Forward programming in a cell aggregate culture

Abstract

The invention relates to an ex vivo method for culturing stem cells in conditions that allow for aggregates to form.

Description

[0001] BIT-C-P3787PCT

[0002] 1

[0003] FORWARD PROGRAMMING IN A CELL AGGREGATE CULTURE

[0004] FIELD OF THE INVENTION

[0005] The invention relates to methods for culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions. This invention results in aggregates comprising cells that are lineage-restricted compared with the starting cells.

[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.

[0009] Traditionally, forward programming protocols are carried out in a 2D culture environment; i.e. where the cells are grown in a flat plane on top of a flat surface (be that an inanimate surface such as a polymer surface of a flask or a living surface such as on top of a feeder layer of cells). Whilst this is a straightforward way to manipulate cells in an ex vivo environment, this has disadvantages in that there are limitations of how quickly cells can be scaled up.

[0010] An alternative is to use a 3D cell culture, where cells are cultured within a three-dimensional space. 3D cultures can involve the cells grafted onto a 3D substrate, such as a scaffold, bead BIT-C-P3787PCT

[0011] 2 or matrix. In some cases the cells themselves act as the substrate, and clusters or aggregates of cells can form.

[0012] However, this is a very different culture environment compared with 2D culture in terms of the cell adhesion dynamics (adhering to a curved surface in 3D compared with adhering to a flat surface in 2D), in terms of the movement of the cells in 3D compared with 2D (added shear stresses to the cells in a 3D culture), and in terms of the vastly varied access to nutrients in a 3D culture (with cells at the centre of an aggregate having limited access to nutrients). Moreover cell signaling can be detrimentally affected by the 3D environment. All of these factors can clearly affect the cells and may impact on whether they can be forward programmed in the same manner as cells grown in 2D.

[0013] There is therefore a need in the art to provide methods for forward programming cells in a three-dimensional, aggregate environment.

[0014] SUMMARY OF THE INVENTION

[0015] According to a first aspect of the invention, there is provided an ex vivo method for culturing stem cells, preferably pluripotent stem cells, more preferably induced pluripotent stem cell, in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions.

[0016] In one embodiment, the forward programming comprises 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 the stem cells.

[0017] In one embodiment, the cells are mammalian cells, preferably human cells.

[0018] In one embodiment, the culture takes place in the absence of a substrate.

[0019] In one embodiment, the culture takes place in a spinner flask, in a shaker flask or in a bioreactor, preferably in a bioreactor.

[0020] In one embodiment, the culturing takes place in a culture vessel able to accommodate at least 10mL, 30 mL, 75 mL, 100 mL, 200 mL, 250 mL, 500 mL, 800 mL, 1 L, 2 L, 5 L or 10 L of culture media. BIT-C-P3787PCT

[0021] 3

[0022] According to a further aspect of the present invention, there is provided an ex vivo method for culturing stem cells, comprising the steps of: a) insertion, preferably targeted insertion, of a gene encoding a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and b) insertion, preferably targeted insertion, of one or more nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or one or more transcription factors, operably linked to an inducible promoter into one or more second genomic safe harbour sites of the stem cell, wherein said inducible promoter is regulated by the transcriptional regulator protein; and c) culturing the stem cells comprising the insertions in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions.

[0023] According to a further aspect of the present invention, there is provided an ex vivo method for culturing stem cells, comprising the steps of: a) insertion, preferably targeted insertion, of a gene encoding a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and b) insertion, preferably targeted insertion, of one or more nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or one or more transcription factors into one or more second genomic safe harbour sites of the stem cell, wherein the transcription of the one or more nucleotide sequences is regulated by the transcriptional regulator protein; and c) culturing the stem cells comprising the insertions in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions.

[0024] According to a further aspect of the present invention, there is provided a method of preparing a cell or tissue suitable for therapeutic or in vivo diagnostic purposes, comprising: (i) culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions; (ii) collecting a lineage-restricted cell or lineage restricted cell aggregate; and (iii) preparing a therapy or diagnostic comprising the lineage-restricted cell or lineage-restricted cell aggregate. BIT-C-P3787PCT

[0025] 4

[0026] According to a further aspect of the present invention, there is provided a cell made by any of the methods described herein.

[0027] According to a further aspect of the present invention, there is provided an aggregate of cells made by any of the methods described herein.

[0028] According to a further aspect of the present invention, there is provided a method of drug screening comprising contacting the lineage-restricted cells or an aggregate of lineage- restricted cells generated using any of the methods described herein, or any of the cells or aggregate of cells described herein, with a drug and observing a change in the cells induced by the drug.

[0029] According to a further aspect of the present 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 or aggregates of cells generated using any of the methods described herein, or any of the cells or aggregates of cells described herein.

[0030] BRIEF DESCRIPTION OF THE FIGURES

[0031] Figure 1. Human induced pluripotent stem cells grown in aggregates allows for a 10-fold expansion in cell number within 4 days of culture; media was exchanged every 24 hours ± 2 hours, (a) Live cell count on harvest in 3D-adapted stemscale media ± NaCCh buffer in 30 mL ABLE Biott vessels; (b) Morphology of cell aggregates at Day 4 of culture without NaCCh in 30 mL Biott vessels (scale bar corresponds to 200 pm); (c) morphology of cell aggregates at Day 4 of culture with NaCCh in 30 mL Biott vessels (scale bar corresponds to 200 pm) (d) viability of cells upon harvest in 30 mL Biott vessels; (e) pH of culture media in 30 mL Biott vessels; (f) glucose concentration in culture media in 30 mL Biott vessels; (g) lactate concentration in culture media in 30 mL Biott vessels; and (h) cell viability comparison in 30 mL, 100 mL and 500 mL Biott vessels.

[0032] Figure 2. Brightfield images of the cells throughout the production process. The cells aggregate within 24 hours (figure 2a) and are circular as with iPSC 3D culture. Day 5 aggregates (figure 2b) are larger and begin to reduce in circularity. Day 10 aggregates (figure 2c) are at their largest and most irregular. Day 14 aggregates (figure 2d) become translucent and regain more circularity. Day 20 aggregates (figure 2e) borders are smooth and circular BIT-C-P3787PCT

[0033] 5 and translucent across a wider portion of the population. Pictures were taken with a Leica DM IL LED microscope from LEICA Microsystems. The scale bar corresponds to 200 pm.

[0034] Figure 3. Albumin secretion from hepatocytes derived from the 3D aggregates (two repeats in Stemscale media) compared with 2D culture (in a T225 flask, a Hyperflask and a Hyperstack) at Day 20 of differentiation.

[0035] Figure 4. Brightfield images of the hepatocyte aggregates at day 20 of production process. Pictures were taken with a Leica DMIL LED microscope from LEICA Microsystems. The scale bar corresponds to 800 pm. Aggregates were generated at a seeding density of 150,000 cells / mL, a media exchange of 50% (15 out of 30 mL) and an impeller speed of 80 rpm (0.134 m / s tip speed) (Figure 4a), or at a seeding density of 300,000 cells / mL, a media exchange of 50% and an impeller speed of 80 rpm (0.134 m / s tip speed) (Figure 4b), or at a seeding density of 300,000 cells / mL, a media exchange of 80% (24 out of 30 mL) and an impeller speed of 80 rpm (0.134 m / s tip speed) (Figure 4c), or at a seeding density of 150,000 cells / mL, a media exchange of 50% and an impeller speed of 90 rpm (0.151 m / s tip speed) (Figure 4d), or at a seeding density of 300,000 cells / mL, a media exchange of 50% and an impeller speed of 90 rpm (0.151 m / s tip speed) (Figure 4e) or at a seeding density of 300,000 cells / mL, a media exchange of 80% and an impeller speed of 90 rpm (0.151 m / s tip speed) (Figure 4f).

[0036] Figure 5. Brightfield images of induced pluripotent stem cell aggregates at day 4 of culture. Representative images of cultures that have been cultured with gas control via overlay (Figure 5a) and gas control maintained with sparging (Figure 5b) in a 250 mL Ambr bioreactor.

[0037] Figure 6. Live cell count / mL (a) and percentage viability (b) on harvest of varying conditions (cultures that have been cultured with gas control via overlay and gas control maintained with sparging) including 30 mL ABLE biott (non-bioreactor) control culture.

[0038] Figure 7. Brightfield images of induced pluripotent stem cell aggregates at day 4 of culture. Culture conditions comprised a 70 rpm impeller speed (0.117 m / s tip speed) with a media change 24 hours post-seed in an ABLE biott (non-bioreactor) (“control”, figure 7a), a 125 rpm impeller speed (0.196 m / s tip speed) with no media change in Ambr bioreactor (“delay”, figure 7b), a 150 rpm impeller speed (0.236 m / s tip speed) with a media change 24 hours post-seed in an Ambr bioreactor (“150 rpm”, figure 7c) and a 125 rpm impeller speed BIT-C-P3787PCT

[0039] 6

[0040] (0.196 m / s tip speed) for the first 24 hours followed by a 150 rpm impeller speed (0.236 m / s tip speed) with a media change 24 hours post-seed in an Ambr bioreactor (“ramp”, figure 7d).

[0041] Figure 8. Albumin secretion by hepatocytes generated through forward programming in a bioreactor aggregate culture at 20-day. Culture conditions were as set out in Table 4 below.

[0042] Figure 9. Brightfield images of cell aggregates at day 1 (a), day 4 (b), day 8 (c) and day 10 after the cells are plated in 2D (d). The scale bars for Figures 9a, 9b and 9c correspond to 200 pm.

[0043] Figure 10. Immunofluorescence imaging of microglia obtained from a 2D forward programming culture ten days after doxycycline addition (left hand column) and from a 3D aggregate forward programming culture eight days after of doxycycline addition (right hand column). DAPI, P2RY12 and IBA1 stains were used.

[0044] Figure 11. Yield comparison of a typical 2D culture, in T75 flasks, unadapted culture (where expansion takes place in 2D, then forward programming takes place in a 3D aggregate) and 3D aggregate culture (where both expansion and forward programming takes place in 3D aggregates). Values are corrected for material loss via sampling.

[0045] Figure 12. Representative brightfield images showing the morphological changes during the pre-freeze induction of iPSCs to GABAergic neurons from Day -4 to Day 0 (top six images, 3D culture pre-freeze) and Day 1 and Day 12 (bottom six images, 2D culture post-freeze). Induction seeding densities of 100,000 cells / mL (left column), 300,000 cells / mL (middle column) and 600,000 cells / mL (right column) were analysed. Scale bars correspond to 200 pm in the top six images and 400 pm for the bottom six images.

[0046] Figure 13. Total number of cells recovered post-freeze (Figure 13a) and pre-freeze cell viability (Figure 13b) of GABAergic neurons produced in 3D cultures following induction at 100,000 cells / mL, 300,000 cells / mL or 600,000 cells / mL. Error bars represent standard deviation, with individual data points overlaid (n=3).

[0047] Figure 14. Viability of GABAergic neurons measured immediately before freezing (light grey) and after thawing (dark grey) across induction seeding densities of 100,000 cells / mL, 300,000 cells / mL or 600,000 cells / mL, compared against 2D production WCB (working cell BIT-C-P3787PCT

[0048] 7 bank) control. Data represents mean ± standard deviation, with exact values annotated above the bars.

[0049] Figure 15. Gene expression profile of GABAergic neurons at Day 3 (D3) and Day 12 (D12) post-thaw across induction seeding densities of 100,000 cells / mL, 300,000 cells / mL or 600,000 cells / mL, compared against 2D control (2D CTRL) and undifferentiated iPSCs. The following genes were analysed: POLI5F1 (Figure 15a), NANOG (Figure 15b), ASCL1 (Figure 15c), DLX1 (Figure 15d), DLX2 (Figure 15e), GAD1 (Figure 15f), GAD2 (Figure 15g), TUBB3 (Figure 15h) and VGAT (Figure 15i). The expression is shown on the y axis in relation to the expression of the housekeeping gene GAPDH. Data represents mean ± standard deviation.

[0050] Figure 16. Representative inucyte images show GABAergic neurons cultured in 2D (Figure 16a) and 3D (Figure 16b), with segmentation overlays generated using NeuroTrack analysis software. Lighter masks indicate cell body clusters and darker lines trace neurite extensions. Insets display magnified regions from the main images, highlighting finer neurite networks and branching patterns typical of each condition. Scale bars correspond to 400 pm in the main images and 100 pm for the inset images.

[0051] Figure 17. Number of neurite branch points per cell body cluster count over time across induction seeding densities of 600,000 cells / mL (top line), 300,000 cells / mL (second from top line) or 100,000 cells / mL (third from top line), compared against 2D control (bottom, dashed line). Analysis was performed using NeuroTrack’s standard metrics; neurite length, branch points and cell body cluster count.

[0052] DETAILED DESCRIPTION

[0053] The present invention provides methods for forward programming stem cells in a 3D aggregate culture environment. The inventors have surprisingly found that forward programming of cells is still achievable even in an aggregate culture environment.

[0054] Definitions

[0055] 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.

[0056] 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 BIT-C-P3787PCT

[0057] 8

[0058] “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.

[0059] 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.

[0060] 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 forward programming into the desired cell type.

[0061] 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 programmed 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 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 to induce pluripotency 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, GATA3, GATA6 and these factors may be used singly, or in combination of two or more kinds BIT-C-P3787PCT

[0062] 9 thereof. In particular, the programming factors may comprise at least the Yamanaka factors, i.e., OCT3 / 4, S0X2, KLF4 and C-MYC. These programming factors may also be used in combination with the transcription factors of interest in the present invention.

[0063] References herein to “somatic” refer to any type of cell that makes up the body of an organism, excluding germ cells. Somatic cells therefore include, for example, skin, heart, muscle, bone or blood cells and their stem cells. Somatic cells may also be referred to as differentiated cells. In one embodiment, the somatic cell may be an adult cell or a cell derived from an adult which displays one or more detectable characteristics of an adult or non-embryonic cell.

