic acid (i.e., polynucleotide, e.g., messenger RNA (mRNA), coding DNA sequence), a protein, an aptamer and small molecule, ribosome, RNAi agent, guide RNA (gRNA) and peptide nucleic acid (PNA) and analogues or variants thereof. In one embodiment, the agent is a transcriptional activation system (e.g., a gRNA for use in a gene activation system such as CRISPR / Cas or TALEN) for increasing the expression of the endogenous transcription factors.

[0470] The method of inducing differentiation of the cell population (i.e., source cells), may comprise delivering to the cells a nucleic acid comprising an open reading frame encoding one or more of the polypeptides having transcription factor activity and / or one or more of the transcription factors (e.g., in an expression cassette), the transcription factor protein and / or polypeptides themselves, or an activator of transcription of the open reading frame encoding the polypeptide and / or transcription factor. This results in the amount of the transcription factor in the cells being increased, and the cells differentiate to form lineage-restricted cells. Said open reading frame may be part of a recombinant expression cassette.

[0471] In one embodiment, the nucleic acid comprises a recombinant or exogenous expression cassette comprising the transcription factor sequences (or genes) in a sufficient number to cause cellular reprogramming of source cells to lineage-restricted cells. The exogenous expression cassette may comprise an externally inducible transcriptional regulatory element for inducible expression of the one or more transcription factors, such as an inducible promoter, e.g., comprising a tetracycline response element or variant thereof. This externally inducible transcriptional regulatory element may also be a blocker sequence that prevents a promoter facilitating the expression of transcription factor sequences, wherein the blocker sequence may be removable using, for example, a recombinase, and wherein removal then allows the expression of the transcription factor sequences.

[0472] If expression of the transcription factors is increased by introducing an exogenous sequence encoding the transcription factor (e.g., the transcription factor gene), then it would be understood that any suitable system for delivering the sequence may be used. The gene delivery system may be a transposon system; a viral gene delivery system; an episomal gene BIT-C-P3860PCT1

[0473] 96 delivery system; or a homologous recombination system such as utilizing a zinc finger nuclease, a transcription activator-like effector nuclease (TALENs), a meganuclease, or CRISPR / Cas, or the like.

[0474] Alternatively, introduction of a nucleic acid, such as DNA or RNA, into cells may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection (including microinjection), by electroporation, by calcium phosphate precipitation, by using DEAE-dextran followed by polyethylene glycol, by direct sonic loading, by liposome mediated transfection, by receptor- mediated transfection, by microprojectile bombardment, by agitation with silicon carbide fibers, by Agrobacterium-mediated transformation, and any combination of such methods. Through the application of these techniques, cells may be stably or transiently transformed.

[0475] In an alternative embodiment, the cell population is contacted with one or more agents that has the same effect as activating or increasing the expression or amount of the transcription factors (i.e. an indirect method of increasing the expression transcription factor). In this aspect of the invention, the method comprises introducing an exogenous agent which mimics the effect of increasing the expression of the transcription factors described herein. For example, such a method may comprise introducing a protein (e.g. an engineered zinc finger nuclease) that has a DNA-binding activity analogous to the transcription factor. For instance, PPAR proteins bind to peroxisome proliferator responsive elements, so the activity of these transcription factors could be reproduced by a zinc finger nuclease engineered to bind the same domain.

[0476] It will be understood that a combination of one or more of the methods for expressing the polypeptides having transcription factor activity or increasing the expression of the transcription factors may be used where the combination overall results in activity necessary for the forward programming or transdifferentiation to lineage-restricted cells.

[0477] Vectors

[0478] In one embodiment, the polypeptides having transcription factor activity or the transcription factors themselves (e.g., combinations of polypeptides and / or transcription factors) are introduced into the cell population using a vector. One of skill in the art would be well equipped to construct a vector through standard recombinant techniques. Vectors include but are not BIT-C-P3860PCT1

[0479] 97 limited to plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).

[0480] In one embodiment, a nucleic acid sequence encoding one or more transcription factors is introduced into a cell by a transposon system (i.e. involving a transposon plasmid). A transposon delivery system is comprised of two plasmids, one encoding the transposase and one encoding the transcription factor(s). The transposase protein mediates random integration of the transcripts encoded in the transposon plasmid into the genome. In one embodiment, the transposon system is selected from a PiggyBac or Sleeping Beauty transposon system. The transposon plasmid encodes a payload flanked by two ITRs (internal terminal repeats). The payload may comprise a Tet inducible promoter, the transcription factor(s), and optionally a selection marker, e.g. an antibiotic selection cassette under a constitutive promoter.

[0481] In one embodiment, the transposase and transposon plasmids are delivered by nucleofection or lipofection into the cells. The number of integration events, and therefore the number of copies of payload per cell can be in part controlled by adjusting the total and relative amounts of transposase and transposon plasmid DNA. This allows the combinatorial delivery of transcription factors at a single cell level.

[0482] In one embodiment, the vector is a viral vector. The viral gene delivery system may be an RNA-based or DNA-based viral vector. Viral vectors include retroviral vectors, lentiviral vectors (e.g., derived from HIV-1 , HIV-2, SIV, BIV, FIV etc.), gammaretroviral vectors, adenoviral (Ad) vectors (including replication competent, replication deficient and gutless forms thereof), adeno-associated virus-derived (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumour virus vectors, Rous sarcoma virus vectors and Sendai virus vectors. In a further embodiment, the viral vector is selected from: a lentiviral vector, an adeno-associated virus vector or a Sendai virus vector. In a yet further embodiment, the viral vector is a lentiviral vector.

[0483] Lentiviral vectors are well known in the art. Lentiviral vectors are complex retroviruses capable of integrating randomly into the host cell genome, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (e.g., accessory genes Vif, Nef, Vpu, Vpr). Lentiviral vectors have the advantage of being able to infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviral vector capable of BIT-C-P3860PCT1

[0484] 98 infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat.

[0485] In one embodiment, the vector is a self-replicating RNA vector expression system. For example, the system may comprise self-replicating RNA vectors that remain ectopic to the host cell genome and encode the transcription factors that induce forward programming or transdifferentiation. Self-replicating RNA vectors are known in the art and many are based on positive strand RNA viruses, such as alphaviruses.

[0486] In one embodiment, the viral vector is used at a high multiplicity of infection (MOI). A high MOI helps to ensure that more than one transcription factor is introduced into the source cell. In one embodiment, the MOI is greater than 0.5, such as 1 .0 or above.

[0487] In one embodiment, a nucleic acid sequence encoding the one or more polypeptides having transcription factor activity and / or transcription factors is introduced into a cell by a plasmid. In one embodiment, at least one nucleic acid sequence encoding the polypeptides having transcription factor activity and / or the transcription factors is introduced into a cell on a single plasmid.

[0488] In one embodiment, the plasmid is episomal. Episomal vectors are able to introduce large fragments of DNA into a cell but are maintained extra-chromosomally, replicated once per cell cycle, partitioned to daughter cells efficiently, and elicit substantially no immune response. In alternative embodiments, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, or a bovine papilloma virus (BPV)-based vector may be used.

[0489] Site-specific delivery

[0490] Any suitable technique for insertion of a nucleic acid sequence into a specific sequence may be used, and several are described in the art. Suitable techniques include any method which introduces a break at the desired location and permits recombination of the vector into the gap. Thus, a crucial first step for targeted site-specific genomic modification is the creation of a double-strand DNA break (DSB) at the genomic locus to be modified. Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, and these are non-homologous end joining repair (NHEJ), which is more prone to error; and homologous recombination repair (HR). BIT-C-P3860PCT1

[0491] 99

[0492] Several techniques exist to allow customized site-specific generation of DSB in the genome. Many of these involve the use of customized endonucleases, such as zinc finger nucleases, TALENs or the CRISPR / CRISPR associated protein (CRISPR / Cas, e.g. CRISPR / Cas9) system.

[0493] Zinc finger nucleases are artificial enzymes which are generated by fusion of a zinc-finger DNA-binding domain to the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific cleavage domain which must dimerise in order to cleave DNA. This means that two zinc finger nuclease monomers are required to allow dimerisation of the Fokl domains and to cleave the DNA. The DNA binding domain may be designed to target any genomic sequence of interest, is a tandem array of Cys2His2 zinc fingers, each of which recognises three contiguous nucleotides in the target sequence. The two binding sites are separated by 5-7bp to allow optimal dimerization of the Fokl domains. The enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break.

[0494] Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factor / nucleases. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. TAL effectors are proteins that are secreted by Xanthomonas bacteria, the DNA binding domain of which contains a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing appropriate residues at the two variable positions. TALENs are thus built from arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By selecting the array of modules, almost any sequence may be targeted. Again, the nuclease used may be Fokl or a derivative thereof.

[0495] Three types of CRISPR mechanisms have been identified, of which type II is the most studied. The CRISPR / Cas9 system (type II) utilises the Cas9 nuclease to make a double-stranded break in DNA at a site determined by a short guide RNA. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition BIT-C-P3860PCT1

[0496] 100 is followed by short segments of “protospacer DNA” from previous exposures to foreign genetic elements. CRISPR spacers recognize and cut the exogenous genetic elements using RNA interference. The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules are composed of a variable sequence transcribed from the protospacer DNA and a CRISPR repeat. Each crRNA molecule then hybridizes with a second RNA, known as the trans-activating CRISPR RNA (tracrRNA) and together these two eventually form a complex with the nuclease Cas9. The protospacer DNA encoded section of the crRNA directs Cas9 to cleave complementary target DNA sequences, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs). This natural system has been engineered and exploited to introduce DSB breaks in specific sites in genomic DNA, amongst many other applications. In particular, the CRISPR type II system from Streptococcus pyogenes may be used. At its simplest, the CRISPR / Cas9 system comprises two components that are delivered to the cell to provide genome editing: the Cas9 nuclease itself and a gRNA. The gRNA is a fusion of a customised, site-specific crRNA (directed to the target sequence) and a standardised tracrRNA.

[0497] Once a DSB has been made, a donor template with homology to the targeted locus is supplied; the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise insertions to be made.

[0498] Derivatives of this system are also possible. Mutant forms of Cas9 are available, such as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only. Cas9D10A may be used in paired Cas9 complexes designed to generate adjacent DNA nicks in conjunction with two sgRNAs complementary to the adjacent area on opposite strands of the target site, which may be particularly advantageous.

[0499] The elements for making the double-strand DNA break may be introduced in one or more vectors, such as plasmids, for expression in the cell.

[0500] Thus, any method of making specific, targeted double strand breaks in the genome in order to effect the insertion of a gene / inducible cassette may be used in the method of the invention. It may be preferred that the method for inserting the gene / inducible cassette utilises any one or more of zinc finger nucleases, TALENs and / or CRISPR / Cas9 systems or any derivative thereof. BIT-C-P3860PCT1

[0501] 101

[0502] Once the DSB has been made by any appropriate means, the gene / inducible cassette for insertion may be supplied in any suitable fashion as described below. The gene / inducible cassette and associated genetic material form the donor DNA for repair of the DNA at the DSB and are inserted using standard cellular repair machinery / pathways. How the break is initiated will alter which pathway is used to repair the damage, as noted above.

[0503] Other methods in the art for site specific delivery include the use of homologous recombination (HR) and recombinase mediated cassette exchange (RMCE). DNA damage mediated site specific insertion methods (such as CRISPR / Cas) can also be used to perform site specific integration of DNA recognition sequences (‘att' sites) which in turn mediate site specific insertion via the activity of tyrosine and serine recombinases or integrases. These sites (e.g. attP) once inserted into the genome, can mediate site specific HR and RMCE. Insertion of exogenous nucleic acid sequences occurs through homologous recombination between cognate attP and attB sites mediated by the expression of the appropriate and cognate recombinase (e.g. Flp, Cre) or integrase (PhiC31 , Bxb1). Using targeting vectors, as described above, flanked by attB sites, site specific exogenous DNA insertion of transgenes can be achieved.

[0504] Controlled expression

[0505] In one embodiment, expression of the transcription factors is under inducible control. In this aspect of the invention, the transcription and translation (expression) of the polypeptides having transcription factor activity and / or the transcription factors may be controlled within the cell. This permits overexpression of the transcription factor(s), if required.