[0064] Methods of the invention (e.g., forward programming of iPSCs) are for use in generating lineage-restricted cells. By “lineage restricted” it is understood that the cells are, compared with the starting stem cells, limited in terms of the number of different cell types that they can differentiate into (without any further artificial transcription factor manipulation). This definition aligns with the understanding of forward programming in the art (the guided differentiation of cells through the forced expression of polypeptides with transcription factor activity and / or transcription factors). As such, the lineage-restricted cell may be a stem cell itself, but that resultant stem cell would be lineage-restricted compared to the source cell (for example, the forward programming of an iPSC to a mesenchymal stem cell).

[0065] References herein to “culturing” in general include the addition of cells {e.g., the cell population, i.e., the source cells), to media comprising growth factors and / or essential nutrients. It will be appreciated that such culture conditions may be adapted according to the cells or cell population to be generated according to methods of the invention.

[0066] References herein to “two-dimensional culture”, or “2D culture” relate to ex vivo cell culture carried out on a flat plane on top of a flat surface. The surface may be an inanimate surface such as a polymer surface of a flask or well plate. Alternatively the surface may be a living surface such as on top of a feeder layer of cells.

[0067] References herein to “three-dimensional culture”, or “3D culture” relate to, in essence, any form of ex vivo culture not covered in the definition of “2D culture” above. 3D culture relates to cells grown in a 3D space, and includes growth in single cell suspension and in aggregates. 3D culture can involve growth within a gel-like matrix or on or within a solid scaffold, or on a bead, but none of these are necessary. Culture vessels suitable for 3D culture may have an BIT-C-P3787PCT

[0068] 10 inner surface that cannot be adhered to, or at least the cells find difficult to adhere to. Culture vessels may be coated in such a manner that deters cell attachment.

[0069] Variants of the transcription factors described herein may be used in methods of the invention. References to a “variant” when referring to a polypeptide could be, for example, 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 forwardprogram a source cell into a lineage restricted 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. 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.

[0070] 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.

[0071] 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 BIT-C-P3787PCT

[0072] 11

[0073] “promoter” or “control element” includes full-length promoter regions and functional (e.g., controls and / or affects transcription or translation) segments of these regions.

[0074] 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.

[0075] 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 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. BIT-C-P3787PCT

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[0077] 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.

[0078] 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.

[0079] 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.

[0080] It will be understood that any method as described herein may have one or more, or all, steps performed in vitro or ex vivo.

[0081] Transcription factor activity

[0082] The method described herein comprises the forward programming of stem cells. This generally involves increasing the expression (in particular, the protein expression) of a sufficient number of polypeptides having the activity of the transcription factors capable of causing forward programming of a cell population, thereby forward programming the cell population into to lineage-restricted cells.

[0083] Expression may be of the transcription factors themselves. In the context of the present invention, these factors may also be referred to as “programming factors”. Where expression is of the transcription factors, the increased expression may be of one or more exogenous transcription factors, one or more endogenous transcription factors, or a combination of the two. Preferably the increased expression is of exogenous transcription factors.

[0084] Transcription factors used in methods of forward programming to particular cell types have been disclosed in the art. BIT-C-P3787PCT

[0085] 13

[0086] For example, it is known that the forward programming of induced pluripotent stem cells to hepatocytes is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise HNF1A, FOXA3, HNF6 and RORc. This is set out in international patent publication WO2023 / 036983 (incorporated herein by reference).

[0087] It is known that the forward programming of induced pluripotent stem cells to microglia is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise SPI1 and one additional transcription factor such as a CEB protein, for example CEBPB. This is set out in international patent publication W02020 / 239807 (incorporated herein by reference).

[0088] It is known that the forward programming of induced pluripotent stem cells to GABAergic neurons is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise ASCL1 and DLX2. This is set out in international patent publication WO2011 / 091048 (incorporated herein by reference).

[0089] It is known that the forward programming of induced pluripotent stem cells to glutamatergic neurons, is possible through the overexpression of polypeptides having Ngn2 activity and / or Ngn2 itself. This is set out in international patent publication WO2011 / 091048 (incorporated herein by reference).

[0090] It is known that the forward programming of induced pluripotent stem cells to sensory neurons, is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise NGN1 , ISL1 and KLF7.

[0091] It is known that the forward programming of induced pluripotent stem cells to myocytes, such as skeletal myocytes, is possible through the overexpression of polypeptides having MYOD1 activity and / or MYOD1 itself. This is set out in international patent publication WO2018 / 096343 (incorporated herein by reference).

[0092] It is known that the forward programming of induced pluripotent stem cells to oligodendrocytes is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise OLIG2 and BIT-C-P3787PCT

[0093] 14

[0094] SOX10. This is set out in international patent publication WO2018 / 096343 (incorporated herein by reference).

[0095] The forward programming of induced pluripotent stem cells to adipocytes is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise one or more of a PPAR protein, HOXC8, EBF1 , EBF2, ZNF467, ZNF423 ora CEB protein. This is set out in international patent application PCT / GB2024 / 051973, published as WO2025 / 022129 (incorporated herein by reference).

[0096] The forward programming of induced pluripotent stem cells to pancreatic beta cells is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise one or more of GLIS3, PDX1 , NEUROD1 , NKX6-2 or HNF6. This is set out in European patent application EP24386074.9 and in international patent application PCT / GB2024 / 052806, published as WO2025 / 099413 (incorporated herein by reference).

[0097] The forward programming of induced pluripotent stem cells to astrocytes is possible through the overexpression of polypeptides having transcription factor activity and / or one or more transcription factors, wherein the transcription factors comprise the combination of SOX9, NFIA, NFIB and one or more transcription factors selected from the group consisting of: FEZF2, TBR1 , FOXG1 , RORB, LHX2, DBX2. This is set out in European patent application EP24386111.9 (incorporated herein by reference).

[0098] References herein to “HNF1A”and “HNF6” relate to hepatocyte nuclear factors 1 homeobox A and 6. HNF6 is also known as One cut homeobox 1 or ONECUT1. In one embodiment, the HNF protein is a human HNF protein. Wild type human HNF1A is identified by UniProt ID: P20823, and is encoded by the HNF1A gene, identified by Ensembl Gene ID: ENSG00000135100. Wild type human HNF6 is identified by UniProt ID: Q9UBC0, and is encoded by the HNF6 gene, identified by Ensembl Gene ID: ENSG00000169856. HNF1A functions as a homodimer and binds to the inverted palindrome 5'-GTTAATNATTAAC-3'. HNF6 controls the diversification of motor neurons by ensuring the maintenance of Isl1 expression during differentiation.

[0099] References herein to “FOXA3” relate to Forkhead Box A3. FOXA3 is also known as Hepatocyte Nuclear Factor 3-Gamma or HNF3G. In one embodiment the FOXA3 transcription factor is BIT-C-P3787PCT

[0100] 15 human FOXA3. Wild type human FOXA3 is identified by UniProt ID: P55318, and is encoded by the F0XA3 gene, identified by Ensembl Gene ID: ENSG00000170608. FOXA3 is a transcriptional activator for liver-specific transcripts such as albumin and transthyretin, and also interacts with chromatin.

[0101] References herein to “FOXG1” relate to Forkhead Box Protein G1. FOXG1 is also known as BF1 , FKHL1 , HBF-1 and HFK1 . In one embodiment the FOXG1 is human FOXG1 . Wild type human FOXG1 is identified by UniProt ID: P55316, and is encoded by the F0XG1 gene, identified by Ensembl Gene ID: ENSG00000176165. The absence of FOXG1 leads to FOXG1 syndrome characterized by microcephaly and brain malformations. It affects most aspects of development and can cause seizures. FOXG1 is a transcription repression factor that plays an important role in the establishment of the regional subdivision of the developing brain and in the development of the telencephalon.

[0102] References herein to “RORB” relate to RAR-related Orphan Receptor Beta. RORB is also known as NR1 F, RZRB, Nuclear Receptor ROR-Beta, ROR-BETA and RZR-BETA. In one embodiment the RORB is human RORB. Wild type human RORB is identified by UniProt ID: Q92753, and is encoded by the RORB gene, identified by Ensembl Gene ID: ENSG00000198963. RORB has been shown to interact with NM23-2, a nucleoside diphosphate kinase involved in organogenesis and differentiation, and to help regulate the expression of some genes involved in circadian rhythm. The absence of RORB leads to epilepsy. The RORB gene can be expressed in different isoforms, such as isoform 1 (RORB1 , UniProt ID: Q58EYO) or as isoform 2 (RORB2, UniProt ID: A0A1 L4FMK7).

[0103] References to “RORc” relate to RAR Related Orphan Receptor C. RORc is also known as NR1 F3, RZRG, RORG and TOR. In one embodiment the RORc transcription factor is human RORc. Wild type human RORc is identified by UniProt ID: P51449, and is encoded by the RORc gene, identified by Ensembl Gene ID: ENSG00000143365. RORc is essential for lymphoid organogenesis and may play an important regulatory role in thymopoiesis.

[0104] References herein to “SPI1” relate to transcription factor SPI1 (also known as haematopoietic transcription factor PU.1 , Spi-1 proto-oncogene, 31 kDa transforming protein, transcription factor PU.1 , Spleen Focus Forming Virus (SFFV) Proviral Integration Oncogene Spi1 , Spleen Focus Forming Virus (SFFV) Proviral Integration Oncogene, 31 kDa-Transforming Protein, SFPI1 , SPI-1 , SPI-A, PU.1 or OF), which belongs to the Erythroblast Transformation Specific (ETS) family of transcription factors. It has been associated with determining haematopoietic BIT-C-P3787PCT

[0105] 16 cell fate. In one embodiment, the SPI1 transcription factor is human SPI1. Wild type human SPI1 is identified by UniProt ID: P17947 and is encoded by the SPI1 gene which is identified by Ensembl Gene ID: ENSG00000066336.

[0106] Mechanistically, as SPI1 is a member of the ETS family, it has a highly conserved ETS domain of 85 amino acids capable of binding to the DNA sequence 5 -GGAA / T-3'. SPI1 is also known to activate a number of cytokine receptors, such as macrophage colony stimulating factor receptor (M-CSFR), granulocyte colony-stimulating factor receptor (G-CSFR), granulocytemacrophage colony stimulating factor receptor (GM-CSFR), interleukin 3 receptor (IL-3R), fragment, crystallizable gamma receptor (FcyR), the cytokine CD11 b and the chemokine CCL- 22. Furthermore SPI1 regulates gene expression through interaction with other transcription factors such as CEBPa, CEBPp and IRF8.

[0107] References herein to “CEBPa” and “CEBPp” (also known as “CEBPATCEBPB” and “C / EBPa “C / EBPP”) relate to the alpha and beta version of CCAAT / enhancer-binding protein. They have been associated with adipogenesis. In one embodiment, the CEBPa and / or CEBPp transcription factors are human CEBPa and / or CEBPp. Wild type human CEBPa is identified by UniProt ID: P49715 and is encoded by the CEBPa gene which is identified by Ensembl Gene ID: ENSG00000245848. Wild type human CEBPp is identified by UniProt ID: P17676 and is encoded by the CEBP / 3 gene which is identified by Ensembl Gene ID: ENSG00000172216.

[0108] CEBPa is known to regulate other transcription factors such as SP11 , c-Jun, c-Myc, SOX4 and E2F, growth factor receptors such as G-CSF and GM-CSF, primary granule proteins such as myeloperoxidase, secondary granule proteins such as lactoferrin and microRNAs such as miR- 223, miR-34a and miR-30c. CEBPp regulates IL-1 p, IL-6, IL-8, IL-12, TNFa, MCP1 and Th17 expression.

[0109] References to “MYOD1” relate to Myogenic Differentiation 1. It is also known as BHLHcl or Myoblast Determination Protein 1. In one embodiment, the MYOD1 transcription factor is human MYOD1. Wild type human MYOD1 is identified by UniProt ID: P15172 and is encoded by the MY0D1 gene which is identified by Ensembl Gene ID: ENSG00000129152. MYOD1 regulates muscle cell differentiation by inducing cell cycle arrest, a prerequisite for myogenic initiation. BIT-C-P3787PCT

[0110] 17

[0111] References to “Ngn1” relates to Neurogenin 1. It is also known NEUROD3, BHLHa6 and Math4C. In one embodiment, the Ngn1 transcription factor is human Ngn1. Wild type human Ngn1 is identified by UniProt ID: Q92886 and is encoded by the Ngn1 gene which is identified by Ensembl Gene ID: ENSG00000181965. Ngn1 plays a role in cranial nerve development and hard palate morphogenesis.

[0112] References to “Ngn2” relates to Neurogenin 2. It is also known NEUROG2, BHLHa8, Math4A and Atoh4. In one embodiment, the Ngn2 transcription factor is human Ngn2. Wild type human Ngn2 is identified by UniProt ID: Q9H2A3 and is encoded by the Ngn2 gene which is identified by Ensembl Gene ID: ENSG00000178403. Ngn2 plays a role in the differentiation and survival of midbrain dopaminergic neurons.

[0113] References to “OLIG2” relate to Oligodendrocyte Transcription Factor 2. It is also known as BHLHe19, RACK17, PRKCBP2, OLIGO2 and BHLHB1. In one embodiment, the OLIG2 transcription factor is human OLIG2. Wild type human OLIG2 is identified by UniProt ID: Q13516 and is encoded by the OLIG2 gene which is identified by Ensembl Gene ID: ENSG00000205927. OLIG2 is an essential regulator of ventral neuroectodermal progenitor cell fate.

[0114] References herein to “SOX9” relate to SRY-box transcription factor 9. SOX9 is also known as CMD1 , CMPD1 , SRA1 , SRXX2, SRXY10 and SRY-box 9. In one embodiment the SOX9 is human SOX9. Wild type human SOX9 is identified by UniProt ID: P48436, and is encoded by the S0X9 gene, identified by Ensembl Gene ID: ENSG00000125398. SOX9 with steroidogenic factor 1 regulates the transcription of the anti-Mullerian hormone (AMH) gene. SOX9 also works in conjunction with Sf 1 to produce AMH in Sertoli cells to inhibit the creation of the female reproductive system.