[0506] An exogenous expression cassette carrying the transcription factors may comprise an externally inducible transcriptional regulatory element (i.e., an inducible promoter) for rapid induction of protein expression in response to external stimuli, i.e. inducible gene (or transgene) expression. The presence or addition of the appropriate external stimuli (e.g. protein, compound or chemical) to cell culture media modulates the controlled expression of the genetic sequence within the inducible expression cassette; and may be administered continuously or transiently to modulate transcription as required.

[0507] Expression of the transcription factors described herein may be increased using a dual cassette expression system, such as the system described in WO2018096343, which is incorporated herein by reference. In this instance, induced transgene over-expression is BIT-C-P3860PCT1

[0508] 102 achieved by using the Tet-ON system components with transgene expression controlled by doxycycline. The components are split between two genomic safe harbour (GSH) sites to reduce the risk of epigenetic gene silencing. The components are (i) transcriptional activator protein (reverse tetracycline trans-activator (rtTA)), which in the presence of doxycycline binds (ii) tetracycline response element (TRE; multiple TetO repeat sequences & minimal Cytomegalovirus (CMV) promoter). TRE binding by rtTA trans-activates transgene expression. Trans-activatable coding sequences for transgenes may be of human origin.

[0509] Therefore, in one embodiment, a sequence encoding one or more of the polypeptides having transcription factor activity and / or the transcription factors is introduced into the cell population using a method comprising:

[0510] - insertion of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of a source cell present in the cell population; and

[0511] - insertion of one or more inducible cassettes into one or more second genomic safe harbour sites of the source cell, wherein said one or more inducible cassettes comprises said sequence encoding the one or more polypeptides having the activity of one or more transcription factors and / or the transcription factors, and the transcription of said cassette is regulated by the transcriptional regulator protein.

[0512] In an alternative embodiment there is provided a dual expression cassette system. The insertion of the gene encoding a transcriptional regulator protein into the first GSH provides the control mechanism for the expression of the inducible cassette and inserted into a second GSH site. In one embodiment, the first and second GSH are different (i.e. are located at different positions in the genome). It will be understood that if more than one transcription factor is to be introduced into the cell using the dual expression system, then the transcription factors may be introduced into the second GSH site (i.e. a multicistronic cassette at the same GSH site), or into multiple GSH sites (i.e. as separate cassettes across different GSH sites).

[0513] Alternatively, the dual expression cassette system utilises different alleles of the same GSH site. In this embodiment, the inducible cassette may be inserted into one allele of the GSH and the system controlling the expression of the inducible cassette into the other allele of the GSH site (e.g. as described in DeKelver et al., 2010, Genome Res., 20, 1133-43 and Qian et al., 2014, Stem Cells, 32, 1230-8).

[0514] A GSH site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most BIT-C-P3860PCT1

[0515] 103 beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighbouring genes and expression of the inducible cassette minimizes interference with the endogenous transcription programme. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al. (2011) Nature Biotechnology, 29(1): 73-8). These criteria include a site that is (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA (miRNA), (iv) located outside a transcription unit and (v) located outside ultraconserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of the criteria. It is thought that a suitable GSH will satisfy at least 2, 3, 4 or all of these criteria. Any suitable GSH site may be used in the method of the invention, on the basis that the site allows insertion of genetic material without deleterious effects to the cell and permits transcription of the inserted genetic material. Those skilled in the art may use these simplified criteria to identify a suitable GSH, and / or the more formal criteria set out above.

[0516] Insertion of the coding sequence for a transcriptional regulator protein and / or the inducible cassette may be carried out through direct delivery methods as described above. It is understood that although such direct delivery methods may lead to the random insertion of the genetic material, screening may be carried out in order to identify clones that show no deleterious effects, are able to express the genetic material and are able to be forward programmed or reprogrammed to regulatory T cells, and by doing so one is able to confirm that the transcriptional regulator protein I inducible cassette has been inserted into a GSH site.

[0517] In one embodiment the insertion of the transcriptional regulator protein or the inducible cassette is targeted. In a further embodiment, the insertion of the transcriptional regulator protein and the inducible cassette is targeted. “Targeted insertion”, as with site-specific delivery, is understood as the insertion of the genetic material into a pre-chosen GSH site. As discussed above, this can be carried out using techniques known in the art such as zinc finger nucleases, TALENs or the clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR / Cas, e.g. CRISPR / Cas9) system.

[0518] In one embodiment, the first and second GSH sites are selected from (in particular any two) of the ROSA26 locus, the AAVS1 locus, the CLYBL gene, the CCR5 gene or the HPRT gene. Insertions specifically within GSH sites is preferred over random genome integration, since this is expected to be a safer modification of the genome, and is less likely to lead to unwanted side effects such as silencing natural gene expression or random insertional mutagenesis. BIT-C-P3860PCT1

[0519] 104

[0520] The adeno-associated virus integration site 1 locus (AAVS1) is located within the protein phosphatase 1 , regulatory subunit 12C (PPP1 R12C) gene on human chromosome 19, which is expressed uniformly and ubiquitously in human tissues. AAVS1 has been shown to be a favourable environment for transcription, since it comprises an open chromatin structure and native chromosomal insulators that enable resistance of the inducible cassettes against silencing. There are no known adverse effects on the cell resulting from disruption of the PPP1 R12C gene. Moreover, an inducible cassette inserted into this site remains transcriptionally active in many diverse cell types.

[0521] The human ROSA26 (hROSA26) site has been identified on the basis of sequence analogy with a GSH from mice (ROSA26 - reverse oriented splice acceptor site #26). The hROSA26 locus is on chromosome 3 (3p25.3), and can be found within the Ensembl database (GenBank:CR624523). The integration site lies within the open reading frame (ORF) of the THUMPD3 long non-coding RNA (reverse strand). Since the hROSA26 site has an endogenous promoter, the inserted genetic material may take advantage of that endogenous promoter, or alternatively may be inserted operably linked to a promoter.

[0522] Intron 2 of the Citrate Lyase Beta-like (CLYBL) gene, on the long arm of Chromosome 13, was identified as a suitable GSH since it is one of the identified integration hot-spots of the phage derived phiC31 integrase. Studies have demonstrated that randomly inserted inducible cassettes into this locus are stable and expressed. It has been shown that insertion of inducible cassettes at this GSH do not perturb local gene expression (Cerbini et al. (2015) PLOS One, 10(1): e0116032). CLYBL thus provides a GSH which may be suitable for use in the present invention.

[0523] CC 5, which is located on chromosome 3 (position 3p21.31) is a gene which codes for HIV-1 major co-receptor. Interest in the use of this site as a GSH arises from the null mutation in this gene that appears to have no adverse effects, but predisposes to HIV-1 infection resistance. Zinc-finger nucleases that target the third exon have been developed, thus allowing for insertion of genetic material at this locus.

[0524] The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase enzyme that plays a central role in the generation of purine nucleotides through the purine salvage pathway. BIT-C-P3860PCT1

[0525] 105

[0526] Other GSH sites have been described in the art, such as in Sadelain et al. (2012) Nature Reviews 12:51-58 and in WO2021 / 152086, which are herein incorporated by reference.

[0527] GSH sites in other organisms have been identified and include ROSA26, HRPT and Hippl 1 (H11) loci in mice. Mammalian genomes may include GSH sites based upon pseudo attP sites. For such sites, hiC31 integrase, the Streptomyces phage-derived recombinase, has been developed as a non-viral insertion tool, because it has the ability to integrate an inducible cassette-containing plasmid carrying an attB site into pseudo attP sites.

[0528] Technically, the insertions into the first and / or second GSH site may occur on one chromosome, or on both chromosomes. The GSH site exists at the same genetic loci on both chromosomes of diploid organisms. Insertion within both chromosomes is advantageous since it may enable an increase in the level of transcription from the inserted genetic material within the inducible cassette, thus achieving particularly high levels of transcription.

[0529] Specific insertion of genetic material into the particular GSH site based upon customised sitespecific generation of DNA double-strand breaks at the GSH site may be achieved. The genetic material may then be introduced using any suitable mechanism, such as homologous recombination. Any method of making a specific DSB in the genome may be used, but preferred systems include CRISPR / Cas9 and modified versions thereof, zinc finger nucleases and the TALEN system, or via HR or ROME mediated integration or recombination.

[0530] One or more genetic sequences may be controllably transcribed from within the second and / or further GSH sites. Indeed, the inducible cassette may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 genetic sequences (e.g., transcription factor sequences) which it is desired to insert into the GSH and the transcription of which be controllably induced. Therefore, the transcription factors required by the present invention may be included within the same cassette introduced into the second genomic safe harbour site. For example, three or more transcription factors may be included in, for example, three mono-cistronic constructs, one mono-cistronic and one bi- cistronic construct or one tri-cistronic construct. It will be understood that similar combinations of constructs may be used to achieve higher orders of transcription factor expression.

[0531] Alternatively, if a combination of transcription factors is used, the individual transcription factors may be introduced into separate GSH sites, and the transcription of these transcription factors may be regulated by the transcriptional regulator protein. Therefore, in one embodiment, the transcription factors are introduced into separate GSH sites. For example, this may be BIT-C-P3860PCT1

[0532] 106 achieved by utilising three or more different GSH sites for three or more transcription factors (i.e., wherein the transcription factors are introduced as mono-cistronic cassettes). Alternatively, this may be achieved by utilising the fact that a GSH exists at the same genetic loci on both chromosomes of diploid organisms, e.g., introducing one transcription factor into the GSH site on one chromosome and a different transcription factor into the same GSH site on the other chromosome. This embodiment is advantageous if different expression levels or timing of expression of the transcription factors is desired. In one embodiment, the method comprises targeted insertion of the transcription factors into a second, third and fourth genomic safe harbour site of the source cell. The transcription of the transcription factors may all be regulated by the transcriptional regulator protein.

[0533] Alternatively, the transcriptional regulator system may be based on the hepatitis C virus (HCV) NS3 protease domain. NS3 is a serine cis-protease that excises itself from the HCV polyprotein by cleaving recognition sites that flank it at either end. Because it is essential for HCV replication, numerous inhibitors targeting the viral protease have been developed, such as danoprevir and grazoprevir. The protease has been used as a ligand-inducible connection to control the association between modular DNA-binding and transcriptional activation domains. In an initial design, the protease was inserted between minimal DNA-binding and transcriptional activator sequences. In this configuration, the viral protease would serve as a self-immolating connection, excising itself from the fusion construct and, in doing so, separating the DNA-binding and transcriptional activator elements. However, in the presence of an NS3 inhibitor, self-excision of the protease would be blocked, resulting in the preservation of full-length gene capable of activating the expression of targeted genes.

[0534] The transcriptional regulator protein may be activated or deactivated by light. Such proteins are described in, for example, W02023 / 004031 (incorporated herein by reference). The protein may comprise a light-activatable domain that responds to light of a particular wavelength. In some cases, the light-activatable domain, upon stimulation with light of a particular wavelength or within a particular spectral range, dimerizes or oligomerizes (e.g., with another light activatable domain). In some cases, the light-activatable domain may form a homodimer or a heterodimer (e.g., may dimerize with a second, different light-activatable domain). In some cases, the light-activatable domain may exist in a (e.g., homo or hetero) dimer or (e.g., homo or hetero) oligomer (e.g., in the absence of light), and may dissociate into a monomeric form after exposure to light. The light-activatable domain may be derived from a natural source (e.g., a naturally occurring protein) or may be synthetically produced. The light- activatable domain may comprise or may be a functional domain or portion of a naturally BIT-C-P3860PCT1

[0535] 107 occurring protein, such as, by way of example only, the PHR domain of Arabidopsis cryptochrome 2. The light-activatable domain may comprise an amino acid sequence identical to an amino acid sequence of a wildtype protein, or may comprise one or more variants (e.g., amino acid substitutions, deletions, insertions, etc.) relative to a wild-type protein. This domain may activate or deactivate the transcriptional regulator protein.

[0536] In various aspects, a combination of light-activatable domains (e.g., a first light activatable domain and a second light-activatable domain) may be used. In this scenario, the first light- activatable domain and the second light-activatable domain are binding partners, such that upon illumination with light at a particular wavelength or within a particular spectral range, the first and second light-activatable domains heterodimerize or heterooligomerize. This heterodimerization or heterooligomerization may activate or deactivate the transcriptional regulator protein.

[0537] In various aspects, the light-activatable domain comprises a Light-Oxygen-Voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a Cryptochrome (CRY) domain, Blue- light-using FAD (BLLIF) photoreceptor domain, a Phytochrome (PHY) domain, a CIBN (N- terminal domain of CIB1 (cryptochrome-interacting basic-helix-loop-helix protein 1)) domain, a PIF (phytochrome interacting factor) domain, a Dronpa domain, a LIVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin binding domain (CBD), or a combination thereof. In one example, the light-activatable domain is a LOV domain (e.g., such as a LOV domain derived from Vaucheria frigida Aureochrome 1).

[0538] In some instances, a combination of light-activatable domains is used, wherein the first light- activatable domain is cryptochrome 2 (or a variant or a functional portion thereof) and the second-light activatable domain is CIBN (or a variant or a functional portion thereof). In some instances, a combination of light-activatable domains is used, wherein the first light-activatable domain is BphP1 (or a variant or a functional portion thereof) and the second-light activatable domain is QPAS1 (or a variant or a functional portion thereof).

[0539] The light-activatable domain may be fused with a domain that, upon activation, interacts with an inducible promoter to allow expression of the genetic material to take place. Alternatively the light-activatable domain may be fused with a domain that, upon activation, interacts with a recombinase that removes a blocker sequence, allowing expression of the genetic material to take place. The advantage of the combination with a recombinase is that only a short burst of light is necessary in order to switch on expression, rather than constant light exposure. BIT-C-P3860PCT1

[0540] 108

[0541] A transcriptional regulator protein is a protein that binds to DNA, preferably sequence- specifically to a DNA site, and either facilitating the transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor).

[0542] The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or near the promoter of the regulated DNA sequence. Transcriptional activator proteins bind to the response element and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression. Transcriptional repressor proteins bind to the response element and prevent gene expression.

[0543] T ranscriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory proteins, phosphorylation, and / or methylation. The transcriptional regulator protein may be controlled by activation or deactivation.

[0544] If the transcriptional regulator protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated through the addition to the cell of an exogenous substance or light. The supply of an exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulator protein can be controlled. Alternatively, an exogenous substance can be supplied in order to deactivate a transcriptional regulator protein, and then supply withdrawn in order to activate the transcriptional regulator protein.

[0545] If the transcriptional regulator protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires deactivation. Thus, a substance is supplied to prevent the transcriptional repressor protein repressing transcription, and thus transcription is permitted.

[0546] Any suitable transcriptional regulator protein may be used, preferably one that may be activated or deactivated. It is preferred that an exogenous substance or light may be supplied to control the transcriptional regulator protein. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins. BIT-C-P3860PCT1

[0547] 109

[0548] Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline which is more stable). In this system, the transcriptional activator protein is reverse tetracycline-controlled transactivator (rtTa, which may also be referred to as tetracycline - responsive transcriptional activator protein) or a derivative thereof. The rtTA protein is able to bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response element (TRE). There are two forms of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or deactivates (Tet-Off) the rtTA protein.

[0549] In a Tet-Off system, tetracycline or a derivative thereof binds rtTA and deactivates the rtTA, rendering it incapable of binding to TRE sequences, thereby preventing transcription of TRE- controlled genes. This system was first described in Gossen et al. (1992) PNAS 89 (12): 5547- 5551.

[0550] The Tet-On system is composed of two components; (1) the constitutively expressed reverse tetracycline-controlled transactivator (rtTa) and the rtTa-sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes. The use of this may be preferred in the method of the invention.

[0551] Thus, the transcriptional regulator protein may thus be a reverse tetracycline-controlled transactivator (rtTa) protein, which can be activated or deactivated by the antibiotic tetracycline or one of its derivatives, which are supplied exogenously. The exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

[0552] Variants and modified rtTa proteins may also be used in the methods of the invention, these include Tet-On Advanced transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0553] The tetracycline response element (TRE) generally consists of 7 repeats of the 19bp bacterial TetO sequence separated by spacer sequences, together with a minimal promoter. Variants and modifications of the TRE sequence are possible, since the minimal promoter can be any suitable promoter. For example, 2, 3, 4, 5 and 6 repeats of the TetO sequence have been BIT-C-P3860PCT1

[0554] 110 found to be effective also. Preferably the minimal promoter shows no or minimal expression levels in the absence of rtTa binding. The inducible promoter may thus comprise a TRE.

[0555] A modified system based upon tetracycline control is the T-REX System (Thermo-Fisher Scientific), in which the transcriptional regulator protein is a transcriptional repressor protein, TetR. The components of this system include (i) an inducible promoter comprising a strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites, and a Tet repressor (TetR). In the absence of tetracycline, the Tet repressor forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the inducible promoter, and prevent transcription from the promoter. Once added, tetracycline binds with high affinity to each Tet repressor homodimer rendering it unable to bind to the Tet operator. The Tet repressor: tetracycline complex then dissociates from the Tet operator and allows induction of expression. In this instance, the transcriptional regulator protein is TetR and the inducible promoter comprises two TetO2 sites. The exogenously supplied substance is tetracycline or a derivative thereof.

[0556] Other inducible expression systems are known and can be used in the method of the invention. These include the Complete Control Inducible system from Agilent Technologies. This is based upon the insect hormone ecdysone or its analogue ponasterone A (ponA) which can activate transcription in mammalian cells which are transfected with both the gene for the Drosophila melanogaster ecdysone receptor (EcR) and an inducible promoter comprising a binding site for the ecdysone receptor. The EcR is a member of the retinoid-X-receptor (RXR) family of nuclear receptors. In humans, EcR forms a heterodimer with RXR that binds to the ecdysoneresponsive element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

[0557] Thus, the transcriptional regulator protein can be a repressor protein, such as an ecdysone receptor or a derivative thereof. Examples of the latter include the VgEcR synthetic receptor from Agilent technologies which is a fusion of EcR, the DNA binding domain of the glucocorticoid receptor and the transcriptional activation domain of Herpes Simplex Virus VP16. The inducible promoter comprises the EcRE sequence or modified versions thereof together with a minimal promoter. Modified versions include the E / GRE recognition sequence of Agilent Technologies, in which mutations to the sequence have been made. The E / GRE recognition sequence comprises inverted half-site recognition elements for the retinoid-X- receptor (RXR) and GR binding domains. In all permutations, the exogenously supplied BIT-C-P3860PCT1

[0558] 111 substance is ponasterone A, which removes the repressive effect of EcR or derivatives thereof on the inducible promoter, and allows transcription to take place.

[0559] Alternatively, inducible systems may be based on the synthetic steroid mifepristone as the exogenously supplied substance. In this scenario, a hybrid transcriptional regulator protein is inserted, which is based upon a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain (LBD) from the human progesterone receptor and an activation domain (AD) from the human NF-KB. This hybrid transcriptional regulator protein is available from Thermo-Fisher Scientific (Gene Switch™). Mifepristone activates the hybrid protein, and permits transcription from the inducible promoter which comprises GAL4 upstream activating sequences (UAS) and the adenovirus E1 b TATA box. This system is described in Wang et al. (1994) PNAS 91 : 8180-8184.

[0560] The transcriptional regulator protein can thus be any suitable regulator protein, either an activator or repressor protein. Suitable transcriptional activator proteins are tetracyclineresponsive transcriptional activator protein or the Gene Switch hybrid transcriptional regulator protein. Suitable repressor proteins include the Tet-Off version of rtTA, TetR or EcR. The transcriptional regulator proteins may be modified or derivatised as required.

[0561] The inducible promoter can comprise elements which are suitable for binding or interacting with the transcriptional regulator protein. The interaction of the transcriptional regulator protein with the inducible promoter is preferably controlled by the exogenously supplied substance or light.

[0562] The exogenously supplied substance can be any suitable substance that binds to or interacts with the transcriptional regulator protein. Suitable substances include tetracycline (or derivatives thereof, such as doxycycline), ponasterone A and mifepristone.

[0563] Alternatively, the gene encoding the transcriptional regulator protein, the inducible cassette or both the gene encoding the transcriptional regulator protein and the inducible cassette are not inserted into a GSH site. Preferably the gene encoding the transcriptional regulator protein, the inducible cassette or both the gene encoding the transcriptional regulator protein and the inducible cassette are inserted within an endogenous protein-encoding gene locus, wherein expression of the endogenous protein-encoding gene within the locus is modulated as a result of the insertion of the transcriptional regulator protein; and wherein expression of the transcriptional regulator protein is lower than the expression if the same genetic material was BIT-C-P3860PCT1

[0564] 112 inserted into a genomic safe harbour site, operatively linked to a promoter. It is understood that the insertion of the gene encoding the transcriptional regulator protein and / or the inducible cassette may have deleterious effects on the cell, but nevertheless the cell can tolerate these effects. It is also understood that it may be beneficial to disrupt the expression of a gene where the transcriptional regulator protein and / or the inducible cassette have been inserted.

[0565] Alternatively, in one embodiment, a sequence encoding one or more of the polypeptides having transcription factor activity and / or the transcription factors is introduced into the cell population using a method comprising:

[0566] (a) insertion of a coding sequence for a transcriptional regulator protein into a source cell present in the cell population; and

[0567] (b) insertion of one or more inducible cassettes into the source cell, wherein said one or more inducible cassettes comprises said sequence encoding the one or more polypeptides having the activity of one or more transcription factors and / or the transcription factors, and the transcription of said cassette is regulated by the transcriptional regulator protein, wherein the gene encoding the transcriptional regulator protein of (a) and / or the inducible cassette of (b) is inserted within an endogenous protein-encoding gene locus; wherein expression of the endogenous protein-encoding gene within the locus is modulated as a result of the insertion of the transcriptional regulator protein; and wherein expression of the transcriptional regulator protein is lower than the expression if the same genetic material was inserted into a genomic safe harbour site, operatively linked to a promoter.

[0568] In an alternative embodiment, there is provided a dual expression cassette system wherein the transcriptional regulator protein and / or the inducible cassette is inserted within an endogenous protein-encoding gene locus. The insertion of the gene encoding a transcriptional regulator protein provides the control mechanism for the expression of the inducible cassette.

[0569] In one embodiment where the gene encoding the transcriptional regulator protein is inserted in the endogenous protein-encoding gene locus, expression of the gene encoding the transcriptional regulatory protein is regulated by the transcription of the endogenous gene, preferably by the transcription of the endogenous gene and by the transcriptional regulator protein itself. In a preferred embodiment, the transcriptional regulator protein regulates an inducible promoter operatively linked to the gene encoding the transcriptional regulator protein. In a preferred embodiment, the promoter is in the form of an intron. BIT-C-P3860PCT1

[0570] 113

[0571] In one embodiment where the gene encoding the transcriptional regulator protein and the inducible cassette are both inserted within the endogenous protein-encoding gene locus in the cell, expression of the gene encoding the transcriptional regulatory protein is regulated by the transcription of the endogenous gene, preferably by the transcription of the endogenous gene and by the transcriptional regulator protein itself. In a preferred embodiment, the transcriptional regulator protein regulates an inducible promoter operatively linked to the gene encoding the transcriptional regulator protein and to the genetic sequence of the inducible cassette. In a preferred embodiment, the promoter and the genetic sequence are in the form of an intron.

[0572] In one embodiment where the gene encoding the transcriptional regulator protein and the inducible cassette are both inserted within the endogenous protein-encoding gene locus in the cell, the gene encoding the transcriptional regulator protein and the genetic sequence form part of an inducible bidirectional cassette comprising a promoter operatively linked and flanked by the gene encoding a transcriptional regulator protein at one end and the genetic sequence at the other end, wherein the transcription of the inducible bidirectional cassette is regulated by the transcriptional regulator protein and wherein the inducible bidirectional cassette is inserted within the endogenous protein-encoding gene locus in the cell such that expression of the endogenous protein-encoding gene leads to the expression of the transcriptional regulator protein. In a preferred embodiment, the promoter and the genetic sequence are in the form of an intron.