[0115] References to “SOX10” relate to SRY-Box Transcription Factor 10. It is also known as WS2E, DOM and WS4. In one embodiment, the SOX10 transcription factor is human SOX10. Wild type human SOX10 is identified by UniProt ID: P56693 and is encoded by the SOX10 gene which is identified by Ensembl Gene ID: ENSG00000100146. SOX10 acts as a nucleocytoplasmic shuttle protein and is important for neural crest and peripheral nervous system development.

[0116] References herein to a “peroxisome proliferator-activated receptor” (PPAR) or a “PPAR protein” refer to transcription factor in the group of nuclear receptor proteins known as BIT-C-P3787PCT

[0117] 18 peroxisome proliferator-activated receptors. There are three types of PPAR in the group: PPAR alpha (PPARA), PPAR beta / delta (PPARD) and PPAR gamma (PPARG). All PPARs heterodimerize with the retinoid X receptor (RXR) and bind to peroxisome proliferator hormone response elements (specific regions on the DNA of target genes). These specific regions have a DNA consensus sequence of AGGTCANAGGTCA (SEQ ID NO: 1), with N being any nucleotide.

[0118] In one embodiment, the PPAR is selected from the group consisting of: PPARA and PPARG. In one embodiment, the PPAR is PPARA. In an alternative embodiment, the PPAR is PPARG. It will be understood that if the expression of one or more polypeptide having the activity of one or more transcription factor and / or of more than one transcription factor is increased, this may include one or more PPAR protein. Therefore, in one embodiment, the method comprises increasing the expression of one or more polypeptide having the activity of PPARA and PPARG and / or of PPARA and PPARG themselves.

[0119] References herein to “PPARA” or “PPARa” or “Peroxisome Proliferator Activated Receptor Alpha” relates to a member of the PPAR subfamily of nuclear hormone receptors. In one embodiment, the PPARA is human PPARA. Wild type human PPARA is identified by UniProt ID: Q07869, and is encoded by the PPARA gene, identified by Ensembl Gene ID: ENSG00000186951.

[0120] References herein to “PPARG” or “PPARy” or “Peroxisome Proliferator Activated Receptor Gamma” relates to a member of the PPAR subfamily of nuclear hormone receptors, and includes all isoforms of PPARG, such as PPARG1 and PPARG2. In one embodiment, the PPARG is human PPARG. Wild type human PPARG is identified by UniProt ID: P37231 , and is encoded by the PPARG gene, identified by Ensembl Gene ID: ENSG00000132170.

[0121] References herein to “HOXC8” relates to Homeobox Protein Hox-C8. In one embodiment, the HOXC8 is human HOXC8. Wild type human HOXC8 is identified by UniProt ID: P31273, and is encoded by the HOXC8 gene, identified by Ensembl Gene ID: ENSG00000037965. HOXC8 belongs to the homeobox family of genes that encode a highly conserved family of transcription factors. HOXC8 is involved in the regulation of cartilage differentiation, HOXC8 is involved with cell junction organization and the regulation of CDH11 expression and function.

[0122] References herein to “EBF1” relates to Early B Cell Factor 1 (also known as Transcription factor COE1). In one embodiment, the EBF1 is human EBF1. Wild type human EBF1 is BIT-C-P3787PCT

[0123] 19 identified by UniProt ID: Q9LIH73, and is encoded by the EBF1 gene, identified by Ensembl Gene ID: ENSG00000164330. EBF1 is involved in the olfactory signalling pathway and in nervous system development. EBF1 activates B-cell-specific genes such as BCR or CD40 and represses genes associated with T cell fates, such as GATA3 and TCF7.

[0124] References herein to “EBF2” relates to Early B Cell Factor 2 (also known as Transcription factor COE2). In one embodiment, the EBF2 is human EBF2. Wild type human EBF2 is identified by UniProt ID: Q9HAK2, and is encoded by the EBF2 gene, identified by Ensembl Gene ID: ENSG00000221818. EBF2 regulates osteoclast differentiation by activating the decoy receptor for RANKL, TNFRSF11 B.

[0125] References herein to “ZNF467” or “Zinc Finger Protein 467” relates to a zinc finger protein. In one embodiment, the ZNF467 is human ZNF467. Wild type human ZNF467 is identified by UniProt ID: Q7Z7K2, and is encoded by the ZNF467 gene, identified by Ensembl Gene ID: ENSG00000181444. ZNF467 binds to STAT3 at the consensus sequence 5'- CTTCTGGGAAGA-3' (SEQ ID NO: 2).

[0126] References herein to “ZNF423” relates to Zinc Finger Protein 423. In one embodiment, the ZNF423 is human ZNF423. Wild type human ZNF423 is identified by UniProt ID: Q2M1 K9, and is encoded by the ZNF423 gene, identified by Ensembl Gene ID: ENSG00000102935. ZNF423 plays a central role in BMP signaling and olfactory neurogenesis. ZNF423 acts as a transcriptional repressor via its interaction with EBF1 , a transcription factor involved in terminal olfactory receptor neurons differentiation. ZNF423 is involved in olfactory neurogenesis by participating in a developmental switch that regulates the transition from differentiation to maturation in olfactory receptor neurons.

[0127] References herein to “GLIS3” relates to GLIS Family Zinc Finger 3. In one embodiment, the GLIS3 is human GLIS3. Wild type human GLIS3 is identified by UniProt ID: Q8NEA6, and is encoded by the GLIS3 gene, identified by Ensembl Gene ID: ENSG00000107249. GLIS3 functions as a bifunctional mediator of the Sonic Hedgehog (SHH) pathway, providing activating and repressing transcription functions.

[0128] References herein to “PDX1” relates to Pancreatic and Duodenal homeobox 1 (also known as insulin promoter factor 1). In one embodiment, the PDX1 is human PDX1. Wild type human PDX1 is identified by UniProt ID: P52945, and is encoded by the PDX1 gene, identified by Ensembl Gene ID: ENSG00000139515. PDX1 is required for the formation of the endocrine BIT-C-P3787PCT

[0129] 20 pancreas. PDX1 targets PDX1 itself (as a form of auto-regulation), regulatory factor X 6 (RFX6), hepatocyte nuclear factor 1 homeobox B (HNF1 B) and Meis homeobox 1 (MEIS1).

[0130] References herein to “NEUROD1” relates to Neurogenic differentiation 1 (also known as p2). In one embodiment, the NEUROD1 is human NEUROD1. Wild type human NEUROD1 is identified by UniProt ID: Q13562, and is encoded by the NEUROD1 gene, identified by Ensembl Gene ID: ENSG00000162992. NEUROD1 is a basic helix-loop-helix (bHLH) transcription factor that forms heterodimers with other bHLH proteins and activates the transcription of genes containing an E-box DNA sequence.

[0131] References herein to “NKX6-2” relates to Homeobox protein Nkx-6.2 (also known as NK6 homeobox 2). In one embodiment, the NKX6-2 is human NKX6-2. Wild type human NKX6-2 is identified by UniProt ID: Q9C056, and is encoded by the NKX6-2 gene, identified by Ensembl Gene ID: ENSG00000148826. NKX6-2 is transcriptional repressor of factors such as developing brain homeobox protein 1 (DBX1).

[0132] References herein to “ASCL1” relates to Achaete-Scute Family BHLH Transcription Factor 1 (also known as HASH1 and ASH1). In one embodiment, the ASCL1 is human ASCL1. Wild type human ASCL1 is identified by UniProt ID: P50553, and is encoded by the ASCL1 gene, identified by Ensembl Gene ID: ENSG00000139352. ASCL1 activates transcription by binding to the E box (5'-CANNTG-3'). Dimerization with other BHLH proteins is required for efficient DNA binding.

[0133] References herein to “DLX2” relates to Distal-Less Homeobox 2 (also known as TES-1). In one embodiment, the DLX2 is human DLX2. Wild type human DLX2 is identified by UniProt ID: Q07687, and is encoded by the DLX2 gene, identified by Ensembl Gene ID: ENSG00000115844. DLX2 plays a role in forebrain and craniofacial development.

[0134] References herein to “ISL1” relates to ISL LIM Homeobox 1 (also known as Islet-1). In one embodiment, the ISL1 is human ISL1. Wild type human ISL1 is identified by UniProt ID: P61371 , and is encoded by the ISL1 gene, identified by Ensembl Gene ID: ENSG00000016082. ISL1 binds to the enhancer region of the insulin gene, among others and plays an important role in regulating insulin gene expression.

[0135] References herein to “KLF7” relates to Kruppel-Like Factor 7. In one embodiment, the KLF7 is human KLF7. Wild type human KLF7 is identified by UniProt ID: 075840, and is encoded by BIT-C-P3787PCT

[0136] 21 the KLF7 gene, identified by Ensembl Gene ID: ENSG00000118263. KLF7 contains three C2H2 zinc fingers at the C-terminus that mediate binding to GC-rich sites. KLF7 contributes to the progression of type 2 diabetes by inhibiting insulin expression and secretion in pancreatic beta-cells and by deregulating adipocytokine secretion in adipocytes.

[0137] References herein to “NFIA” relate to nuclear factor 1 A-type. NFIA is also known as NF1a, NF1-A and NFI-A. In one embodiment the NFIA is human NFIA. Wild type human NFIA is identified by UniProt ID: Q12857, and is encoded by the NFIA gene, identified by Ensembl Gene ID: ENSG00000162599. Diseases associated with NFIA include bilateral polymicrogyria.

[0138] References herein to “NFIB” relate to nuclear factor 1 B-type. NFIB is also known as NF1 b, NF1-B and NFI-B. In one embodiment the NFIB is human NFIB. Wild type human NFIB is identified by UniProt ID: 000712, and is encoded by the NFIB gene, identified by Ensembl Gene ID: ENSG00000147862. Diseases associated with NFIB include adenoid cystic carcinoma. NFIB also plays an important role in lung development. NFIB downregulates TGF- 1 and Shh.

[0139] References herein to “FEZF2” relate to Forebrain Embryonic Zinc Finger-Like Protein 2. FEZF2 is also known as FEZL, FKSG36, Zfp312, Znf312 and TOP. In one embodiment the FEZF2 is human FEZF2. Wild type human FEZF2 is identified by UniProt ID: Q8TBJ5, and is encoded by the FEZF2 gene, identified by Ensembl Gene ID: ENSG00000153266. Diseases associated with FEZF2 include uterine inversion and ceftriaxone allergy. FEZF2 is required for the specification of corticospinal motor neurons and other subcerebral projection neurons.

[0140] References herein to “TBR1” relate to T-box Brain Transcription Factor 1. TBR1 is also known as T-box brain protein and T-brain. In one embodiment the TBR1 is human TBR1. Wild type human TBR1 is identified by UniProt ID: Q16650, and is encoded by the TBR1 gene, identified by Ensembl Gene ID: ENSG00000136535. TBR1 plays an important role in neuronal migration and axonal projection. TBR1 is associated with intellectual development, and its absence can lead to autism and speech delay.

[0141] References herein to “LHX2” relate to LIM Homeobox Protein 2. LHX2 is also known as LH2. In one embodiment the LHX2 is human LHX2. Wild type human LHX2 is identified by UniProt ID: P50458, and is encoded by the LHX2 gene, identified by Ensembl Gene ID: ENSG00000106689. LHX2 carries a unique cysteine-rich zinc-binding LIM domain. Diseases BIT-C-P3787PCT

[0142] 22 associated with LHX2 include Septooptic Dysplasia. LHX2 is important in olfactory signaling and nervous system development.

[0143] References herein to “DBX2” relate to Developing Brain Homeobox Protein 2. In one embodiment the DBX2 is human DBX2. Wild type human DBX2 is identified by UniProt ID: Q6ZNG2, and is encoded by the DBX2 gene, identified by Ensembl Gene ID: ENSG00000185610. Diseases associated with DBX2 include Miles-Carpenter Syndrome, glioblastoma and endometrial cancer. DHX2 plays an important role in the development of the neural tube and brain.

[0144] Once the activity of the transcription factor is appreciated, the endogenous transcription machinery can be modulated using not only the transcription factors themselves, but also 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 by reference.

[0145] 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.

[0146] 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. BIT-C-P3787PCT

[0147] 23

[0148] Methods of the invention encompass the use of variants of the transcription factors of interest. 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.

[0149] Cell Types

[0150] The method may be used on any type of stem cell. The generation of lineage-restricted cells using the method is referred to as “forward programming”, i.e., the stem cell is forward programmed into a lineage-restricted cell. The resultant cells that are generated after carrying out the methods of the present invention, (i.e. the lineage-restricted cells) may be referred to as the “converted” cells. “Forward programming” relates to the artificial overexpression of molecular cues forcing a change of cell fate to a more lineage-restricted cell type. This is distinctly different from differentiation that takes place naturally or “directed differentiation”, where the environment of the cell culture is manipulated to direct cell fate. Forward programming methods are known in the art to be faster than directed differentiation methods, provide higher target cell yields and provide a higher level of cellular homogeneity.

[0151] The source stem cells may be pluripotent stem cells, for example 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-P3787PCT

[0152] 24

[0153] In one embodiment, the cell population is of human origin. The source cell may be of human origin. It is well known that, compared with non-human pluripotent cells, genome engineering in human pluripotent stem cells is challenging due to, for example, partially due to low transfection / transduction efficiency and high apoptosis under stresses such as low-density culture, drug-selection and sorting (Cerbini et al., PLOS ONE, 10(1), e0116032).

[0154] 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.

[0155] 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.

[0156] 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 programming efficiency, like Nanog, Lin28, Klf4, or c-Myc; specific sets of programming 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.

[0157] In one embodiment, the cell population comprises stem cells, such as induced pluripotent stem cells, embryonic stem cells, progenitor cells, hematopoietic stem cells (preferably induced pluripotent stem cells).

[0158] 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 BIT-C-P3787PCT

[0159] 25 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.

[0160] Delivery

[0161] It will be understood that methods for expressing polypeptides having transcription factor activity or increasing the expression of the transcription factors in the cells to be forward programmed 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.

[0162] In one embodiment, the expression of the polypeptides having transcription factor activity or the transcription factors is carried out by contacting the cell population with the polypeptides or the transcription factors (i.e. , the proteins encoding the transcription factors). Delivery of the transcription factors may occur using direct electroporation of polypeptides and / or transcription factor proteins to the cells.