[0573] In one embodiment where the transcriptional regulator protein and the inducible cassette are inserted separately, the inducible cassette comprises a further gene encoding the transcriptional regulator protein, preferably wherein the transcriptional regulator protein regulates the transcription of both the further gene encoding the transcriptional regulator protein and the genetic sequence.

[0574] In one embodiment where the transcriptional regulator protein and the inducible cassette are inserted separately, the inducible cassette is a bidirectional cassette comprising a promoter operatively linked and flanked at one end by a further gene encoding the transcriptional regulator protein and at the other end by a gene sequence.

[0575] In one embodiment, the endogenous protein-coding gene within the locus is not transcribed after forward programming or transdifferentiation has taken place. BIT-C-P3860PCT1

[0576] 114

[0577] One embodiment provides a method for controlling transcription of a genetic sequence suitable for forward programming or transdifferentiation in a cell, the method comprising insertion in a source cell of:

[0578] (a) a gene encoding a transcriptional regulator protein; and

[0579] (b) an inducible cassette comprising a genetic sequence, wherein the transcription of the inducible cassette is regulated by the transcriptional regulator protein; wherein the gene encoding the transcriptional regulator protein of (a) and / or the inducible cassette of (b) is inserted within an endogenous protein-encoding gene locus that is not transcribed after forward programming or transdifferentiation has taken place.

[0580] One embodiment provides a cell with a modified genome that comprises:

[0581] (a) an inserted gene encoding a transcriptional regulator protein; and

[0582] (b) an inserted inducible cassette comprising a genetic sequence suitable for forward programming or transdifferentiation, wherein the transcription of the inducible cassette is regulated by the transcriptional regulator protein; wherein the gene encoding the transcriptional regulator protein of (a) and / or the inducible cassette of (b) is inserted within an endogenous protein-encoding gene locus that is not transcribed after forward programming or transdifferentiation has taken place.

[0583] It is preferred that the gene encoding the transcriptional regulator protein is operably linked to a constitutive promoter. Alternatively, the first GSH site or endogenous protein-encoding gene can be selected such that it already has a constitutive promoter that can also drive expression of the transcriptional regulator protein gene and any associated genetic material. Constitutive promoters ensure sustained and high-level gene expression. Commonly used constitutive promoters, including the human p-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1a, (EF1a), phosphoglycerate kinase (PGK) and ubiquitin C (UbC). The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression and was constructed from the following sequences: (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene.

[0584] Obtaining lineage-restricted cells

[0585] It is clear that the methods of the present invention may be used for the forward programming or transdifferentiation to the lineage-restricted cells as previously set out. These lineage- restricted cells may be: BIT-C-P3860PCT1

[0586] 115 glial cells, such as oligodendrocyte precursor cells (that differentiate to form mature oligodendrocytes), astrocytes or Schwann cells; retinal cells, such as photoreceptor cells, retinal ganglion cells, amacrine cells (including glycinergic amacrine cells) or Muller cells; kidney cells, such as podocytes, loop of Henle epithelial cells (including the thin descending limb and the thick ascending limb), collecting duct principal cells, interstitial fibroblasts, papillary tip cells, proximal tubule epithelial cells or parietal epithelial cells; lung cells, such as respiratory epithelial cells (including basal epithelial cells of the tracheobronchial tree and epithelial cells of the lower respiratory tract), type 1 pneumocytes, type 2 pneumocytes or club cells; muscle cells, such as pericytes, cardiac muscle cells, skeletal muscle cells (including skeletal satellite muscle cells) or smooth muscle cells (including enteric smooth muscle cells or bronchial smooth muscle cells); intestinal epithelial cells;

[0587] T cells, such as regulatory T cells or gamma delta T cells; endocrine cells, such as enteroendocrine cells or neural endocrine cells (including lung neural endocrine cells; ciliated cells, such as ependymal cells or ciliated cells of the trachea or oesophagus; neutrophils; endocardial cells; endothelial cells, such as endothelial cells of the lymph vessel or sinusoidal endothelial cells (including endothelial cells of the pericentral hepatic sinusoid); neurons, such as excitatory neurons (including glutamatergic neurons), inhibitory neurons (including GABAergic neurons), enteric neurons or peripheral nervous system neurons; mesothelial cells; hepatoblasts; trophoblasts, such as extravillous trophoblasts, trophoblast giant cells or placental villous trophoblasts; erythroblasts; melanocytes; transit amplifying cells; prostate epithelial cells; basement membrane fibroblasts; mammary gland cells, such as luminal epithelial cells of the mammary gland or myoepithelial cells of the mammary gland; mesenchymal stem cells; BIT-C-P3860PCT1

[0588] 116 goblet cells, including respiratory, intestinal or conjunctival goblet cells; migratory enteric neural crest cells; or neuronal brush cells.

[0589] Glial cells, also known as neuroglia, are cells that support and protect neurons. Oligodendrocytes, for example, are cells that produce myelin, the insulating coating for nerve fibres, and are generated from oligodendrocyte precursor cells. Astrocytes, easily recognizable through their star-shaped morphology, comprise many processes that envelop neuronal synapses, and through this interaction perform many functions including the regulation of axonal growth and support, blood-brain barrier formation, immune responses as well as functioning in higher cognitive functions, including memory. Schwann cells are essential glial cells of the peripheral nervous system, responsible for supporting neurons, forming the myelin sheath around axons, guiding nerve growth, and aiding in nerve repair after injury by clearing debris and promoting regeneration. For the purposes of the present invention, ependymal cells are categorised as ciliated cells rather than glial cells based on the presence of cilia on the cells and the common transcription factor profile found to relate to all ciliated cells.

[0590] Retinal cells comprise any cells found in the retina of the eye. Retinal cells include photoreceptor cells, a neuroepithelial cell found in the retina capable of visual phototransduction (converting visible light into signals that stimulate biological processes). Preferably the retinal cells are photoreceptor cells. Retinal ganglion cells are involved with sending visual information from the retina to the brain via the optic nerve. Amacrine cells, found in the retina, integrate, modulate, and add a temporal domain to visual information sent to the retinal ganglion cells. Glycinergic amacrine cells are inhibitory interneurons in the retina that use the neurotransmitter glycine to process visual signals, forming crucial links within the inner plexiform layer to modulate bipolar, ganglion, and other amacrine cells. Muller cells support the neurons present in the retinal. For the purposes of the present invention, photoreceptor cells, retinal ganglion and amacrine cells are categorised as retinal cells rather than neurons based on the specialist role of the cells in the retina and the common transcription factor profile found to relate to all retinal cells. Similarly, for the purposes of the present invention, Muller cells are categorised as retinal cells rather than a glial cell based on the specialist role of the cells in the retina and the common transcription factor profile found to relate to all retinal cells.

[0591] Kidney cells comprise any cells found at least predominately in the kidney organs. Kidney cells include podocytes, the cells of the Bowman’s capsule that help maintain the glomerular filtration barrier. Preferably the kidney cells are podocytes. Loop of Henle epithelial cells are BIT-C-P3860PCT1

[0592] 117 cells involved with reabsorbing water and sodium chloride from the glomerular filtrate. Collecting duct principal cells are cells involved with sodium reabsorption and potassium secretion. Interstitial fibroblasts control systemic oxygen delivery to the kidneys by producing renin and erythropoietin. Papillary tip cells, located in the renal papilla, are involved with the formation of nephrons. Proximal tubule epithelial cells are involved with the reabsorption of nutrients such as glucose, water and low-molecular weight proteins from the glomerular filtrate and help control acid-base balance. Parietal epithelial cells form a monolayer on the parietal basement membrane surrounding the glomerular tuft and act as a barrier to glomerular filtrate.

[0593] Lung cells comprise any cells found at least predominately in the lung organs. Lung cells include respiratory epithelial cells, the cell lining the upper respiratory airways and the lower respiratory tract. These cells moisten and protect the airways. Preferably the lung cells are respiratory epithelial cells. These respiratory epithelial cells may be basal epithelial cells of the tracheobronchial tree. The basal cells provide an attachment site for ciliated and goblet cells to the basal lamina. Type 1 pneumocytes are specialized cells that line the alveoli that exchange gas and form the air-blood barrier with the pulmonary capillary endothelium, and also secrete surfactant. Type 2 pneumocytes also line the alveoli but synthesise surfactant that reduces the surface tension in the alveoli. Club cells, also known as bronchial exocrine cells, secrete bioactive compounds that detoxify harmful substances and repair the airways after injury. For the purposes of the present invention, ciliated cells are categorised separately based on the presence of cilia on the cells and the common transcription factor profile found to relate to all ciliated cells. Similarly bronchial smooth muscle cells have been categorised as muscle cells based on the function of these cells and the common transcription factor profile found to relate to other muscle cells.

[0594] Muscle cells are cells able to contract, creating a pulling force able to stabilise or move parts of the body. Muscle cells include pericytes, cells that wrap around microvessels and play a key role in vascular homeostasis and disease. Preferably the muscle cells are pericytes. Cardiac muscle cells are specialized cells that aid heart contraction. Skeletal muscle cells connect to the skeleton and aid the movement of the skeleton. Skeletal muscle cells comprise skeletal satellite muscle cells that are the primary stem cells in adult skeletal muscle. Smooth muscle is involuntary, non-striated muscle. Smooth muscle cells may be enteric smooth muscle that forms part of the intestinal wall or bronchial smooth muscle found in the walls of the respiratory tree. BIT-C-P3860PCT1

[0595] 118

[0596] Intestinal epithelial cells are cells that line the surface of the intestinal epithelium and perform key functions including nutrient absorption, maintaining a barrier against harmful microorganisms, presenting antigens (as part of the intestinal immune system) and producing mucus.

[0597] T cells, also known as thymocytes, are a type of lymphocyte produced from or processed by the thymus gland and comprising a T cell receptor on the surface. T cells include regulatory T cells, a subset of T helper cells that maintain a tolerance to self-antigens by downregulating the induction and proliferation of effector T cells, and in doing so prevent autoimmune disease. Regulatory T cells also shut down the immune responses after an invading microorganism has successfully been eliminated. By contrast, gamma delta T cells (y<5 T cells) are T cells that have a T cell receptor and are found predominantly in the gut mucosa. They have a prominent role in lipid antigen recognition.

[0598] Endocrine cells are cells associated with the endocrine system, a network of glands that produce hormones that regulate many of the body's functions. Endocrine cells include enteroendocrine cells, cells of the gastrointestinal tract and pancreas that produce hormones and peptides in response to stimuli, such as somatostatin, motilin, cholecystokinin, neurotensin, vasoactive intestinal peptide and enteroglucagon. By contrast, neural endocrine cells are associated with the neuroendocrine system, a network of nerves and glands that work together to produce and release hormones into the bloodstream. Lung neural endocrine cells, also known as pulmonary neuroendocrine cells, function as sensory cells that detect stimuli like oxygen levels, mechanical stress, and inflammatory molecules, and respond by releasing neurotransmitters and hormones.

[0599] Ciliated cells are cells that comprise short, hair-like projections, called cilia, that extend from the cell surface. Ciliated cells include those found in the trachea and oesophagus, as well as ependymal cells.

[0600] Neutrophils are a form of phagocytic granulocyte that form a part of the innate immunity.

[0601] Endocardial cells are specialized cells forming the innermost layer of the heart. For the purposes of this invention endocardial cells have been categorised separately from other forms of endothelial cells due to the specialized nature of the cells. BIT-C-P3860PCT1

[0602] 119

[0603] Endothelial cells are cells that line the inside of blood vessels and lymph vessels. They form a single layer of cells called the endothelium. Endothelial cells include endothelial cells of the lymph vessel and sinusoidal endothelial cells. Sinusoidal endothelial cells are specialized cells that line the hepatic sinusoids and play critical roles in blood filtering, endocytosis, immune regulation, coagulation and hepatic regeneration. Sinusoidal endothelial cells may be endothelial cells of the pericentral hepatic sinusoid.