[0163] In an alternative embodiment, the expression of endogenous transcription factors is increased by introducing a promoter (e.g. a strong promoter) ahead of an endogenous gene encoding the transcription factor(s).

[0164] In a further alternative embodiment, the expression of polypeptides having transcription factor activity and / or transcription factors is increased by contacting the cell population with one or more agents that activate or increase the expression amount of the 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.

[0165] 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) BIT-C-P3787PCT

[0166] 26 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 one or more endogenous transcription factors.

[0167] The method of inducing forward programming 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 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 convert to form lineage restricted cells. Said open reading frame may be part of a recombinant expression cassette.

[0168] In one embodiment, the nucleic acid comprises a recombinant or exogenous expression cassette (preferably a single multicistronic 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.

[0169] 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 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.

[0170] 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, BIT-C-P3787PCT

[0171] 27 by Agrobacterium-mediated transformation, and any combination of such methods. Through the application of these techniques, cells may be stably or transiently transformed.

[0172] Further, the expression cassette (e.g., an inducible recombinant expression cassette) may include cleavable sequences. Such sequences are sequences that are recognised by an entity capable of specifically cutting DNA, and include restriction sites, which are the target sequences for restriction enzymes or sequences for recognition by other DNA cleaving entities, such as nucleases, recombinases, ribozymes or artificial constructs. At least one cleavable sequence may be included, but preferably two or more are present. These cleavable sequences may be at any suitable point in the cassette, such that a selected portion of the cassette, or the entire cassette, can be selectively removed if desired. The cleavable sites may thus flank the part / al I of the genetic sequence that it may be desired to remove. The method may therefore also comprise removal of the expression cassette and / or the genetic material.

[0173] 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.

[0174] It will be understood that a combination of one or more of the methods for expressing the polypeptides having transcription factor activity and increasing the expression of the transcription factors may be used, where the combination results in forward programming towards the desired lineage-restricted cells.

[0175] Vectors

[0176] In one embodiment, the polypeptides having transcription factor activity or the transcription factors themselves 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 limited to plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). BIT-C-P3787PCT

[0177] 28

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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 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. BIT-C-P3787PCT

[0182] 29

[0183] 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 polypeptides having transcription factor activity or the transcription factors that induce reprogramming. Self-replicating RNA vectors are known in the art and many are based on positive strand RNA viruses, such as alphaviruses.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] Site-specific Delivery

[0188] 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).

[0189] 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, BIT-C-P3787PCT

[0190] 30

[0191] TALENs or the clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR / Cas, e.g. CRISPR / Cas9) system.

[0192] 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.

[0193] 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.

[0194] 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 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 BIT-C-P3787PCT

[0195] 31 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] Thus, any method of making specific, targeted double strand breaks in the genome in order to affect 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-P3787PCT

[0200] 32

[0201] 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.

[0202] 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.

[0203] Controlled expression

[0204] 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 polypeptide having transcription factor activity and / or the transcription factors may be controlled within the cell. This permits expression of the polypeptides and overexpression of the transcription factor(s).

[0205] 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.

[0206] 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 achieved by using the Tet-ON system components with transgene expression controlled by BIT-C-P3787PCT

[0207] 33 doxycycline. The components are split between two genomic safe harbour sites (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.

[0208] Therefore, in one embodiment, a sequence encoding one or more of the polypeptides having transcription factor activity and / or the transcription factors are introduced into the stem cell using a method comprising:

[0209] - insertion, preferably targeted insertion, of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and

[0210] - insertion, preferably targeted insertion, of one or more inducible cassettes into one or more second genomic safe harbour sites of the stem cell, wherein said one or more inducible cassettes comprises said sequence encoding one or more of the polypeptides and / or transcription factors operably linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein.

[0211] Alternatively viewed, 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:

[0212] - 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

[0213] - 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.

[0214] According to another aspect of the invention, there is provided an ex vivo method for culturing stem cells, comprising the steps of: a) insertion, preferably targeted insertion, of a gene encoding a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and b) insertion, preferably targeted insertion, of one or more nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or one or more transcription factors, operably linked to an inducible promoter into one or BIT-C-P3787PCT

[0215] 34 more second genomic safe harbour sites of the stem cell, wherein said inducible promoter is regulated by the transcriptional regulator protein; and c) culturing the stem cells comprising the insertions in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are more lineage- restricted in such culturing conditions.

[0216] This embodiment of the invention provides a dual expression cassette system. The insertion of the gene encoding a transcriptional regulator protein into the first GSH site provides the control mechanism for the expression of the inducible cassette which is operably linked to the inducible promoter and inserted into a second GSH site. In one embodiment, the first and second GSH sites are different (i.e. are located at different positions in the genome). It will be understood that if more than one gene for a polypeptide having transcription factor activity and / or transcription factor is to be introduced into the cell using the dual expression system, then they 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).

[0217] This dual expression cassette system produces high levels of homogeneity within the population of the desired lineage-restricted cells compared with, for example, directed differentiation methods. Without wishing to be bound by theory, these high levels of homogeneity are understood to contribute to the stability and viability of the cell aggregates and so aids the transfer of the forward programming procedure from 2D to 3D.

[0218] 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 site and the system controlling the expression of the inducible cassette into the other allele of the FSH 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).

[0219] 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, or any deleterious effects on the ability of the cells to capable of being forward programmed or reprogrammed to microglial cells. Most 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 BIT-C-P3787PCT

[0220] 35 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.

[0221] 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 microglial 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. Screening can also determine whether the gene encoding a transcriptional regulator protein is in a GSH site that is different from the GSH site containing the inducible cassette.

[0222] 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. 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.

[0223] In one embodiment, the first and second genomic safe harbour (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. BIT-C-P3787PCT

[0224] 36

[0225] 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.

[0226] 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.

[0227] 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.

[0228] CCR5, 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.

[0229] 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-P3787PCT

[0230] 37

[0231] Other GSH 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.

[0232] 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.

[0233] Technically, the insertions into the first and / or second GSH sites may occur on one chromosome, or on both chromosomes. The GSH site exists at the same genomic 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.

[0234] 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.

[0235] One or more genetic sequences may be controllably transcribed from within the second and / or further GSH. 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.

[0236] Alternatively, if a combination of transcription factors is used, the individual transcription factors may be introduced into separate GSHs and / or under the control of the same, different or orthogonal inducible promoters. Therefore, in one embodiment, the transcription factors are introduced into separate GSH sites. For example, this may be achieved by utilising three or BIT-C-P3787PCT

[0237] 38 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 genomic 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, each operably linked to an inducible promoter into a second, third and fourth genomic safe harbour site of the source cell. The inducible promoter may be the same of each transcription factor and therefore are all regulated by the transcriptional regulator protein.

[0238] 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.

[0239] 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- BIT-C-P3787PCT

[0240] 39 activatable domain may comprise or may be a functional domain or portion of a naturally 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.

[0241] 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.

[0242] 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).

[0243] 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).

[0244] 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 BIT-C-P3787PCT

[0245] 40 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.

[0246] A transcriptional regulator protein is a protein that binds to DNA, preferably sequence- specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor).

[0247] 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.

[0248] 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.

[0249] 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. 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.

[0250] 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.

[0251] Any suitable transcriptional regulator protein may be used, preferably one that may be activated or deactivated. It is preferred that an exogenous substance may be supplied to BIT-C-P3787PCT

[0252] 41 control the transcriptional regulator protein. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.

[0253] Tetracycline-Controlled Transcriptional Activation is a method of inducible 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.

[0254] 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.

[0255] 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.

[0256] 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. If the transcriptional regulator protein is rtTA, then the inducible promoter inserted into the second GSH site includes the tetracycline response element (TRE). The exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

[0257] 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). BIT-C-P3787PCT

[0258] 42

[0259] 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. Preferably the minimal promoter shows no or minimal expression levels in the absence of rtTa binding. The inducible promoter inserted into the second GSH may thus comprise a TRE.

[0260] 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 prevents 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.

[0261] 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.

[0262] 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 BIT-C-P3787PCT

[0263] 43 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 substance is ponasterone A, which removes the repressive effect of EcR or derivatives thereof on the inducible promoter and allows transcription to take place.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] It is preferred that the gene encoding the transcriptional regulator protein is operably linked to a constitutive promoter. Alternatively, the first GSH site can be selected such that it already has a constitutive promoter than 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 BIT-C-P3787PCT

[0269] 44

[0270] P-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1 a, (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.

[0271] It will be understood that this aspect of the invention may be used with any of the combinations of polypeptides having transcription factor activity or transcription factors described herein.

[0272] Obtaining lineage-restricted cells

[0273] It is clear that the 3D culturing methods of the present invention may be used for the forward programming to any lineage-restricted cell desired. In one embodiment, the methods are used to generate nerve cells (including neural stem cells, peripheral nervous system neurons, migratory enteric neural crest cell, neural crest cell, enteric neurons, glutamatergic neurons, GABAergic neurons, sensory neurons, motor neurons, dopaminergic neurons, medium spiny neurons, cortical neurons, interneurons, brainstem neurons, cholinergic neurons, hippocampal neurons, projection neurons, Lewy body-containing neurons, pyramidal neurons, cerebellar neurons, serotonergic neurons, thalamic neurons, neuromuscular junction cells, ganglion cells, oligodendrocyte precursors, oligodendrocytes, astrocytes, Purkinje cells, Muller cells, Granule cells, photoreceptors (rods and cones), retinal ganglion cells, retinal precursor cells, retinal pigment epithelium and Bruch's membrane cells), myocytes (including myoblasts, myogenic progenitors, skeletal myocytes, smooth muscle cells, satellite cells and cardiomyocytes), osteocytes (such as osteoclasts and osteoblasts), chondrocytes, adipocytes (including preadipocytes, brown adipocytes, beige adipocytes, white adipocytes and epicardial adipocytes), pericytes, hepatocytes (including hepatic stellate cells), kidney cells (such as papillary tips cells, podocytes and mesangial cells), respiratory cells (such as airway epithelial cells, club cells and ciliated cells), megakaryocytes, epithelial cells (such as melanocytes and placental villous trophoblasts), mesothelial cells (such as epicardium), endothelial cells (such as endocardial cells, keratinocytes and trabecular meshwork cells), secretary cells (such as pancreatic beta cells, pancreatic alpha cells, pancreatic acinar cells, pancreatic ductal cells and chromaffin cells), gastrointestinal cells (such as intestinal endocrine cell, intestinal epithelial cells, enteroendocrine cells, goblet cells, submucosal gland cells, Paneth cells and enterocytes), fibroblasts, myofibroblasts, mesodermal cells, mesenchymal cells (such as mesenchymal stem cells) and / or blood cells (including hematopoietic stem cells, erythrocytes, platelets, plasma cells and immune cells, such as neutrophils, glial cells, microglia, dendritic BIT-C-P3787PCT

[0274] 45 cells, T cells, such as CD4+ T helper cells, CD8+ cytotoxic T cells, regulatory T cells and gamma delta T cells, B cells, macrophages, Kupffer cells, innate lymphoid cells, eosinophils, mast cells, monocytes, Langerhans cells and natural killer cells). In one further embodiment the methods are used to generate mature cells selected from the list consisting of hepatocytes, microglia, glutamatergic neurons, GABAergic neurons, sensory neurons and pancreatic beta cells. In yet a further embodiment, the methods are used to generate hepatocytes, microglia or GABAergic neurons.

[0275] The methods described above may produce a population of lineage-restricted cells that is substantially free from other cell types. In other words, the methods described above may produce a homogenous (or substantially homogenous) population of lineage-restricted cells. For example, a population produced by a method described herein may contain 80% or more, 85% or more, 90% or more, or 95% or more of a specific lineage-restricted cell type following culture. Preferably the population of a specific lineage-restricted cell type is sufficiently free of other cell types that no purification is required. If required, the population of a specific lineage- restricted cell type may be purified by any convenient technique including FACS.

[0276] Forward programming methods are known to be more effective in general than directed differentiation methods at creating a homogenous population of specific lineage-restricted cells, and more effective at generating lineage-restricted cells that more closely resemble their primary counterparts.

[0277] In one embodiment, the method additionally comprises monitoring the cell population for at least one characteristic of the desired lineage-restricted cell. Cells may be monitored throughout culturing to identify expression of key lineage markers.

[0278] 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. BIT-C-P3787PCT

[0279] 46

[0280] 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.

[0281] 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.

[0282] Examples of fluorescent protein genes which may be used as markers include: a green fluorescent protein (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.

[0283] 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.

[0284] 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).

[0285] The method may therefore comprise a selection or enrichment step for the desired lineage- restricted cells provided from the methods described herein. In one embodiment, the method comprises the step of sorting the lineage-restricted cells using fluorescence activated cell sorting (FACS) or immunomagnetic sorting methods based on the expression of cell markers BIT-C-P3787PCT

[0286] 47 specific to the desired lineage-restricted cells and / or absence of cell markers not indicative of the desired lineage-restricted cells (such as pluripotency markers). In one embodiment, the desired lineage-restricted cells are selected or enriched by removing cells which express pluripotency markers, such as Ki67, TRA-1-60, SOX2, OCT4, NANOG or SSEA4, in particular Ki67, NANOG or POLI5F1 . 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 antibody or a fragment thereof, a peptide or a synthetic binder such as a plastic antibody, or an aptamer or oligonucleotide, capable of specific binding to an epitope. The binding agent may be labelled with a detectable marker, such as a luminescent, fluorescent (e.g. fluorochrome), enzyme or radioactive marker; alternatively or additionally an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. In one embodiment, fluorochrome conjugated antibodies targeting cell surface proteins (e.g. microglial cell markers) may be used to sort target cells.

[0287] In another embodiment, the desired lineage-restricted cells are enriched by drug-resistance selection from genetically engineered source cells expressing an antibiotic-resistance gene under the control of a microglial cell-specific promoter.

[0288] The method may generate cells (i.e. , converted cells) exhibiting at least one characteristic of the desired lineage-restricted cells. One or more characteristics may be used to select for the lineage-restricted cells generated by the methods of the invention.

[0289] Characteristics include but are not limited to the detection or quantitation of expressed cell markers, enzymatic activity, and the characterization of morphological features and intercellular signaling. The biological function of the desired lineage-restricted cell may also be evaluated, for example using functional assays, e.g. flow cytometry.

[0290] The cell markers indicative of the desired lineage-restricted cells may be markers obtained by transcriptome analysis. For example, single cell RNA sequencing has been used to provide detailed transcriptional profiles of human cells obtained from primary human tissues. This information can be used to identify cells generated by the methods described herein. Additional resources, such as Human Cell Atlas and CellTypist may also be used to identify markers of specific lineage-restricted cells.

[0291] The method may comprise assaying the converted cells obtained by the method described herein and determining a set of transcribed genes; comparing the set of transcribed genes of BIT-C-P3787PCT

[0292] 48 the converted cells to one or more reference sets of transcribed genes from one or more reference cells; and identifying a match between the converted cells and a reference cell.

[0293] In one embodiment, the method comprises the step of identifying converted cells as a desired lineage-restricted cell by assaying morphological features of the converted cells and matching the morphological features to a reference tissue or cell's morphological features.

[0294] In one embodiment, the method comprises the step of identifying converted cells as a desired lineage-restricted cell by assaying protein marker expression of the converted cells and matching the protein marker expression to a reference cell protein marker expression.

[0295] In one embodiment, the method comprises the step of identifying converted cells as a desired lineage-restricted cell by assaying a function and matching the function to a function of a reference cell.

[0296] In one embodiment, the cells obtained by the methods of the invention express a particular cell phenotype. The target cells may also be negative for markers of pluripotency. This may include cells that do not express any one or more of the transcription factors known to induce pluripotency (any member of the Sox family, any member from Oct family, Nanog, Lin28, Klf4, or c-Myc).

[0297] Alternatively, certain converted cells may be sorted from other converted cells and from cells on the basis of their expression of a lineage-specific cell surface antigen. Yet another means is by assessing expression at the RNA level, e.g., by RT-qPCR methods or by single cell RNA sequencing without any sorting or pre-selection step. Such techniques are known in the art.

[0298] Hepatocytes

[0299] Where the methods described herein are used to generate hepatocytes, those hepatocytes in the population may be functionally mature. A functionally mature hepatocyte may display a mature hepatocyte phenotype.

[0300] Hepatocytes produced by a method described herein may express the hepatocyte markers albumin (ALB), alphal -antitrypsin (AAT, A1AT or SERPINAI), CYP2A6, CYP3A4, CYP2C8, CYP2C9, UGT1A1 , ApoA1 , FASN, NR1 H4, G6PC, UGT1A6, PCK1 , PPRa / g and / or RORg. BIT-C-P3787PCT

[0301] 49

[0302] Other hepatocyte markers may include fumarylacetoacetase (FAH), cytokeratin 8 (CK8), cytokeratin 18 (CK18), asialoglycoprotein Receptor (ASGR), alcohol dehydrogenase 1 , arginase type I and liver-specific organic anion transporter (LST-1).

[0303] The hepatocytes may express the hepatocyte markers at the same level or substantially the same level as primary adult human hepatocytes. For example, the expression level in the hepatocytes may be the same or higher or lower than the expression level in primary adult human hepatocytes by 20% or less, 10% or less or 5% less.

[0304] The hepatocytes may not express progenitor markers, such as AFP, CK18 and Sox17, or may express them at low levels. For example, the level of expression of progenitor markers in the hepatocytes may be less than 20%, less than 10%, less than 5% or less than 1 % of the level of expression of the above hepatocyte markers.

[0305] Hepatocytes produced by a method described herein may be capable of performing the functions of primary adult human hepatocytes. For example, the hepatocytes may be capable of one or more of storing glycogen and LDL, synthesising and secreting AAT and / or albumin (ALB), uptaking LDL and fatty acids and detoxifying xenobiotics via the CytP450 pathway. The hepatocytes may be able to produce one or more of bile, thrombopoietin, angiotensinogen, urea and cholesterol; and perform one or more of glycogenolysis, gluconeogenesis, glycogenesis and lipogenesis.

[0306] Hepatocyte functions may be performed by hepatocytes produced by a method described herein at the same activity or substantially the same activity as primary adult human hepatocytes. For example, the amount of activity in the hepatocytes may be the same as the amount of activity in primary adult human hepatocytes or may be higher or lower by 20% or less, 10% or less or 5% less.

[0307] Hepatocytes produced by a method described herein may be capable of in vivo engraftment and the liver colonisation in model systems, for example murine mouse models, such as the humanised FRG mouse (Strom et al (2010) Methods Mol Biol 640: 491-509).

[0308] Hepatocytes produced by a method described herein may display the same or substantially the same gene expression profile of mature primary human hepatocytes (PHHs). BIT-C-P3787PCT

[0309] 50

[0310] Hepatocytes produced by the present methods may display one or more of the following hepatocyte morphological characteristics: cobblestone morphology, occasional binucleity; glycogen deposits; apical microprotrusions; rough and smooth endoplasmic reticulum (ER) and a prominent Golgi body.

[0311] Microglia

[0312] Where the methods described herein are used to generate microglia, those microglia in the population may be functionally mature. Functionally mature microglia may display a mature microglia phenotype.

[0313] Microglia produced by a method described herein may express one or more of the microglia markers P2RY12, IBA1 , TREM2, CX3CR1 , CD11 b, CD45, CD14 and TMEM119.

[0314] The microglia may express the microglia markers at the same level or substantially the same level as primary adult human microglia. For example, the expression level in the microglia may be the same or higher or lower than the expression level in primary adult human microglia by 20% or less, 10% or less or 5% less.

[0315] Microglia produced by a method described herein may be capable of performing the functions of primary adult human microglia. For example, the microglia may be capable of one or more of mediating an inflammatory response, disposing of unwanted materials through for example phagocytosis and carrying out immune surveillance.

[0316] Microglia functions may be performed by microglia produced by a method described herein at the same activity or substantially the same activity as primary adult human microglia. For example, the amount of activity in the microglia may be the same as the amount of activity in primary adult human microglia or may be higher or lower by 20% or less, 10% or less or 5% less.

[0317] Microglia produced by a method described herein may display the same or substantially the same gene expression profile of mature primary human microglia.

[0318] Glutamatergic neurons

[0319] Where the methods described herein are used to generate glutamatergic neurons (also known as excitatory neurons), those glutamatergic neurons in the population may be functionally BIT-C-P3787PCT

[0320] 51 mature. A functionally mature glutamatergic neurons may display a mature glutamatergic neuron phenotype.

[0321] Glutamatergic neurons produced by a method described herein may express one or more of the glutamate transporter genes VGLLIT1 or VGLLIT2. Glutamatergic neurons may also be MAP2-positive and / or Til BBS-positive.

[0322] The glutamatergic neurons may express the glutamatergic neuron markers at the same level or substantially the same level as primary adult human glutamatergic neurons. For example, the expression level in the glutamatergic neurons may be the same or higher or lower than the expression level in primary adult human glutamatergic neurons by 20% or less, 10% or less or 5% less.

[0323] Glutamatergic neurons produced by a method described herein may display the same or substantially the same gene expression profile of mature primary human glutamatergic neurons.

[0324] GABAergic neurons

[0325] Where the methods described herein are used to generate GABAergic neurons (also known as inhibitory neurons), those GABAergic neurons in the population may be functionally mature. A functionally mature GABAergic neurons may display a mature GABAergic neuron phenotype.

[0326] GABAergic neurons produced by a method described herein may express one or more of the following markers: GAD1 , GAD2, VGAT, DLX1 or DLX2.

[0327] The GABAergic neurons may express the GABAergic neurons markers at the same level or substantially the same level as primary adult human GABAergic neurons. For example, the expression level in the GABAergic neurons may be the same or higher or lower than the expression level in primary adult human GABAergic neurons by 20% or less, 10% or less or 5% less.

[0328] GABAergic neurons produced by a method described herein may display the same or substantially the same gene expression profile of mature primary human GABAergic neurons.

[0329] Sensory neurons BIT-C-P3787PCT

[0330] 52

[0331] Where the methods described herein are used to generate sensory neurons, those sensory neurons in the population may be functionally mature. A functionally mature sensory neurons may display a mature sensory neuron phenotype.

[0332] Sensory neurons produced by a method described herein may express one or more of the following markers: PRPH, BRN3A, ISL1 and TLIBB3.

[0333] The sensory neurons may express the sensory neuron markers at the same level or substantially the same level as primary adult human sensory neurons. For example, the expression level in the sensory neurons may be the same or higher or lower than the expression level in primary adult human sensory neurons by 20% or less, 10% or less or 5% less.

[0334] Sensory neurons produced by a method described herein may display the same or substantially the same gene expression profile of mature primary human sensory neurons.

[0335] Sensory neurons produced by a method described herein may be capable of performing the functions of primary adult human sensory neurons. For example, the sensory neurons (if showing a nociceptor phenotype) may be responsive to one or more of the responsive agonists TRPV1 , TRPM3 or TRPM8.

[0336] Sensory neuron functions may be performed by sensory neurons produced by a method described herein at the same activity or substantially the same activity as primary adult human sensory neurons. For example, the amount of activity in the sensory neurons may be the same as the amount of activity in primary adult human sensory neurons or may be higher or lower by 20% or less, 10% or less or 5% less.

[0337] Pancreatic beta cells

[0338] Where the methods described herein are used to generate pancreatic beta cells, those pancreatic beta cells in the population may be functionally mature. A functionally mature pancreatic beta cells may display a mature pancreatic beta cell phenotype.

[0339] Pancreatic beta cells produced by a method described herein may express one or more of the following markers: proinsulin, MAFA or insulin c-peptide. BIT-C-P3787PCT

[0340] 53

[0341] The pancreatic beta cells may express the pancreatic beta cell markers at the same level or substantially the same level as primary adult human pancreatic beta cells. For example, the expression level in the pancreatic beta cells may be the same or higher or lower than the expression level in primary adult human pancreatic beta cells by 20% or less, 10% or less or 5% less.

[0342] Pancreatic beta cell produced by a method described herein may display the same or substantially the same gene expression profile of mature primary human pancreatic beta cells.

[0343] Pancreatic beta cells produced by a method described herein may be capable of performing the functions of primary adult human pancreatic beta cells. For example, the pancreatic beta cells may secrete insulin and / or amylin.

[0344] Pancreatic beta cell functions may be performed by pancreatic beta cells produced by a method described herein at the same activity or substantially the same activity as primary adult human pancreatic beta cells. For example, the amount of activity in the pancreatic beta cells may be the same as the amount of activity in primary adult human pancreatic beta cells or may be higher or lower by 20% or less, 10% or less or 5% less.

[0345] Cell culturing

[0346] In one embodiment, the method includes culturing the cell population in a 3D environment for a sufficient time and under conditions to allow forward programming to the desired lineage- restricted cells. In one embodiment, the stem cells are present in the form of a single cell suspension before forward programming takes place. In this circumstance, the cells would aggregate during the forward programming process and / or after the forward programming process is complete.

[0347] The stem cells form aggregates in the 3D environment. These aggregates may form on or within a substrate, such as a scaffold, matrix or bead. These substrates may have a surface that promotes cell adhesion, encouraging the cells to bind to it rather than bind to another surface, such as the inner surface of a culture vessel.

[0348] However, it is preferable that the aggregates form in the absence of such substrates. As discussed in, for example, Liu et al (Stem Cell Res. & Ther. (2024) 15:244), 3D culture is often carried out using a substrate such as Matrigel, but such materials introduce ill-defined, animal- derived constituents to the culture environment, meaning that the cultured cells would then be BIT-C-P3787PCT

[0349] 54 unsuitable for therapeutic purposes. Use of such ill-defined constituents also lead to a lack of reproducibility. The inventors surprisingly found that forward programming was possible in a 3D environment even in the absence of such substrates.

[0350] In one embodiment, the culturing takes place at an initial cell seeding density of less than 500,000 cells / mL, 450,000 cells / mL, 400,000 cells / mL, 350,000 cells / mL or 325,000 cells / mL. These cell seeding densities are lower than the densities typically used for meat production, where the proportion of viable cells does not need to be as high. In one embodiment, the culturing takes place at an initial cell seeding density of greater than 10,000 cells / mL, 25,000 cells / mL, 50,000 cells / mL, 100,000 cells / mL or 125,000 cells / mL.

[0351] The terms "cluster" and "aggregate" may be used interchangeably and refer to multiple cells having grouped together so that adjacent cells are in close proximity and / or direct contact with each other, and wherein the adjacent cells have an affinity towards each other so as to maintain the three-dimensional structure of the cluster. Such aggregates would have a defined boundary. Aggregates may be spherical in shape, although a skilled person will readily recognize that a cluster of cells inherently will never form a perfectly round geometrical shape in the three-dimensional space. The cluster may for example be elongated in spheroid-like structure or protrude or depress in certain areas.

[0352] In one embodiment, the aggregates are less than 800 pm in diameter. In a preferred embodiment, the aggregates are less than 700 pm in diameter. In a preferred embodiment, the aggregates are less than 600 pm in diameter. In a preferred embodiment, the aggregates are less than 500 pm in diameter. In a preferred embodiment, the aggregates are less than 400 pm in diameter.

[0353] In one embodiment, the aggregates are more than 30 pm in diameter. In one embodiment, the aggregates are more than 40 pm in diameter. In one embodiment, the aggregates are more than 50 pm in diameter. In a preferred embodiment, the aggregates are more than 60 pm in diameter. In a preferred embodiment, the aggregates are more than 70 pm in diameter. In a preferred embodiment, the aggregates are more than 80 pm in diameter. In a preferred embodiment, the aggregates are more than 90 pm in diameter. In a preferred embodiment, the aggregates are more than 100 pm in diameter.