[0604] Neurons are the fundamental units of the brain and nervous system responsible for transforming or relaying electrical signals. Neurons include excitatory neurons (neurons that produce excitatory neurotransmitters, such as glutamatergic neurons producing glutamate) and inhibitory neurons (neurons that produce inhibitory neurotransmitters, such as GABAergic neurons producing GABA (y-aminobutyric acid)). Neurons may also be enteric neurons; neurons that form a neural network within the gastrointestinal tract, part of the autonomic nervous system. Neurons may also be peripheral nervous system neurons, which are neurons found outside the brain and spinal cord.

[0605] Mesothelial cells are pavement-like cells that line the body’s internal organs and serous cavities, forming a protective, non-adhesive mesothelium layer.

[0606] Hepatoblasts are liver stem cells that retain the ability to self-renew and proliferate to provide liver progenitor cells. These liver progenitor cells can differentiate into hepatocytes or cholangiocytes.

[0607] Trophoblasts are cells forming the outer layer of a blastocyst, which provides nutrients to the embryo, and develops into a large part of the placenta. Trophoblasts include extravillous trophoblasts, a form of differentiated trophoblast cells of the placenta, establishing a critical tissue connection in the placental-uterine interface. Trophoblasts also include trophoblast giant cells, cells that forward the outermost layer of the placenta’s extraembryonic compartment, involved with the regulation of the maternal immune and endocrine systems. Placental villous trophoblasts are specialized cells forming the outer layer of placental villi, crucial for maternal- fetal exchange of nutrients, gases, and waste, comprising inner cytotrophoblasts and outer syncytiotrophoblasts, with roles in hormone production (like hCG) and anchoring the placenta.

[0608] Erythroblasts are immature polychromatic nucleated cells that synthesise haemoglobin and form erythrocytes. BIT-C-P3860PCT1

[0609] 120

[0610] Melanocytes are cells capable of forming melanin. Melanocyte are found predominantly in the skin, but are found in the eyes also.

[0611] Transit amplifying cells are differentiated, proliferating cell population in transition between stem cells and differentiated cells. Transit amplifying cells are involved with tissue homeostasis and tissue regeneration after insult.

[0612] Prostate epithelial cells are cells that form part of the male reproductive system and produce exocrine secretions, specifically part of the fluid that makes up semen.

[0613] Basement membrane fibroblasts are cells that contribute to the development of the basement membrane, a thin layer of extracellular matrix that supports tissue function and integrity. Basement membrane fibroblasts contribute to collagen IV assembly in the basement membrane.

[0614] Mammary gland cells are cells that form the mammary gland, part of the milk-producing system in female mammals. The mammary gland cells may be luminal epithelial cells that line the mammary ducts and alveoli, and are involved with milk production, storage and secretion. The mammary glands may be myoepithelial cells that surround the mammary gland’s secretory units and contract to move the milk towards the lactiferous sinus.

[0615] Mesenchymal stem cells are multipotent stem cells found in the bone marrow, skin and fat tissue. They play an important role in making and repairing skeletal tissues such as bone, cartilage, muscle and fat.

[0616] Goblet cells are specialized epithelial cells that produce and secrete mucus, that lubricates and protects the lining of organs. Goblet cells are found in respiratory and gastrointestinal tracts, and also in the conjunctiva of the eye.

[0617] Migratory enteric neural crest cells are migratory cells that originate in the neural tube and travel along the gastrointestinal tract to form the enteric nervous system.

[0618] Neuronal brush cells are a unique type of sensorial cell found primarily within the mammalian respiratory and gastrointestinal tracts, especially in the throat and nose regions. Despite their alternative nomenclature, these cells are not neurons per se, but rather, they exhibit neuronlike properties and structure. The name 'brush cell' is derived from their distinctive morphology, BIT-C-P3860PCT1

[0619] 121 characterized by blunt, brush-like microvilli protruding from the apical surface of the cell. The primary function of neuronal brush cells is detecting extracellular changes and stimuli, acting as intermediaries between sensory neuronal signals and their targets. They act as chemosensors, responding to changes in the chemical environment. It is thought that brush cells detect chemical contents in the mucosal lining of the nasal and oral cavities and convert this to a chemical signal which is then conveyed to the brain, contributing to the mechanics of smell and taste. This might also suggest a role in monitoring bacterial populations and regulating inflammatory reactions within the respiratory and gastrointestinal tracts.

[0620] In one embodiment, the method additionally comprises monitoring the cell population for at least one characteristic of a lineage-restricted cell. Cells may be monitored throughout culturing to identify expression of key lineage markers.

[0621] For example, monitoring may be through the use of engineered ‘reporter’ cell lines (i.e. endogenously tagged proteins or positive selection markers under the control of promoters specific to the lineage-restricted cells of interest) or immunostaining and detection, using fluorescence microscopy or flow cytometry. Such material includes genes for markers or reporter molecules, such as genes that induce visually identifiable characteristics including fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue / UV light, luciferase, which catalyses a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed.

[0622] The cell may further comprise a positive selection marker and / or selectable reporter expression cassette, e.g., comprising a promoter specific to the lineage-restricted cells of interest operably linked to a reporter gene.

[0623] Selectable markers may include resistance genes to antibiotics or other drugs. Examples of drug resistance genes may include: a puromycin resistance gene, an ampicillin resistance gene, a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene or a chloramphenicol resistance gene. Cells can be cultured on a medium containing the appropriate drug (i.e., a selection medium) and only those cells which incorporate and express the drug resistance gene will survive. Therefore, by culturing cells using a selection medium, it is possible to select for cells comprising and expressing a drug resistance gene, positively enriching for a target cell population. BIT-C-P3860PCT1

[0624] 122

[0625] Examples of fluorescent protein genes which may be used as markers include: a GFP gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene or aequorin gene. Cells expressing the fluorescent protein can be detected using a fluorescence microscope and fluorescence activated cell sorting (FACS) used to identify and select cell populations based on the expression of fluorescent proteins.

[0626] Fluorescent protein genes may be tagged with a nuclear localization signal peptide to confine expression of the fluorescent proteins to the nucleus. This may be helpful in cell types with a high lipid content which may not be suitable for FACS. This allows end-point fluorescence- activated cell sorting to be carried out on either whole cell populations, or purified nuclei which maintain an intact fluorescent signal.

[0627] Examples of chromogenic enzyme genes which may be used as markers, and known in the art, include but are not limited to: p-galactosidase gene, p-glucuronidase gene, alkaline phosphatase gene, or secreted alkaline phosphatase SEAP gene. Cells expressing these chromogenic enzyme genes can be detected by applying the appropriate chromogenic substrate (e.g., X-gal for p galactosidase) so that cells expressing the marker gene will produce a detectable colour (e.g., blue in a blue-white screen test).

[0628] The method may therefore comprise a selection or enrichment step for the specific lineage- restricted cells of interest provided from the methods described herein. In one embodiment, the method comprises the step of sorting the lineage-restricted cells using FACS or immunomagnetic sorting methods based on the expression of markers specific to the lineage- restricted cells of interest and / or absence of markers that are not specific to the lineage- restricted cells of interest. A labelled binding agent directed to target cell surface proteins may be used. Any binding agent capable of specific binding to a particular epitope may be used for this purpose, for example an anti...

Claims

BIT-C-P3860PCT1166CLAIMS1. A method of generating muscle cells, such as pericytes, cardiac muscle cells, skeletal muscle cells, skeletal satellite muscle cells, smooth muscle cells, enteric smooth muscle cells or bronchial smooth muscle cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain muscle cells, wherein the transcription factors are selected from the group consisting of: MEF2C, MEF2D, MYF6, MYOD1 , ESRRG and NKX2.3, preferably in combination with one or more of PHOX2B, SOX9, or ESRRB, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, MYF6, MYOD1 ESRRB, ESRRG, NKX2.3, PHOX2B and SOX9, or wherein the transcription factors are selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9 and ESRRB in combination with one or more of DPF1 , EN1 , FERD3L, FOXF2, HOXC4, KLF10, LEUTX, MESP2, MYOG, NR3C2, SMYD2, SOX15, ZNF429, ZNF619, NEUROD2 or TFEB, wherein the transcription factors do not consist of (i) ESRRB and TFEB, (ii) NEUROD2 and PHOX2B or (iii) SOX9 and SOX15, or variants thereof.

2. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: MEF2C, MEF2D, MYF6, MYOD1 , ESRRG and NKX2.3, preferably in combination with one or more of PHOX2B, SOX9 or ESRRB, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MEF2C, MEF2D, MYF6, MYOD1 ESRRB, ESRRG, NKX2.3, PHOX2B and SOX9, wherein the transcription factors are selected from the group consisting of: MEF2C, MEF2D, ESRRG, NKX2.3, MYF6, MYOD1 , PHOX2B, SOX9 and ESRRB in combination with one or more of DPF1 , EN1 , FERD3L, FOXF2, HOXC4, KLF10, LEUTX, MESP2, MYOG, NR3C2, SMYD2, SOX15, ZNF429, ZNF619, NEUROD2 and TFEB, wherein the transcription factors do not consist of (i) ESRRB and TFEB, (ii) NEUROD2 and PHOX2B or (iii) SOX9 and SOX15,BIT-C-P3860PCT1167 or variants thereof.

3. The method as defined in claim 1 or the cell as defined in claim 2, wherein the transcription factors are selected from the group consisting of: MEF2D, MOYD1 , MEF2C, MYF6 and ESRRG, preferably in combination with ESRRB.

4. The method or the cell as defined in claim 3, wherein the transcription factors comprise:(i) MEF2C, ESRRB and ESRRG;(ii) MYOD1 , MEF2D and MYF6;(iii) MYOD1 , MEF2D, MYF6, MEF2C, ESRRB and ESRRG;(iv) MEF2D and ESRRB, preferably in combination with MYF6;(v) MYF6, ESRRG and MEF2C; or(vi) ESRRG, MEF2D and MYOD1.

5. The method as defined in claim 1 or the cell as defined in claim 2, wherein the transcription factors comprise NKX2.3, preferably in combination with one or more of MYOD1 , SOX9 or MYF6, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: MYOD1 , NKX2.3, SOX9 and MYF6 wherein the transcription factors do not consist of MYOD1 and MYF6, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: NKX2.3, MYOD1 , SOX9 and MYF6, or wherein the transcription factors are selected from the group consisting of: NKX2.3, MYOD1 , SOX9 and MYF6 in combination with one or more of MYOG, NR3C2, DPF1 , LEUTX, ZNF619, EN1 , NEUROD1 of TFEB.

6. The method or the cell as defined in claim 5, wherein the transcription factors comprise NKX2.3, preferably in combination with MYOD1 , SOX9 or MYOD1 and SOX9, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: NKX2.3, MYOD1 and SOX9, or wherein the transcription factors are selected from the group consisting of: NKX2.3, MYOD1 and SOX9 in combination with one or more of DPF1 , LEUTX, ZNF619, NEUROD1 or TFEB.BIT-C-P3860PCT11687. The method of the cell as defined in claim 6, wherein the transcription factors comprise MYOD1 and / or SOX9 in combination with TFEB.

8. The method as defined in claim 1 or the cell as defined in claim 2, wherein the transcription factors comprise MEF2D and / or PHOX2B, preferably in combination with one or more of FERD3L, FOXF2, HOXC4, KLF10, MESP2, SMYD2, SOX15 or ZNF429.

9. The method or the cell as defined in claim 8, wherein the transcription factors comprise MEF2D, preferably in combination with one or more of MESP2, FERD3L, KLF10, SMYD2, SOX15 or ZNF429.

10. The method or the cell as defined in claim 9, wherein the transcription factors comprise MEF2D and MESP2.

11. A method of generating glial cells, such as oligodendrocyte precursor cells, oligodendrocytes astrocytes or Schwann cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain glial cells, wherein the transcription factors are selected from the group consisting of: LMX1A, SOX9, SOX10 and SOX8, preferably in combination with one or more of NEUROD1 , FOXJ1 or SOXI , wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX1 , SOX9, SOX10, SOX8, NEUROD1 and FOXJ1 , wherein the transcription factors do not consist of SOX1 and NEU ROD 1 , wherein the transcription factors are three or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 and FOXJ1 , or wherein the transcription factors are selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 and FOXJ1 in combination with one or more of HEY1 , ZBTB37 or REST, or variants thereof.

12. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors,BIT-C-P3860PCT1169 wherein the transcription factors are selected from the group consisting of: LMX1A, SOX9, SOX10 and SOX8, preferably in combination with one or more of NEUROD1 , FOXJ1 or SOXI , wherein the transcription factors are two or more transcription factors selected from the group consisting of: LMX1A, SOX1 , SOX9, SOX10, SOX8, NEUROD1 and FOXJ1 , wherein the transcription factors do not consist of SOX1 and NEU ROD 1 , wherein the transcription factors are three or more transcription factors selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 and FOXJ1 , or wherein the transcription factors are selected from the group consisting of: LMX1A, SOX8, SOX9, SOX10, SOX1 , NEUROD1 and FOXJ1 in combination with one or more of HEY1 , ZBTB37 or REST, or variants thereof.

13. The method as defined in claim 9 or the cell as defined in claim 10, wherein the transcription factors are selected from the group consisting of: LMX1A, SOX9 and SOX10.

14. The method or the cell as defined in claim 11 , wherein the transcription factors comprise SOX9, SOX10 or both SOX9 and SOX10.

15. The method as defined in claim 9 or the cell as defined in claim 10, wherein the transcription factors comprise SOX8, preferably in combination with NEUROD1 , FOXJ1 or NEUROD1 and FOXJ1 or wherein the transcription factors are two or more transcription factors selected from the group consisting of: SOX8, NEUROD1 and FOXJ1 , or wherein the transcription factors are selected from the group consisting of: SOX8, NEUROD1 and FOXJ1 in combination with one or more one or more of ZBTB37, REST or SOX1 , wherein the transcription factors do not consist of NEUROD1 and SOX1 .

16. A method of generating photoreceptor cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain photoreceptor cells, wherein the transcription factors are selected from the group consisting of: NEUROD1 , NEUROD2, ATOH1 , HOXB2 and SOX1 , or variants thereof.BIT-C-P3860PCT117017. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: NEUROD1 , NEUROD2, ATOH1 , HOXB2, and SOX1 , or variants thereof.

18. A method of generating retinal ganglion cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain retinal ganglion cells, wherein the transcription factors comprise KLF4 in combination with ASCL1 , ZNF282 or both ASCL1 and ZNF282, or wherein the transcription factors are selected from the group consisting of: ZNF282, KLF4 and ASCL1 in combination with one or more of NR2F1 , CDX2, HOXB2, MYT 1 or SOX3, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and HOXB2, (iii) ZNF282 and CDX2, (iv) ZNF282 and HOXB2, (v) ASCL1 , CDX2 and HOXB2, (vi) ZNF282, CDX2 and HOXB2, (vii) ASCL1 , ZNF282 and CDX2, (viii) ASCL1 , ZNF282 and HOXB2 or (ix) ASCL1 , ZNF282, CDX2 and HOXB2, or variants thereof.

19. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise KLF4 in combination with ASCL1 , ZNF282 or both ASCL1 and ZNF282, or wherein the transcription factors are selected from the group consisting of: ZNF282, KLF4 and ASCL1 in combination with one or more of NR2F1 , CDX2, HOXB2, MYT 1 or SOX3, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and HOXB2, (iii) ZNF282 and CDX2, (iv) ZNF282 and HOXB2, (v) ASCL1 , CDX2 and HOXB2, (vi) ZNF282, CDX2 and HOXB2, (vii) ASCL1 , ZNF282 and CDX2, (viii) ASCL1 , ZNF282 and HOXB2 or (ix) ASCL1 , ZNF282, CDX2 and HOXB2, or variants thereof.

20. The method of claim 18 or the cell of claim 19, wherein the transcription factors comprise:BIT-C-P3860PCT1171(i) CDX2, ASCL1 and KLF4, or(ii) MYT1 in combination with ASCL1 , ZNF282 or both ASCL1 and ZNF282.

21. A method of generating amacrine cells, preferably glycinergic amacrine cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain amacrine cells, wherein the transcription factors comprise ASCL1 , preferably in combination with TCF3, HOXB2 or both HOXB2 and TCF3, wherein the transcription factors are two or more transcription factors selected from the group consisting of: ASCL1 , HOXB2 and TCF3, or wherein the transcription factors are selected from the group consisting of: ASCL1 , HOXB2 and TCF3 in combination with one or more of FOXN2, ZNF263, ZNF282 or CDX2.

22. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ASCL1 , preferably in combination with TCF3, HOXB2 or both HOXB2 and TCF3, wherein the transcription factors are two or more transcription factors selected from the group consisting of: ASCL1 , TCF3 and HOXB2, or wherein the transcription factors are selected from the group consisting of: ASCL1 , HOXB2 and TCF3 in combination with one or more of FOXN2, ZNF263, ZNF282 and CDX2.

23. The method as defined in claim 20 or the cell as defined in claim 21 , wherein the transcription factors comprise:(i) ZNF282 in combination with ASCL1 , HOXB2 or both ASCL1 and HOXB2; or(ii) ASCL1 and TCF3.

24. A method of generating podocytes comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain podocytes, wherein the transcription factors comprise CREBZF, preferably in combination with GATA4, IRF5 or both GATA and IRF5, orBIT-C-P3860PCT1172 wherein the transcription factors are two or more transcription factors selected from the group consisting of: CREBZF, IRF5 and GATA4, or variants thereof.

25. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise CREBZF, preferably in combination with GATA4, IRF5 or both GATA4 and IRF5, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: CREBZF, IRF5, and GATA4, or variants thereof.

26. A method of generating loop of Henle epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain loop of Henle epithelial cells, wherein the transcription factors are selected from the group consisting of: BARHL2, NR0B1 , POLI6F2 and TFAP2E, preferably in combination with one or more of SOX15, NFIC, or HNFIA, wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, SOX15, NFIC and HNF1A, or wherein the transcription factors are selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, NFIC, SOX15 and HNF1A in combination with FAM200B, SOX9 or both FAM200B and SOX9, or variants thereof.

27. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: BARHL2, NR0B1 , POLI6F2 and TFAP2E, preferably in combination with one or more of SOX15, NFIC or HNFIA, wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFAP2E, BARHL2, NFIC, NR0B1 , POU6F2, SOX15 and HNF1A, orBIT-C-P3860PCT1173 wherein the transcription factors are selected from the group consisting of: TFAP2E, BARHL2, NR0B1 , POU6F2, NFIC, SOX15 and HNF1A in combination with FAM200B, SOX9 or both FAM200B and SOX9, or variants thereof.

28. A method of generating collecting duct principal cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain collecting duct principal cells, wherein the transcription factors comprise IRF5 and RREB1 , or variants thereof.

29. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise IRF5 and RREB1 , or variants thereof.

30. A method of generating interstitial fibroblasts comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain interstitial fibroblasts, wherein the transcription factors are selected from the group consisting of: CXXC4, FOXC2, MEF2B and TBX2, preferably in combination with MEF2D, SOX3 or both MEF2D and SOX3, wherein the transcription factors are two or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3, or wherein the transcription factors are selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3 in combination with HOXB9, ZNF570 or both HOXB9 and ZNF570, or variants thereof.31 . A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors,BIT-C-P3860PCT1174 wherein the transcription factors are selected from the group consisting of: FOXC2, CXXC4, MEF2B and TBX2, preferably in combination with MEF2D, SOX3 or both MEF2D and SOX3, wherein the transcription factors are two or more transcription factors selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3, or wherein the transcription factors are selected from the group consisting of: CXXC4, FOXC2, MEF2B, TBX2, MEF2D and SOX3 in combination with HOXB9, ZNF570 or both HOXB9 and ZNF570, or variants thereof.

32. A method of generating papillary tip cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain papillary tip cells, wherein the transcription factors comprise KLF2 in combination with one or more of KLF1 , TBX22, TFAP2B or GRHL3, wherein the transcription factors do not consist of KLF2 and GRHL3.

33. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise KLF2 in combination with one or more of KLF1 , TBX22, TFAP2B or GRHL3, wherein the transcription factors do not consist of KLF2 and GRHL3.

34. A method of generating proximal tubule epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain proximal tubule epithelial cells, wherein the transcription factors comprise HNF1A in combination with ANHX or both ANHX and HNF1 B.

35. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors,BIT-C-P3860PCT1175 wherein the transcription factors comprise HNF1A in combination with ANHX or both ANHX and HNF1 B.

36. A method of generating parietal epithelial cells comprising expressing one or more polypeptides having the activity of FOXP1 and / or increasing the expression of FOXP1 in a cell population and culturing the cell population to obtain parietal epithelial cells.

37. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of FOXP1 and / or encoding FOXP1 .

38. A method of generating respiratory epithelial cells, preferably epithelial cells of the lower respiratory tract, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain respiratory epithelial cells, wherein the transcription factors comprise FOXA2, preferably in combination with one or more of HEY2, PRDM6, ZBTB18, FOXA1 , GFI1 , NFIX or SOXI , or variants thereof.

39. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise FOXA2, preferably in combination with one or more of HEY2, PRDM6, ZBTB18, FOXA1 , GFI1 , NFIX or SOXI , or variants thereof.

40. A method of generating basal epithelial cells of the tracheobronchial tree comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain basal epithelial cells of the tracheobronchial tree, wherein the transcription factors are selected from the group consisting of: GBX1 , WIZ, HOXD12 and KLF2, preferably in combination with IRF5, MYC or both IRF5 and MYC, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: GBX1 , HOXD12, WIZ, IRF5, MYC and KLF2, or variants thereof.BIT-C-P3860PCT117641 . A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: GBX1 , WIZ, KLF2 and HOXD12, preferably in combination with IRF5, MYC or both IRF5 and MYC, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: GBX1 , HOXD12, WIZ, IRF5, MYC and KLF2, or variants thereof.

42. A method of generating type 1 pneumocytes comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain type 1 pneumocytes, wherein the transcription factors comprise GRHL3 and / or NFIC, preferably in combination with one or more of CEBPB, MYC, KLF2 and NFIX, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX, wherein the transcription factors do not consist of (i) CEBPB and KLF2 or (ii) KLF2 and MYC, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX, or wherein the transcription factors are selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX in combination with ZNF441 , or variants thereof.

43. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise GRHL3 and / or NFIC, preferably in combination with one or more of CEBPB, MYC, KLF2 or NFIX, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: CEBPB, MYC, KLF2, NFIX, GRHL3 and NFIC, wherein the transcription factors do not consist of CEBPB and KLF2 or KLF2 and MYC, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX, or wherein the transcription factors are selected from the group consisting of: GRHL3, NFIC, CEBPB, MYC, KLF2 and NFIX in combination with ZNF441 ,BIT-C-P3860PCT1177 or variants thereof.

44. A method of generating club cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain club cells, wherein the transcription factors comprise CEBPB and / or RREB1 , preferably in combination with one or more of GFI1 B, FOXA1 , GFI1 , KLF2 or FOXA2, or wherein the transcription factors comprise FOXA1 in combination with one or more of CEBPB, RREB1 , GFI1 B, GFI1 , KLF2 or FOXA2, wherein the transcription factors do not consist of (i) FOXA1 and FOXA2, (ii) FOXA1 and GFI1 or (iii) FOXA1 , FOXA2 and GFI1 , or wherein the transcription factors are selected from the group consisting of: CEBPB, GFI1 B, FOXA1 , GFI1 , KLF2, RREB1 and FOXA2 in combination with MAF.

45. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise CEBPB and / or RREB1 , preferably in combination with one or more of GF11 B, FOXA1 , GF11 , KLF2 or FOXA2, or wherein the transcription factors comprise FOXA1 in combination with one or more of GFI1 B, FOXA2, CEBPB, GFI1 , KLF2 and RREB1 , wherein the transcription factors do not consist of (i) FOXA1 and FOXA2, (ii) FOXA1 and GFI1 or (iii) FOXA1 , FOXA2 and GFI1 , or wherein the transcription factors are selected from the group consisting of: GFI1 B, FOXA1 , FOXA2, CEBPB, GFI1 , KLF2 and RREB1 in combination with MAF.