[0354] The step of aggregating the cells may be carried out passively or actively as long as the cells form clusters. Accordingly, in an embodiment, the cells are aggregated by gravitational settling BIT-C-P3787PCT

[0355] 55 of the cells in the single cell suspension. In another embodiment, the cells are aggregated by spin-aggregation in the single cell suspension. In such an embodiment, the spin-aggregation clusters the cells. The spin may be generated by an impeller present within, for example, a spinner flask or a bioreactor. The speed of the spin can be measured in tip speed, this being the speed at which the tip of the impeller travels within the culture vessel. The tip speed may be between 0.05 m / s and 0.40 m / s, 0.08 m / s and 0.32 m / s or 0.10 m / s and 0.30 m / s. However, it is typically found that the impellers are larger in spinner flasks compared with bioreactors (in relation to the size of the culture vessel), and as a result a greater tip speed is generally required in a bioreactor setup compared with a non-bioreactor, such as a spinner flask, setup. As such, the tip speed may be between 0.05 m / s and 0.20 m / s, 0.07 m / s and 0.18 m / s, 0.09 m / s and 0.17 m / s or 0.10 m / s and 0.16 m / s where the culture vessel is not a bioreactor. Alternatively, the tip speed may be between 0.10 m / s and 0.40 m / s, 0.12 m / s and 0.36 m / s, 0.14 m / s and 0.32 m / s, 0.16 m / s and 0.30 m / s or 0.19 m / s and 0.28 m / s where the culture vessel is a bioreactor.

[0356] In an embodiment, the cells are added as a cell suspension and left under stationary conditions, thereby allowing the cells in suspensions to settle by the force of gravity into microwells, which subsequently form aggregates.

[0357] Throughout the methods according to the present invention, the cells may be maintained in any suitable cell culture vessel, which supports three-dimensional culturing of the cells. As used herein, the term "cell culture vessel" is defined as a container specifically designed to support the growth and propagation of cells in culture. The container may vary in size, shape, coating, and the presence or absence of a lid. Non-limiting examples of cell culture vessels include cell culture tubes, plates (such as wells), flasks bottles and bags.

[0358] For the step of aggregating the stem cells the cell culture vessel must accommodate the formation of the aggregates and the cells may be transferred to such. In an embodiment, the stem cells are seeded in a cell culture vessel accommodating the formation of aggregates subsequent to dissociation. In an embodiment, the method comprises the additional step of transferring the stem cells to a cell culture well suitable for maintaining an aggregate prior to the step of aggregating the stem cells. In a further embodiment, the cell culture well is suitable for maintaining the aggregate in a static non-adherent culture. As used herein, by the term "well" is meant a space suitable for one or more aggregate to be maintained in a static nonadherent culture. By the term "static non-adherent culture" is meant that cells are placed in a condition where they are not adhered to a surface and suspended in a media solution but one BIT-C-P3787PCT

[0359] 56 which does not employ motion, with forces of gravity being the only means to hold cells in position. Cell culture vessels may comprise a well, which functions as a parent housing, wherein one or more additional smaller wells, optionally referred to as microwells, are comprised. Accordingly, in an embodiment, the cell culture well is a microwell. In an embodiment, the cell culture vessel suitable for maintaining the aggregate in a static nonadherent culture has a surface with low cell attachment properties. By "low cell attachment properties" is meant that a material has a low affinity for adhesion with cells and prevents their direct binding and growth on said material. Further to this, in an embodiment the surface with low cell attachment properties is low-adherent plastic and / or plastic treated with a low-adherent agent. Vessels with the aforementioned properties are readily available. In an embodiment, multiple aggregates are obtained simultaneously.

[0360] In a preferred embodiment, the cell culture vessel features low cell attachment properties. Such properties are typical for non-treated plastics that have not been exposed to plasma gasses, which would typically add oxygen-containing functional groups such as hydroxyl and carboxyl to modify the hydrophobic plastic surface, thereby making the surface more hydrophilic. It is believed that the low-attachment properties of the cell culture vessel facilitate the maturation process as it prevents aggregate attachment to the plastic, which would otherwise allow cells to migrate. Furthermore, in an embodiment, the cell culture vessel is not coated with an extracellular matrix component. It follows that in a preferred embodiment, the surface of the cell culture vessel is free of an extracellular matrix.

[0361] In one embodiment, the culturing takes place in a culture vessel able to accommodate at least 10 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 30 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 75 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 100 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 200 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 250 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 500 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 800 mL of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 1 L of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 2 L of culture media. In a preferred embodiment, the culturing takes BIT-C-P3787PCT

[0362] 57 place in a culture vessel able to accommodate at least 5 L of culture media. In a preferred embodiment, the culturing takes place in a culture vessel able to accommodate at least 10 L of culture media. It is understood that scaling up culture environments into larger culture vessels, whilst beneficial in terms of being able to generate cells at a faster rate, brings with it complications that impact on cell viability. For example, spinning and shaker arrangements create greater shear stresses on cells when the size of the culture vessel is increased. This is particularly the case when the stem cells are mammalian stem cells, as such cells are known to be sensitive to shear stresses.

[0363] The culture vessel may be a flask, such as a spinner flask or a shaker flask. Preferably the culture vessel is a bioreactor. A bioreactor is a device that actively supports a biological environment comprising probes that may monitor parameters such as temperature, pH, dissolved oxygen and carbon dioxide concentration, and nutrient levels and through a feedback mechanism supply heat, pH, nutrient and / or gas adjustments. This ensures that the monitored parameters are always optimal. Such bioreactors can be set up in a continuously operating manner that provides a regular supply of lineage-restricted cells of interest in the effluent. The bioreactor may be pressurized. Bioreactors are available in the art, such as AMBR 250 (Sartorius) and DASBOX reactor (Eppendorf). As with the complications of increasing the culture vessel size as discussed above, using a bioreactor can detrimentally impact on cell viability. This can be due to the cells being exposed to excessive shear stresses. These shear stresses can result from the sparging of gases in the bioreactor, the probes in the bioreactor disrupting the flow, and the increased impact of eddies that can results from larger impeller sizes.

[0364] With the aforementioned methods the present inventors aim at providing an aggregate, wherein the entire volume of aggregate consists of cells. A person skilled in the art will recognize that the aggregate according to the present invention comprises living cells and that this inherently introduces variability into the product. In particular, the cells may vary in size by growing or shrinking during different stages, the number of cells in the aggregate may not be constant due to proliferation or apoptosis. Dead cells may be broken down and cell membranes destroyed, thus rupturing the cell and releasing cellular contents. The cells may also secrete cellular material during different stages of development. Accordingly, it is to be understood that an aggregate comprising cells or consisting of cells may also comprise cellular material originating from the cells. Therefore, in an embodiment, is provided an aggregate, wherein the entire volume of the aggregate consists of cells and optionally cellular material originating from the cells. As used herein, the term "cellular material originating from the cells" means secreted BIT-C-P3787PCT

[0365] 58 proteins or other molecules and includes cellular debris originating from dead cells. Due to the aforementioned nature of the aggregate comprising living cells the aggregate product may also be defined as an aggregate, wherein the entire volume of the aggregate substantially consists of cells. Further to this, an embodiment relates to an aggregate, wherein the entire volume of the aggregate substantially consists of cells.

[0366] In an embodiment, at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the volume of the aggregate comprises cells, preferably at least 90%, more preferably at least 95%. In an embodiment, at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the volume of the aggregate comprises cells and optionally cellular material originating from the cells, preferably at least 90% of the volume of the aggregate comprises neural cells and optionally cellular material originating from the cells, more preferably at least 95% of the volume of the aggregate comprises cells and optionally cellular material originating from the cells. In a preferred embodiment, the volume of the aggregate substantially consists of cells.

[0367] Generally, cells of the present invention are cultured in a culture medium, which is a nutrientrich buffered solution capable of sustaining cell growth. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as growth factors and small molecules, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art. For example, the media may comprise Basal Medium (e.g. DMEM / F12 or STEMPRO-34) supplemented with GLUTAMAX, antibiotics (such as penicillin or streptomycin), B27 supplement and / or N2 supplement (all available from Thermo Fisher Scientific). Any suitable growth medium containing sufficient quantities of vitamins, minerals, salts, glucose, amino acids and carrier proteins desirable for cell forward programming or reprogramming may be used, in particular TESR-E8 medium (available from STEMCELL Technologies). The media may then be further supplemented at different time points during the culturing process. For example, one or more peptide hormones and / or cytokines can be added at 2, 4 and / or 10 days during the culturing process.

[0368] The term "growth factor" means a signaling molecule that controls cell activities in an autocrine, paracrine or endocrine manner. As used herein, in the context of the present invention, the term "growth factor" may be used interchangeably with "cytokine". Growth factors or cytokines are produced by different cell types of the organism and exert their biological functions by binding to specific receptors and activating associated downstream signaling pathways which in turn, regulate gene transcription in the nucleus and ultimately stimulate a biological BIT-C-P3787PCT

[0369] 59 response, including regulatory cellular processes like cell division, cell survival, cell differentiation, adhesion and migration.

[0370] The term "small molecule" means a bioactive molecule that is naturally or artificially produced and is capable of diffusion through the cell membrane and is able to regulate signaling pathways. Small molecules, which are preferably used within the present invention, may inhibit phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3, respectively like LY294002 and CHIR99021.

[0371] In one embodiment, the culture media comprises one or more components selected from the group consisting of: Activin A, bone morphogenetic protein (BMP) such as BMP4, fibroblast growth factor (FGF) such as FGF2, vascular endothelial growth factor (VEGF), such as VEGF- A, stem cell factor (SCF), interleukins, such as IL-3, IL-4, IL-6 and IL-34, colony-stimulating factor (CSF), such as CSF1 and CSF2, CD200, CX3CL1 , transforming growth factor p, such as TGF i , I DE1 , interferons, such as IFNy, and FLT3 ligand (FLT3). In a further embodiment, the culture media comprises one or more components selected from the group consisting of BMP4, FGF2, FLT3 CSF2, IL-4 and IFNy.

[0372] Activin A is also known as Activin beta-A chain, EDF, Erythroid differentiation protein, FRP, FSH-releasing protein, INHBA, Inhibin beta-A chain or Inhibin beta-1. The protein encoded by this gene is a member of the TGF family of proteins produced by pluripotent stem cells, endoderm, and mesoderm.

[0373] BMP4 is also known as ZYME, BMP2B or BMP2B1. The protein encoded by this gene is a member of the bone morphogenetic protein family, which is part of the TGFp superfamily.

[0374] VEGF is also known as VPF, VEGF or MVCD1. The protein encoded by this gene is a member of the PDGF / VEGF growth factor family and a heparin-binding protein. This growth factor induces proliferation and migration of vascular endothelial cells and is essential for both physiological and pathological angiogenesis.

[0375] SCF is also known as known Kit ligand, Mast cell growth factor or Steel factor. The protein encoded by this gene is an early-acting cytokine that plays a pivotal role in the regulation of embryonic and adult hematopoiesis. BIT-C-P3787PCT

[0376] 60

[0377] IL-3 is also known as MCGF (Mast cell growth factor), Multi-CSF, HCGF, P-cell stimulation factor, MGC79398 or MGC79399. The protein encoded by this gene is a growth promoting cytokine.

[0378] IL-6 is also known as B-Cell Stimulatory Factor 2, CTL Differentiation Factor, Hybridoma Growth Factor, Interferon Beta-2, IFN-Beta-2, IFNB2, BSF-2, GDF, Interferon, Beta 2, B-Cell Differentiation Factor, Interferon, Beta 2, Interleukin BSF-2, BSF2, HGF, or HSF. The protein encoded by this gene is a cytokine that functions in inflammation and the maturation of B cells.

[0379] IL-34 is also known as C16 orf77. The protein encoded by this gene is a cytokine that promotes the differentiation and viability of monocytes and macrophages through the colony-stimulating facto r-1 receptor.

[0380] CSF1 is also known as Macrophage Colony Stimulating Factor 1 , Macrophage Colony Stimulating Factor 1 , Lanimostim, MCSF, MCSF and the protein encoded by this gene is a cytokine that controls the production, differentiation, and function of macrophages.

[0381] CSF2 is also known as Sargramostim, Colony Stimulating Factor 2 (Granulocyte- Macrophage), Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF), Molgramostin and Molgramostim. The protein encoded by this gene is a cytokine that controls the production, differentiation, and function of granulocytes and macrophages.

[0382] CD200 is also known as OX-2 Membrane Glycoprotein, MOX1 , MOX2, OX-2 and MRC. The protein encoded by this gene is a type I membrane glycoprotein containing two extracellular immunoglobulin domains, a transmembrane and a cytoplasmic domain.

[0383] CX3CL1 is also known as C-X3-C Motif Chemokine Ligand 1 , Small Inducible Cytokine Subfamily D (Cys-X3-Cys), Member 1 (Fractalkine, Neurotactin), Chemokine (C-X3-C Motif) Ligand 1 , CX3C Membrane-Anchored Chemokine, Small-Inducible Cytokine D1 , C-X3-C Motif Chemokine 1 , Neurotactin, Fractalkine, or SCYDI , NTT, Small Inducible Cytokine Subfamily D (Cys-X3-Cys), Member-1 , C3Xkine, ABCD-3, CXC3C, CXC3, NTN or FKN. The protein encoded by this gene belongs to the CX3C subgroup of chemokines, characterized by the number of amino acids located between the conserved cysteine residues.

[0384] TGFpi is also known as Latency-Associated Peptide, IBDIMDE, DPD1 , CED and LAP. The protein encoded by this gene is a secreted ligand of the TGFp superfamily of proteins. BIT-C-P3787PCT

[0385] 61

[0386] A known advantage of forward programming over directed differentiation is that the desired lineage-restricted cell type can be obtained much more quickly with forward programming. The desired lineage restricted cells may be obtained using methods of the invention at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after culturing. In one embodiment, the method comprises culturing under suitable conditions for at least 3 days, such as at least 6 days or about 14 days. In further embodiments, method comprises culturing cells for a duration (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., from 5 days to 40 days, from 7 days to 35 days, from 14 days to 28 days, or about 21 days) which is sufficient to generate lineage restricted cells. In some embodiments, the cells are cultured for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 days). In one embodiment, the method comprises culturing the cells for at least about 5, 10, 15 or 20 days to produce lineage restricted cells. In one embodiment, the cells are cultured for a period of between 4 and 25 days, such as between 6 and 14 days.