46. The method as defined in claim 41 or the cell as defined in claim 42, wherein the transcription factors comprise:(i) CEBPB and GFI1 B;(ii) CEBPB in combination with one or more of FOXA2, GFI1 or KLF2; or(iii) RREB1 and KLF2.

47. A method of generating intestinal epithelial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain intestinal epithelial cells,BIT-C-P3860PCT1178 wherein the transcription factors comprise HMX1 and / or GATA5, preferably in combination with one or more of RREB1 , ESRRA, HNF1A, FOXQ1 , GATA4, GATA6 or KLF2, or wherein the transcription factors comprise FOXQ1 and / or KLF2 in combination with one or more of HMX1 , GATA5, RREB1 , ESRRA, HNF1A, GATA4 or GATA6, wherein the transcription factor do not consist of RREB1 and KLF2, or variants thereof.

48. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise HMX1 and / or GATA5, preferably in combination with one or more of RREB1 , ESRRA, HNF1A, FOXQ1 , GATA4, GATA6 or KLF2, or wherein the transcription factors comprise FOXQ1 and / or KLF2 in combination with one or more of HMX1 , GATA5, RREB1 , ESRRA, HNF1A, GATA4 or GATA6, wherein the transcription factor do not consist of RREB1 and KLF2, or variants thereof.

49. A method of generating enteroendocrine cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain enteroendocrine cells, wherein the transcription factors comprise ASCL1 , preferably in combination with one or more of GF11 , FOXA2 or PTF1 A, or wherein the transcription factors are selected from the group consisting of: ASCL1 , GF11 , PTF1A and FOXA2 in combination with one or more of GLIS1 , NKX3.2, RFX6, CDX2, KLF1 , KLF4, TCF3 or GFU B, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and KLF4, (iii) ASCL1 , CDX2 and KLF4, (iv) ASCL1 and TCF3, (v) FOXA2 and KLF4, (vi) GFI1 and KLF4, (vii) KLF1 and FOXA2, (viii) KLF1 and GFI1 , (ix) KLF1 and PTF1A, (x) KLF1 , FOXA2 and GFI1 , (xi) KLF1 , FOXA2 and PTF1A, (xii) KLF1 , GFI1 and PTF1A or (xiii) KLF1 , FOXA2, GFI1 and PTF1A, or variants thereof.

50. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors,BIT-C-P3860PCT1179 wherein the transcription factors comprise ASCL1 , preferably in combination with one or more of GFI1 , FOXA2 or PTF1A, or wherein the transcription factors are selected from the group consisting of: ASCL1 , GFI1 , PTF1A and FOXA2 in combination with one or more of GLIS1 , NKX3.2, RFX6, CDX2, KLF1 , KLF4, TCF3 or GFU B, wherein the transcription factors do not consist of (i) ASCL1 and CDX2, (ii) ASCL1 and KLF4, (iii) ASCL1 , CDX2 and KLF4, (iv) ASCL1 and TCF3, (v) FOXA2 and KLF4, (vi) GFI1 and KLF4, (vii) KLF1 and FOXA2, (viii) KLF1 and GFI1 , (ix) KLF1 and PTF1A, (x) KLF1 , FOXA2 and GFI1 , (xi) KLF1 , FOXA2 and PTF1A, (xii) KLF1 , GFI1 and PTF1A or (xiii) KLF1 , FOXA2, GFI1 and PTF1A, or variants thereof.

51. The method as defined in claim 48 or the cell as defined in claim 49, wherein the transcription factors comprise ASCL1 and GFI1.

52. A method of generating neural endocrine cells, preferably lung neural endocrine cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain neural endocrine cells, wherein the transcription factors comprise ASCL1 in combination with GLI2 or both GLI2 and RFX6, or variants thereof.

53. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ASCL1 in combination with GLI2 or both GLI2 and RFX6, or variants thereof.

54. A method of generating ciliated cells, such as multiciliated cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain ciliated cells, wherein the transcription factors comprise FOXJ1 , preferably in combination with FOXQ1 , or variants thereof.BIT-C-P3860PCT118055. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise FOXJ1 , preferably in combination with FOXQ1 , or variants thereof.

56. A method of generating endocardial cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endocardial cells, wherein the transcription factors comprise TBXT, preferably in combination with one or more of ETS1 , ETV4 or TAL1 , or wherein the transcription factors are selected from the group consisting of: TBXT, ETS1 ETV4 and TAL1 in combination with one or more of PATZ1 , ZNF84, ZSCAN9 or ETV2, wherein the transcription factors do not consist of (i) ETV2 and ETS1 , (ii) ETV2 and ETV4, (iii) ETV2 and TAL1 , (iv) ETV2, ETS1 and ETV4, (v) ETV2, ETS1 and TAL1 , (vi) ETV2, ETV4 and TAL1 , or (vii) ETV2, ETS1 , ETV4 and TAL1 , or or variants thereof.

57. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise TBXT, preferably in combination with one or more of ETS1 , ETV4 or TAL1 , or wherein the transcription factors are selected from the group consisting of: TBXT, ETS1 ETV4 and TAL1 in combination with one or more of PATZ1 , ZNF84, ZSCAN9 or ETV2, wherein the transcription factors do not consist of (i) ETV2 and ETS1 , (ii) ETV2 and ETV4, (iii) ETV2 and TAL1 , (iv) ETV2, ETS1 and ETV4, (v) ETV2, ETS1 and TAL1 , (vi) ETV2, ETV4 and TAL1 , or (vii) ETV2, ETS1 , ETV4 and TAL1 , or or variants thereof.

58. A method of generating endothelial cells, such as endothelial cells of the lymph vessel, sinusoidal endothelial cells and endothelial cells of the pericentral hepatic sinusoid, comprising expressing one or more polypeptides having the activity of one or more transcription factorsBIT-C-P3860PCT1181 and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain endothelial cells, wherein the transcription factors are selected from the group consisting of: ARID5A, ETS1 , ETS2, SNAI1 , ETV2, ETV4, MSX1 , ERG, FLI1 and ZNF467, preferably in combination with TAL1 , HNFI B or both TAL1 and HNF1 B, wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS1 , ETS2, SNAI1 , ETV2, ETV4, TAL1 , HNF1 B, MSX1 , ERG, FLI1 and ZNF467, or wherein the transcription factors are selected from the group consisting of: ARID5A, ETS1 , ETS2, SNAI1 , ETV2, ETV4, MSX1 , ERG, FLI1 , ZNF467, HNF1 B and TAL1 , in combination with ZNF711 , TBX6 or both ZNF711 and TBX6, or variants thereof.

59. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are selected from the group consisting of: ARID5A, ETS1 , ETS2, SNAI1 , ETV2, ETV4, MSX1 , ERG, FLI1 and ZNF467, preferably in combination with TAL1 , HNFI B or both TAL1 and HNF1 B, wherein the transcription factors are two or more transcription factors selected from the group consisting of: ARID5A, ETS1 , ETS2, SNAI1 , ETV2, ETV4, TAL1 , HNF1 B, MSX1 , ERG, FLI1 and ZNF467, or wherein the transcription factors are selected from the group consisting of: ARID5A, ETS1 , ETS2, SNAI1 , ETV2, ETV4, MSX1 , ERG, FLI1 , ZNF467, HNF1 B and TAL1 , in combination with ZNF711 , TBX6 or both ZNF711 and TBX6, or variants thereof.

60. The method as defined in claim 58 or the cell as defined in claim 59, wherein the transcription factors comprise:(i) ETV2, ERG and ETS1 ;(ii) ETV2, ERG, ETS1 , ETV4, ETS2 and TAL1 , or(iii) ETS1 , ETV2, ETV4 and TAL1 .

61. The method as defined in claim 58 or the cell as defined in claim 59, wherein the transcription factors are selected from the group consisting of: ETV2, ETV4 and FLI1 , preferably in combination with ERG.BIT-C-P3860PCT118262. The method as defined in claim 58 or the cell as defined in claim 59, wherein the transcription factors are selected from the group consisting of: ARID5A, ETS1 , ETS2 and SNAI1 , preferably in combination with HNF1 B.

63. The method or the cell as defined in claim 62, wherein the transcription factors comprise ARID5A, preferably in combination with HNF1 B.

64. A method of generating glutamatergic neurons comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain glutamatergic neurons, wherein the transcription factors comprise SOX10 in combination with one or more of ZNF131 , GLIS3, GFI1 or NEUROG1 , or variants thereof.

65. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise SOX10, in combination with one or more ofZNF131 , GLIS3, GFI1 or NEUROG1 , or variants thereof.

66. A method of generating GABAergic neurons comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain GABAergic neurons, wherein the transcription factors comprise LBX1 and / or NR2E1 , preferably in combination with one or more of NEUROG3, NEUROG1 , NEUROG2, NEUROD1 , NEUROD2, HOXB2 or MYT1 , or variants thereof.

67. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors,BIT-C-P3860PCT1183 wherein the transcription factors comprise LBX1 and / or NR2E1 , preferably in combination with one or more of NEUROG3, NEUROG1 , NEUROG2, NEUROD1 , NEUROD2, HOXB2 or MYT1 , or variants thereof.

68. A method of generating enteric neurons comprising expressing one or more polypeptides having the activity of NEUROD4 and / or increasing the expression of NEUROD4 in a cell population and culturing the cell population to obtain enteric neurons.

69. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of NEUROD4 and / or encoding NEUROD4.

70. A method of generating peripheral nervous system neurons comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain peripheral nervous system neurons, wherein the transcription factors comprise INSM1 , preferably in combination with one or more of NEUROD1 , ATOH1 or MYT1 , or wherein the transcription factors are selected from the group consisting of: INSM1 , NEUROD1 , ATOH1 and MYT1 in combination with one or more of AHCTF1 , DLX1 , DLX3, PRDM4, PURG, ZBTB7A, ASCL1 , MEF2C, NEUROG1 , NEUROG2, NEUROG3, ZNF282 or ZNF746, wherein the transcription factors do not consist of (i) MYT1 and ASCL1 , (ii) MYT1 and ZNF282, (iii) MYT1 , ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1 , (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1 , (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1 , (ix) NEU ROD 1 and NEUROG2, (x) ATOH1 , MYT1 and NEUROG1 , (xi) ATOH1 , MYT1 and NEUROG2, (xii) ATOH1 , NEUROD1 and NEUROG1 , (xiii) ATOH1 , NEUROD1 and NEUROG2, (xiv) MYT1 , NEUROD1 and NEUROG1 , (xv) MYT1 , NEUROD1 and NEUROG2, (xvi) ATOH1 , NEU ROG 1 and NEUROG2, (xvii) MYT1 , NEU ROG 1 and NEUROG2, (xviii) NEUROD1 , NEUROG1 and NEUROG2, (xix) ATOH1 , MYT1 , NEUROD1 and NEUROG1 , (xx) ATOH1 , MYT1 , NEUROD1 and NEUROG2, (xxi) ATOH1 , MYT1 , NEUROG1 and NEUROG2, (xxii) ATOH1 , NEUROD1 , NEUROG1 and NEUROG2, (xxiii) MYT1 , NEUROD1 , NEUROG1 and NEUROG2, or (xxiv) ATOH1 , MYT1 , NEUROD1 , NEUROG1 and NEUROG2, or variants thereof.BIT-C-P3860PCT118471 . A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise INSM1 , preferably in combination with one or more of NEUROD1 , ATOH1 or MYT1 , or wherein the transcription factors are selected from the group consisting of: INSM1 , NEUROD1 , ATOH1 and MYT1 in combination with one or more of AHCTF1 , DLX1 , DLX3, PRDM4, PURG, ZBTB7A, ASCL1 , MEF2C, NEUROG1 , NEUROG2, NEUROG3, ZNF282 or ZNF746, wherein the transcription factors do not consist of (i) MYT1 and ASCL1 , (ii) MYT1 and ZNF282, (iii) MYT1 , ASCL1 and ZNF282, (iv) ATOH1 and NEUROG1 , (v) ATOH1 and NEUROG2, (vi) MYT1 and NEUROG1 , (vii) MYT1 and NEUROG2, (viii) NEUROD1 and NEUROG1 , (ix) NEU ROD 1 and NEUROG2, (x) ATOH1 , MYT1 and NEUROG1 , (xi) ATOH1 , MYT1 and NEUROG2, (xii) ATOH1 , NEUROD1 and NEUROG1 , (xiii) ATOH1 , NEUROD1 and NEUROG2, (xiv) MYT1 , NEUROD1 and NEUROG1 , (xv) MYT1 , NEUROD1 and NEUROG2, (xvi) ATOH1 , NEUROG1 and NEUROG2, (xvii) MYT1 , NEUROG1 and NEUROG2, (xviii) NEUROD1 , NEUROG1 and NEUROG2, (xix) ATOH1 , MYT1 , NEUROD1 and NEUROG1 , (xx) ATOH1 , MYT1 , NEUROD1 and NEUROG2, (xxi) ATOH1 , MYT1 , NEUROG1 and NEUROG2, (xxii) ATOH1 , NEUROD1 , NEUROG1 and NEUROG2, (xxiii) MYT1 , NEUROD1 , NEUROG1 and NEUROG2, or (xxiv) ATOH1 , MYT1 , NEUROD1 , NEUROG1 and NEUROG2, or variants thereof.

72. A method of generating hepatoblasts, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain hepatoblasts, wherein the transcription factors comprise ESRRA, or variants thereof.

73. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise ESRRA, or variants thereof.

74. A method of generating trophoblasts, such as extravillous trophoblasts, trophoblast giant cells or placental villous trophoblasts, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain trophoblasts,BIT-C-P3860PCT1185 wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4, wherein the transcription factors do not consist of (i) CEBPB and KLF4 or (ii) KLF4 and ZNF282, wherein the transcription factors are three or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4, or wherein the transcription factors are selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4 in combination with one or more of ZNF804A, ZSCAN30, DMRT1 , ESRRA, KLF7 or ESRRB, wherein the transcription factors do not consist of ESRRA and GATA4, or variants thereof.

75. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4, wherein the transcription factors do not consist of (i) CEBPB and KLF4 or (ii) KLF4 and ZNF282, wherein the transcription factors are three or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4, or wherein the transcription factors are selected from the group consisting of: TFEB, GATA4, LMX1A, ZNF282, CEBPB and KLF4 in combination with one or more of ZNF804A, ZSCAN30, DMRT1 , ESRRA, KLF7 and ESRRB, wherein the transcription factors do not consist of ESRRA and GATA4, or variants thereof.

76. The method as defined in claim 74 or the cell as defined in claim 75, wherein the transcription factors are two or more transcription factors selected from the group consisting of: TFEB, GATA4, LMX1A and ZNF282, or wherein the transcription factors are one or more selected from the group consisting of: TFEB, GATA4, LMX1A and ZNF282 in combination with one or more of ZNF804A, ZSCAN30, DMRT1 , ESRRA or KLF7, wherein the transcription factors do not consist of ESRRA and GATA4.

77. The method as defined in claim 74 or the cell as defined in claim 75, wherein the transcription factors comprise TFEB in combination with KLF4, CEBPB, or both CEBPB and KLF4, preferably KLF4.BIT-C-P3860PCT118678. A method of generating transit amplifying cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain transit amplifying cells, wherein the transcription factors comprise KLF2 and RREB1 , preferably in combination with IRF5, or wherein the transcription factors are selected from the group consisting of: KLF2, RREB1 and IRF5 in combination with one or more of FOXP3, ZBTB7B or MYC, wherein the transcription factors do not consist of (i) MYC and IRF5, (ii) MYC and KLF2, or (iii) MYC, IRF5 and KLF2, or variants thereof.

79. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise KLF2 and RREB1 , preferably in combination with IRF5, or wherein the transcription factors are selected from the group consisting of: KLF2, RREB1 and IRF5 in combination with one or more of FOXP3, ZBTB7B or MYC, wherein the transcription factors do not consist of (i) MYC and IRF5, (ii) MYC and KLF2, or (iii) MYC, IRF5 and KLF2, or variants thereof.

80. A method of generating prostate epithelial cells, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain prostate epithelial cells, wherein the transcription factors comprise CEBPB and / or GFI1 in combination with one or more of MAF, IRF7, SPDEF, ZNF552, ZNF614, FOXA2, KLF1 , KLF2, MYOD1 , PTF1A or ESRRB, wherein the transcription factors do not consist of (i) CEBPB and FOXA2, (ii) CEBPB and KLF2, (iii) GFI1 and FOXA2, (iv) GFI1 and KLF2, (v) CEBPB, GFI1 and FOXA2, (vi) CEBPB, GFI1 and KLF2, (vii) CEBPB, FOXA2 and KLF2, (viii) GFI1 , FOXA2 and KLF2, (ix) CEBPB, GFI1 , FOXA2 and KLF2, (x) CEBPB and PTF1A and (xi) CEBPB and ESRRB, or variants thereof.BIT-C-P3860PCT118781 . A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise CEBPB and / or GFI1 in combination with one or more of MAF, IRF7, SPDEF, ZNF552, ZNF614, FOXA2, KLF1 , KLF2, MYOD1 , PTF1A or ESRRB, wherein the transcription factors do not consist of (i) CEBPB and FOXA2, (ii) CEBPB and KLF2, (iii) GFI1 and FOXA2, (iv) GFI1 and KLF2, (v) CEBPB, GFI1 and FOXA2, (vi) CEBPB, GFI1 and KLF2, (vii) CEBPB, FOXA2 and KLF2, (viii) GFI1 , FOXA2 and KLF2, (ix) CEBPB, GFI1 , FOXA2 and KLF2, (x) CEBPB and PTF1A and (xi) CEBPB and ESRRB, or variants thereof.

82. The method as defined in claim 82 or the cell as defined in claim 83, wherein the transcription factors comprise CEBPB and KLF1.

83. A method of generating mammary gland cells, such as luminal epithelial cells of the mammary gland or myoepithelial cells of the mammary gland, comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain mammary gland cells, wherein the transcription factors comprise KLF16 and / or PLIRA, preferably in combination with one or more of KLF4, GFI1 B or WIZ, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ, wherein the transcription factors do not consist of KLF4 and WIZ, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ, or wherein the transcription factors are selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2 or TEAD3, or variants thereof.

84. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise KLF16 and / or PLIRA, preferably in combination with one or more of KLF4, GF11 B or WIZ, orBIT-C-P3860PCT1188 wherein the transcription factors are two or more transcription factors selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ, wherein the transcription factors do not consist of GFI1 and WIZ, or wherein the transcription factors are three or more transcription factors selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ, or wherein the transcription factors are selected from the group consisting of: KLF16, PLIRA, KLF4, GFI1 B and WIZ in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2 or TEAD3, or variants thereof.

85. The method as defined in claim 84 or the cell as defined in claim 85, wherein the transcription factors comprise KLF4 and / or GFI1 B.

86. The method as defined in claim 84 or the cell as defined in claim 85, wherein the transcription factors comprise KLF16 and / or PLIRA, preferably in combination with WIZ, or wherein the transcription factors are selected from the group consisting of: KLF16, PLIRA and WIZ in combination with one or more of HAND1 , KLF11 , LHX8, OVOL2 or TEAD3.

87. A method of generating goblet cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain goblet cells, wherein the transcription factors comprise KLF4 and / or ZNF853, preferably in combination with CEBPB, REST or both CEBPB and REST, or wherein the transcription factors are two or more transcription factors selected from the group consisting of: REST, ZNF853, CEBPB and KLF4, or wherein the transcription factors are selected from the group consisting of: ZNF853, KLF4, CEBPB and REST in combination with one or more of DBX1 , KLF17, ZNF503 or RUNX1 , or variants thereof.

88. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise KLF4 and / or ZNF853, preferably in combination with CEBPB, REST or both CEBPB and REST, orBIT-C-P3860PCT1189 wherein the transcription factors are two or more transcription factors selected from the group consisting of: REST, ZNF853, CEBPB and KLF4, or wherein the transcription factors are selected from the group consisting of: ZNF853, KLF4, CEBPB and REST in combination with one or more of DBX1 , KLF17, ZNF503 or RUNX1 , or variants thereof.

89. A method of generating neuronal brush cells comprising expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of one or more transcription factors in a cell population and culturing the cell population to obtain neuronal brush cells, wherein the transcription factors comprise NEUROD1 , NEUROD2, ATOH1 and NEUROG1 , or variants thereof.

90. A cell comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or encoding one or more transcription factors, wherein the transcription factors comprise NEUROD1 , NEUROD2, ATOH1 and NEUROG1 , or variants thereof.

91. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87 or 89, or the cell as defined in any one of claims 2 to 10, 12 to 15, 17, 19, 20, 22, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 46, 48, 50, 51 , 53, 55, 57, 59 to 63, 65, 67, 69, 71 , 73, 75 to 77, 79, 81 , 82, 84 to 86, 88 or 90, which comprises increasing the expression of one or more additional transcriptional factors as listed in Table 1.

92. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89 or 91 or the cell as defined in any one of claims 2 to 10, 12 to 15, 17, 19, 20, 22, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 46, 48, 50, 51 , 53, 55, 57, 59 to 63, 65, 67, 69, 71 , 73, 75 to 77, 79, 81 , 82, 84 to 86, 88, 90 or 91 , wherein the cell population comprises pluripotent stem cells, in particular induced pluripotent stem cells.

93. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89, 91 or 92, wherein the expression of the transcription factors is increased by contacting the cell population with one or more exogenous expressionBIT-C-P3860PCT1190 cassettes encoding one or more of the genes, or one or more agents that activate or increase the expression or amount of the transcription factors.

94. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89 or 91 to 93 or the cell as defined in any one of claims 2 to 10, 12 to 15, 17, 19, 20, 22, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 46, 48, 50, 51 , 53, 55, 57, 59 to 63, 65, 67, 69, 71 , 73, 75 to 77, 79, 81 , 82, 84 to 86, 88 or 90 to 92, wherein expression of the genes is under controlled transcription.

95. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89 or 91 to 94, wherein a sequence encoding one or more of the transcription factors is introduced into the cell population using a method comprising:- insertion of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of a source cell present in the cell population; and- insertion of an inducible cassette into a second genomic safe harbour site of the source cell, wherein said inducible cassette comprises said sequence encoding one or more transcription factors, and transcription of said inducible cassette is regulated by the transcriptional regulator protein.

96. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89 or 91 to 94, wherein a sequence encoding one or more of the polypeptides having transcription factor activity and / or transcription factors is introduced into the cell population using a method comprising:- insertion of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of a source cell present in the cell population; and- insertion of an inducible cassette into a second genomic safe harbour site of the source cell, wherein said inducible cassette comprises said sequence encoding one or more transcription factors, and transcription of said inducible cassette is regulated by the transcriptional regulator protein.

97. The method as defined in any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89 or 91 to 94, wherein a sequence encoding one or more ofBIT-C-P3860PCT1191 the polypeptides having transcription factor activity and / or the transcription factors is introduced into the cell population using a method comprising:(a) insertion of a coding sequence for a transcriptional regulator protein into a source cell present in the cell population; and(b) insertion of one or more inducible cassettes into the source cell, wherein said one or more inducible cassettes comprises said sequence encoding the one or more polypeptides having the activity of one or more transcription factors and / or the transcription factors, and the transcription of said cassette is regulated by the transcriptional regulator protein, wherein the gene encoding the transcriptional regulator protein of (a) and / or the inducible cassette of (b) is inserted within an endogenous protein-encoding gene locus; wherein expression of the endogenous protein-encoding gene within the locus is modulated as a result of the insertion of the transcriptional regulator protein; and wherein expression of the transcriptional regulator protein is lower than the expression if the same genetic material was inserted into a genomic safe harbour site, operatively linked to a promoter.

98. The method or the cell as defined in any one of claims 95 to 97, wherein the insertion is targeted.

99. A cell made by the method of any one of claims 1 , 3 to 11 , 13 to 16, 18, 20, 21 , 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 47, 49, 51 , 52, 54, 56, 58, 60 to 64, 66, 68, 70, 72, 74, 76 to 78, 80, 82, 83, 85 to 87, 89 or 91 to 98.

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