[0387] After culturing, the cell population may comprise two cell types. For example, such a cell population may have two cell types including the stem cells and lineage-restricted cells. In one embodiment, the cell population comprises up to 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% (or any intermediate ranges) of lineage-restricted cells in the resulting cell population.

[0388] Culturing the cells may either help to induce cells to commit to a more mature phenotype, preferentially promote survival of the mature cells, or have a combination of both these effects.

[0389] According to a further aspect of the invention, there is provided a cell obtainable by any one of the methods defined herein.

[0390] According to a further aspect of the invention, there is provided a cell aggregate obtainable by any one of the methods defined herein.

[0391] As described herein, the exogenous expression cassettes encoding the one, two or three or more polypeptides having transcription factor activity and / or transcription factors may be BIT-C-P3787PCT

[0392] 62 integrated into the genome of the cell. In a further embodiment, exogenous expression cassettes encoding the polypeptides having transcription factor activity and / or the transcription factors are integrated into a site (preferably a targeted site) in the genome of the cell.

[0393] Cell compositions

[0394] According to a further aspect, there is provided a pharmaceutical composition comprising the lineage-restricted cells or cell aggregates produced by the method as described herein and a pharmaceutically acceptable carrier.

[0395] Pharmaceutical compositions may include lineage-restricted cells or cell aggregates as described herein in combination with one or more pharmaceutically or physiologically acceptable carrier, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminium hydroxide); and preservatives. Cryopreservation solutions which may be used in the pharmaceutical compositions of the invention include, for example, DMSO.

[0396] For purposes of manufacture, distribution, and use, the lineage-restricted cells described herein may be supplied in the form of the aforementioned cell aggregates, a cell culture or suspension in an isotonic excipient or culture medium, optionally frozen to facilitate transportation or storage.

[0397] Uses of the lineage-restricted cells

[0398] The cells and cell aggregates produced according to any of the methods of the invention have applications in basic and medical research, diagnostic and therapeutic methods. The cells and cell aggregates may be used in vitro to study cellular development, provide test systems for new drugs, enable screening methods to be developed, scrutinise therapeutic regimens, provide diagnostic tests and the like. These uses form part of the present invention. Alternatively, the cells and / or cell aggregates may be transplanted into a human or animal patient for diagnostic or therapeutic purposes. The use of the cells and cell aggregates in therapy is also included in the present invention.

[0399] According to one aspect of the invention, there is provided a lineage-restricted cell and / or lineage-restricted cell aggregates as defined herein, for use in in vitro diagnostics or drug screening. BIT-C-P3787PCT

[0400] 63

[0401] Lineage-restricted cells and equivalent cell aggregates generated by methods of the invention may find particular use in drug screening. Therefore, in one embodiment, the method additionally comprises contacting the lineage restricted cells and / or lineage-restricted cell aggregates with a test substance and observing a change (e.g., an effect) in the lineage restricted cells and / or aggregate induced by the test substance. The change or effect may be observed using methods known in the art, for example using pharmacological or toxicological assays. In one aspect, the cells and / or cell aggregates may be used in a method of assessing a test substance (e.g., a drug, such as a compound), comprising assaying a pharmacological or toxicological property of the test substance on the lineage-restricted cells or equivalent cell aggregates provided by the methods described herein. The method may comprise: a) contacting the lineage restricted cells and / or equivalent cell aggregates described herein with the test substance; and b) assaying an effect of the test substance on the lineage restricted cells and / or cell aggregates.

[0402] Assessment of the activity of a candidate molecule may involve combining the lineage restricted cells and / or cell aggregates described herein with the candidate molecule, determining any change in the morphology, phenotype, or metabolic activity of the lineage restricted cells and / or cell aggregates that is attributable to the molecule (i.e. , compared with a control, such as untreated cells or cells treated with an inert compound or equivalent cell aggregates), and then correlating the effect of the molecule with the observed change. The screening may be done either because the candidate molecule is designed to have a pharmacological effect on the lineage-restricted cells or cell aggregates, or because the molecule is designed to have effects elsewhere but there is a need to determine if it has any unintended side effects.

[0403] Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and leakage of enzymes into the culture medium. On a macroscopic level, with respect to a cell aggregate, effects of aggregate integrity or aggregate size may be determined. More detailed analysis may be conducted to determine whether a test substance affects cell function without causing toxicity.

[0404] Alternatively, the cells (including cell within aggregates) can be used to assess changes in gene expression patterns caused by a potential drug candidate. In this embodiment, the changes in gene expression pattern from addition of the candidate drug can be compared with BIT-C-P3787PCT

[0405] 64 the gene expression pattern caused by a control drug with a known effect on the lineage- restricted cells and / or cell aggregates.

[0406] Therefore, according to a further aspect, there is provided a method for drug screening (e.g., evaluating drug reactivity), comprising a step of using the lineage restricted cells and / or lineage restricted cell aggregates produced by the method as described herein. According to a further aspect of the invention, there is provided a method of drug screening comprising contacting a lineage restricted cell or lineage-restricted cell aggregate generated using the method as defined herein, or a lineage restricted cell or lineage-restricted cell aggregate as defined herein, with the drug and observing a change in the lineage restricted cell or cell aggregate induced by the drug.

[0407] According to a further aspect of the invention, there is provided the lineage restricted cell or lineage restricted cell aggregate as defined herein for use in therapy.

[0408] In one embodiment, the method additionally comprises transplanting the lineage restricted cells and / or lineage restricted cell aggregates into a patient. In this aspect of the invention, the cells used to generate the lineage restricted cells or lineage restricted cell aggregates may be autologous (i.e. , adult stem cells or mature cells removed, modified and returned to the same individual) or from a donor (i.e., allogeneic, including a stem cell line). Forward programming of cells into lineage restricted cells and / or lineage-restricted cell aggregates is amenable to the production of autologous and allogeneic lineage restricted cells and aggregates.

[0409] Therefore, 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 lineage restricted cells and / or lineage restricted cell aggregates generated using the method as defined herein, or lineage restricted cells and / or lineage restricted cell aggregates as defined herein.

[0410] In a different aspect, the lineage-restricted cells and / or lineage restricted cell aggregates produced according to any of the methods of the invention may be used in tissue engineering. Tissue engineering requires the generation of tissue which could be used to replace tissues or even whole organs of a human or animal. Methods of tissue engineering are known to those skilled in the art, but include the use of a scaffold (an extracellular matrix) upon which the cells or aggregates are applied in order to generate tissues / organs. These methods can be used to generate an “artificial” tissue or organ. Methods of generating tissues may include additive BIT-C-P3787PCT

[0411] 65 manufacturing, otherwise known as three-dimensional (3D) printing, which can involve directly printing cells to make tissues. The present invention thus provides a method for generating tissues using the cells or aggregates produced as described in any aspect of the invention.

[0412] In one aspect, there is provided a method of preparing a cell or tissue suitable for therapeutic or in vivo diagnostic purposes, comprising: (i) culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage- restricted in such culturing conditions; (ii) collecting a lineage-restricted cell or lineage restricted cell aggregate; and (iii) preparing a therapy or diagnostic comprising the lineage- restricted cell or lineage restricted cell aggregate.

[0413] For the drug screening, diagnostic and therapeutic uses set out above, it is clear that the lineage-restricted cells need to meet stringent requirements in terms of batch-to-batch reproducibility and cell viability. Cell viability typically needs to be maintained throughout the drug screening, diagnostic or therapeutic process. When the uses are applied to humans specifically it is also important that the cells are cultured in defined media in the absence of animal-derived constituents, such as fetal bovine serum. Excipients need to not only be compatible with the cells but they also need to not cause any adverse reactions in the patient if the cells are used therapeutically. The donor information of the cells also needs to be taken into account, as that in itself may affect how the cells behave in a drug screening, diagnostic and therapeutic context. All of these requirements add complexity to the culturing process compared with, for example, a culturing process for meat production.

[0414] Hepatocytes

[0415] Where the lineage-restricted cells are hepatocytes, the hepatocytes may be administered to an individual in a genetically manipulated form to produce a therapeutic molecule, for example a drug or growth factor (Behrstock S et al, Gene Ther 2006 Mar;13(5):379-88, Klein SM et al, Hum Gene Ther 2005 Apr;16(4):509-21).

[0416] A population of hepatocytes produced by the methods described herein may be used in methods of drug screening. In particular the hepatocytes may be used to assess any liver toxicity caused by drug candidates of interest.

[0417] A population of hepatocytes produced by the methods described herein may be used in methods of treatment of the human or animal body, for example the treatment of an individual with a liver disorder, liver injury and / or damaged or dysfunctional hepatic tissue. A population BIT-C-P3787PCT

[0418] 66 may also be used in the manufacture of a medicament for use in the treatment of a liver disorder, liver injury and / or damaged or dysfunctional hepatic tissue in an individual. A suitable individual may have an acute liver injury, for example drug induced liver injury; a chronic liver disease, such as hepatitis (e.g. hepatitis A, B, C, D, E, G or K), cirrhosis, hepatocellular carcinoma, non-alcoholic fatty liver disorder, alcoholic liver disorder, autoimmune liver disorder or an inherited metabolic disorder, such as Alpha 1 Antitrypsin deficiency, a Glycogen Storage Disease, for example Glycogen Storage Disease Type 1a, Familial Hypercholesterolemia, Hereditary Tyrosinaemia, Crigler Najjar syndrome, ornithine transcarbamylase deficiency, or factor IX deficiency or other haemophilia, haemochromatosis, Wilson's disease, Dubin- Johnson syndrome, familial amyloidosis, or Refsum’s disease. For therapeutic applications, the hepatocytes are preferably clinical grade hepatocytes.

[0419] Microglia, Glutamatergic neurons and GABAergic neurons

[0420] Where the lineage-restricted cells are microglia, glutamatergic neurons or GABAergic neurons these mature cells may be used directly for transplantation into a human or animal body to treat a disease. Alternatively, the mature cells may form a test material for research, including the effects of drugs on gene expression and the interaction of drugs with a particular gene. Additionally, it may enable the mature cells to be used to produce large quantities of desirable materials, such as growth factors or cytokines. Further, the present invention is also directed in one embodiment to the use of such a microglia, glutamatergic neuron or GABAergic neuron according to the present invention for in vitro diagnostics of a disease. Preferably, the disease is selected from the group consisting of diseases of the central nervous system, preferably neurodegenerative diseases; more preferably Alzheimer's disease, Parkinson's disease, frontotemporal dementia or Amyotrophic Lateral Sclerosis; neuroinflammatory or autoimmune diseases, preferably Multiple Sclerosis, auto-antibody-mediated encephalitis or infectious diseases, neurovascular diseases; preferably stroke, vasculitis; traumatic brain injury, and cancer.

[0421] Sensory neurons

[0422] Where the lineage-restricted cells are sensory neurons, these mature cells may be used directly for transplantation into a human or animal body to treat a disease. These may include peripheral neuropathies such as chronic or acute pain, nerve regeneration such as spinal cord repair, photoreceptor repair, otic sensory neuron repair or olfactory receptor neuron repair. Alternatively, the mature cells may form a test material for research, including the effects of drugs, such as analgesics, on gene expression and the interaction of drugs with a particular gene. Additionally, it may enable the mature cells to be used to produce large quantities of BIT-C-P3787PCT

[0423] 67 desirable materials, such as growth factors or cytokines. Further, the present invention is also directed in one embodiment to the use of such a sensory neuron according to the present invention for in vitro diagnostics of a disease as set out above.

[0424] Pancreatic beta cells

[0425] Where the lineage-restricted cells are pancreatic beta cells, these mature cells may be used directly for transplantation into a human or animal body to treat a disease. These may include diabetes (e.g., type 1 diabetes or type 2 diabetes), as well as the secondary disorders of pancreas dysfunction (e.g., obesity, hyperlipidemia, hypertension or cardiovascular disease). Alternatively, the mature cells may form a test material for research, including the effects of drugs on gene expression and the interaction of drugs with a particular gene. Additionally, it may enable the mature cells to be used to produce large quantities of desirable materials, such as growth factors or cytokines. Further, the present invention is also directed in one embodiment to the use of such a pancreatic beta cell according to the present invention for in vitro diagnostics of a disease as set out above.

[0426] CLAUSES

[0427] A set of clauses defining the invention and its preferred aspects is as follows:

[0428] Clause 1 . An ex vivo method for culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions.

[0429] Clause 2. The method according to clause 1 , wherein the forward programming comprises 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 the stem cells.

[0430] Clause 3. The method according to clause 1 or clause 2, wherein the stem cells are pluripotent stem cells, more preferably induced pluripotent stem cells.

[0431] Clause 4. The method according to any one of clauses 1 to 3, wherein the cells obtained from the method are lineage restricted specific stem cells, progenitor cells or mature cells, preferably mature cells.

[0432] Clause 5. The method according to any one of clauses 1 to 4, wherein the cells are mammalian cells, preferably human cells. BIT-C-P3787PCT

[0433] 68

[0434] Clause 6. The method according to any one of clauses 1 to 5, which additionally comprises monitoring the cell population for at least one characteristic of a cell that is lineage- restricted.

[0435] Clause 7. The method according to any one of clauses 1 to 6, wherein the lineage- restricted cells are selected from the list consisting of:

[0436] (i) nerve cells, preferably neural stem cells, peripheral nervous system neurons, migratory enteric neural crest cells, neural crest cells, enteric neurons, glutamatergic neurons, GABAergic neurons, sensory neurons, motor neurons, dopaminergic neurons, medium spiny neurons, cortical neurons, interneurons, brainstem neurons, cholinergic neurons, hippocampal neurons, projection neurons, Lewy body-containing neurons, pyramidal neurons, cerebellar neurons, serotonergic neurons, thalamic neurons, neuromuscular junction cells, ganglion cells, oligodendrocyte precursors, oligodendrocytes, astrocytes, Purkinje cells, Muller cells, Granule cells, photoreceptors, such as rod cells and cone cells, retinal ganglion cells, retinal precursor cells, retinal pigment epithelium and Bruch's membrane cells;

[0437] (ii) myocytes, preferably myoblasts, myogenic progenitors, skeletal myocytes, smooth muscle cells, satellite cells and cardiomyocytes;

[0438] (iii) osteocytes, preferably osteoclasts and osteoblasts;

[0439] (iv) chondrocytes;

[0440] (v) adipocytes, preferably preadipocytes, brown adipocytes, beige adipocytes, white adipocytes and epicardial adipocytes;

[0441] (vi) pericytes;

[0442] (vii) hepatocytes, preferably hepatic stellate cells;

[0443] (viii) kidney cells, preferably papillary tips cells, podocytes and mesangial cells;

[0444] (ix) respiratory cells, preferably airway epithelial cells, club cells and ciliated cells;

[0445] (x) megakaryocytes;

[0446] (xi) epithelial cells, preferably melanocytes and placental villous trophoblasts);

[0447] (xii) mesothelial cells, preferably epicardium;

[0448] (xiii) endothelial cells, preferably endocardial cells, keratinocytes and trabecular meshwork cells;

[0449] (xiv) secretary cells, preferably pancreatic beta cells, pancreatic alpha cells, pancreatic acinar cells, pancreatic ductal cells and chromaffin cells; BIT-C-P3787PCT

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[0451] (xv) gastrointestinal cells, preferably intestinal endocrine cell, intestinal epithelial cells, enteroendocrine cells, goblet cells, submucosal gland cells, Paneth cells and enterocytes;

[0452] (xvi) fibroblasts;

[0453] (xvii) myofibroblasts;

[0454] (xviii) mesodermal cells;

[0455] (xix) mesenchymal cells, preferably mesenchymal stem cells;

[0456] (xx) blood cells, preferably hematopoietic stem cells, erythrocytes, platelets, plasma cells and immune cells, such as neutrophils, glial cells, microglia, dendritic cells, T cells, such as CD4+ T helper cells, CD8+ cytotoxic T cells, regulatory T cells and gamma delta T cells, B cells, macrophages, Kupffer cells, innate lymphoid cells, eosinophils, mast cells, monocytes, Langerhans cells and natural killer cells.

[0457] Clause 8. The method according to clause 7, wherein the lineage-restricted cells are selected from the list consisting of hepatocytes, microglia, glutamatergic neurons, GABAergic neurons, sensory neurons and pancreatic beta cells, preferably hepatocytes, microglia and GABAergic neurons.

[0458] Clause 9. The method according to any one of clauses 1 to 8, which comprises selection or enrichment of the cells that are lineage-restricted, for example selecting cells which express a lineage-specific marker and / or removing cells which express pluripotency markers.

[0459] Clause 10. The method according to any one of clauses 1 to 9, wherein the expression of the transcription factors is increased by contacting the cell population with one or more exogenous expression 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.

[0460] Clause 11 . The method according to clause 10, wherein the expression of the transcription factors is increased by contacting the cell population with a single exogenous expression cassette encoding genes for all of the polypeptides having transcription factor activity and / or the transcription factors.

[0461] Clause 12. The method according to clause 10 or clause 11 , wherein expression of the genes is under controlled transcription. BIT-C-P3787PCT

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[0463] Clause 13. The method according to any one of clauses 1 to 12, which comprises culturing under suitable conditions for less than 40, 35, 30 or 25 days.

[0464] Clause 14. The method according to any one of clauses 1 to 13, wherein the aggregates are less than 1000, 900, 800, 700, 600 or 500 pm in diameter.

[0465] Clause 15. The method according to any one of clauses 1 to 14, wherein the aggregates are more than 30, 40, 50, 60, 70 or 80 pm in diameter.

[0466] Clause 16. The method according to any one of clauses 1 to 15, wherein the culturing takes place at an initial cell seeding density of between 50,000 cells / mL and 400,000 cells / mL, between 100,000 cells / mL and 350,000 cells / mL and between 125,000 cells / mL and 325,000 cells / mL.

[0467] Clause 17. The method according to any one of clauses 1 to 16, wherein the culture takes place in the absence of a substrate.

[0468] Clause 18. The method according to any one of clauses 1 to 17, wherein the culture takes place in a spinner flask, in a shaker flask or in a bioreactor, preferably in a bioreactor.

[0469] Clause 19. The method according to any one of clauses 1 to 18, wherein the culture takes place in a culture vessel and:

[0470] (i) where the culture vessel is a bioreactor, the tip speed of the impeller within the bioreactor is between 0.10 m / s and 0.40 m / s, between 0.12 m / s and 0.36 m / s, between 0.14 m / s and 0.32 m / s, between 0.16 m / s and 0.30 m / s or between 0.19 m / s and 0.28 m / s; and

[0471] (ii) where the culture vessel is not bioreactor, the tip speed of an impeller present within the culture vessel is between 0.05 m / s and 0.20 m / s, between 0.07 m / s and 0.18 m / s, between 0.09 m / s and 0.17 m / s or between 0.10 m / s and 0.16 m / s.

[0472] Clause 20. The method according to any one of clauses 1 to 19, wherein the culturing takes place in a culture vessel able to accommodate at least 10mL, 30 mL, 75 mL, 100 mL, 200 mL, 250 mL, 500 mL, 800 mL, 1 L, 2 L, 5 L or 10 L of culture media.

[0473] Clause 21 . The method according to any one of clauses 1 to 20, wherein the stem cells are present in the form of a single cell suspension before forward programming takes place. BIT-C-P3787PCT

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[0475] Clause 22. The method according to any one of clauses 1 to 21 , wherein a sequence encoding one or more polypeptides having transcription factor activity and / or the transcription factors is introduced into the stem cell using a method comprising:

[0476] - insertion, preferably targeted insertion, of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and

[0477] - insertion, preferably targeted insertion, of an inducible cassette into a second genomic safe harbour site of the stem cell, wherein said inducible cassette comprises said sequence, wherein the transcription of said sequence is regulated by the transcriptional regulator protein. Clause 23. An ex vivo method for culturing stem cells, comprising the steps of: a) insertion, preferably targeted insertion, of a gene encoding a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and b) insertion, preferably targeted insertion, of one or more nucleotide sequences encoding one or more polypeptides having the activity of one or more transcription factors and / or one or more transcription factors, into one or more second genomic safe harbour sites of the stem cell, wherein transcription of said one or more nucleotide sequences is regulated by the transcriptional regulator protein; and c) culturing the stem cells comprising the insertions in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions.

[0478] Clause 24. A method of preparing a cell or tissue suitable for therapeutic or in vivo diagnostic purposes, comprising: (i) culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions; (ii) collecting a lineage-restricted cell or lineage restricted cell aggregate; and (iii) preparing a therapy or diagnostic comprising the lineage-restricted cell or lineage-restricted cell aggregate.

[0479] Clause 25. A cell or an aggregate of cells made by the method of any one of clauses 1 to 23.

[0480] Clause 26. The cell or aggregate of cells according to clause 25, wherein nucleotide sequences encoding the one or more polypeptides and / or transcription factors are integrated into the genome of the cell(s). BIT-C-P3787PCT

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[0482] Clause 27. The cell or aggregate of cells according to clause 26, wherein nucleotide sequences encoding the one or more transcription factors are integrated into a target site of the cell(s).

[0483] Clause 28. A cell or aggregate of cells according to any one of clauses 25 to 27, for use in therapy, in vitro diagnostics or drug screening.

[0484] Clause 29. A method of drug screening comprising contacting the lineage-restricted cells or an aggregate of lineage-restricted cells generated using the method according to any one of clauses 1 to 23, or the cells or aggregate of cells according to any one of clauses 25 to 28, with a drug and observing a change in the cells induced by the drug.

[0485] Clause 30. 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 or aggregates of cells generated using the method according to any one of clauses 1 to 23, or the cells or aggregates of cells according to any one of clauses 25 to 28.

[0486] It will be understood that all embodiments described herein may be applied to all aspects of the invention.

[0487] Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples:

[0488] EXAMPLES

[0489] EXAMPLE 1 - Hepatocyte forward programming in aggregate culture conditions

[0490] Methods

[0491] Human induced pluripotent stem cells (hiPSCs) were engineered using an inducible OPTi-OX system as previously described (WO2018 / 096343). Briefly, two genetic safe harbour (GSH) sites were targeted. The hROSA26 locus was targeted with a constitutively expressed transactivator (rtTA), responsive to doxycycline, and the AAVS1 locus was targeted with the transgenes for HNF1A, HNF6, FOXA3 and RORc under a TET-responsive element (TRE). BIT-C-P3787PCT

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[0493] The hiPSCs were detached from 2D culture using TrypLE (Gibco 12093745) and seeded into 30 mL ABLE Biott vessels at 300,000 cells / mL for 3 days or 150,000 cells / mL for 4 days. The hiPSC were cultured in Stemscale (Gibco A4965001) + 10 mM ROCKi (Tocris TB1254), Nutri- Stem or Stem Fit media. ABLE Biotts were incubated at 37 °C with agitation from a magnetic delta-wing shaped impeller (32 mm in diameter) set to between 70 and 90 rpm (with a tip speed of between 0.117 and 0.151 m / s). 50% or 80% media changes were performed every 24 hours and cells were imaged using a light microscope. On day -1 iPSCs were passaged for induction into new 30 mL, 100 mL and 500 mL ABLE Biott vessels by first dissociating into single cell suspension with TrypLE and reseeding into Stemscale + ROCKi at 150,000 - 450,000 cells / mL. 50 or 80% media changes occur every 24 to 48 hours following the schedule listed in table 1 below. Table 1 BIT-C-P3787PCT

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[0495] E6 + DOX- CTS Essential 6 medium (Gibco A4238501) + 1 mg / mL Doxcycline (Sigma D9891) + 1% Poloxamer 188.

[0496] HPZc + OHD - Hepatozyme SFM (Gibco 17705-021) + 2% Chemically defined lipid concentrate (Gibco 11905-031) + 2% MEM Non-essential amino acid solution (Gibco 11140- 050) + 1% L-Glutamine (Gibco 25030-081) + 14 mg / mL insulin solution (Lonza BE02-033E20) + 30 mg / mL Human Apo-transferrin (R&D Systems 3188-AT) + 20 ng / mL Oncostatin M (ProteinTech HZ-1030) + 50 ng / mL Human Growth Factor (ProteinTech HZ-1084) + 1 mg / mL Doxcycline + 1 % Poloxamer 188.

[0497] HLI+OH - Hepatozyme SFM + 2% Chemically defined lipid concentrate + 2% MEM Non- essential amino acid solution + 1% L-Glutamine + 2 mg / mL insulin solution + 30 mg / mL Human Apo-transferrin + 20 ng / mL Oncostatin M + 50ng / mL Human Growth Factor 1% Poloxamer 188.

[0498] During the culture, aggregates were sampled for light microscope imaging. After imaging a sample of media was taken and analysed for glucose and lactate levels (data shown in Figures 1f and 1g).

[0499] At timepoints during the culture cells were harvested, the aggregates were allowed to settle, supernatant removed and aggregates washed with the addition of PBS (Gibco A1287301). The aggregates were allowed to settle again, PBS removed and TrypLE added to dissociate the aggregates to form a single cell suspension, these were the analysed for cell count and viability. A sample of the cells were then plated and an Albumin ELISA performed using an abeam ELISA kit (ab179887). Additionally RNA was produced using Qiagen RNeasy kit (Qiagen 74104), cDNA synthesised using Maxima First strand cDNA kit (Thermo Scientific BIT-C-P3787PCT

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[0501] K1672) and qPCR performed using Taqman assay mixes and TaqMan fast advanced master mix (Thermo Scientific 4444557).

[0502] Results hiPSCs form aggregates of between 100 pm and 400 pm in size. The cells proliferate with these aggregates (Figure 1a). Despite the lack of a sodium bicarbonate buffer leading to a decrease in pH during culturing (Figure 1e), the cells were seen to consume glucose (Figure 1f), release lactate (Figure 1g) and remain viable (Figure 1d) regardless of whether the buffer was present in a 30 mL ABLE Biott system. Impressive levels of cell viability were seen even when the system was scaled up to 100 mL and 500 mL ABLE Biott vessels (Figure 1 h). Statistically insignificant changes in cell viability were seen across 30 mL, 100 mL and 500 mL ABLE Biott sizes.

[0503] Mature hepatocytes were successfully generated through forward programming (inducing the cassette comprising the transcription factors HNF1A, HNF6, FOXA3 and RORc using doxycycline) in a 3D aggregate culture. Viable hepatocyte aggregates were observed after culturing at initial seeding densities of both 150,000 cell / mL and 300,000 cells / mL, at impeller speeds of both 80 (tip speed 0.134 m / s) and 90 rpm (tip speed 0.151 m / s), and at media exchanges of both 50% and 80% every 24 hours (Figure 4). All culture conditions set out in Figure 4 resulted in expression of CYP3a4, CYP3a7, AFP, albumin, Asgrl and Serbianl (all markers for hepatocytes). An initial seeding density of 150,000 cells / ml was found to be preferable for albumin expression. A impeller speed of 90 rpm (tip speed 0.151 m / s) was found to be preferably for Asgrl expression. A seeding density of 150,000 cells / mL, a media exchange of 50% and an impeller speed of 80 rpm (tip speed 0.134 m / s) was preferable for CYP3a4 expression. A seeding density of 300,000 cells / mL, a media exchange of 50% and an impeller speed of 80 rpm (tip speed 0.134 m / s) was preferable for CYP3a7 expression. A seeding density of 300,000 cells / mL, a media exchange of 80% and an impeller speed of 80 rpm (tip speed 0.134 m / s) was particularly preferable for albumin. A seeding density of 150,000 cells / mL, a media exchange of 50% and an impeller speed of 90 rpm (tip speed 0.151 m / s) was preferable for AFP, ASGR1 and SERPINA1 expression. These hepatocytes were able to secrete albumin at a level comparable with hepatocytes that were forward-programmed in a 2D environment (Figure 3).

[0504] Table 2 below shows gene expression levels, determined using qPCR, of various markers after 20 days of hepatocyte forward programming in a 3D aggregate system. Expression levels are shown relative to the housekeeping gene ACTB. In terms of culture conditions, (1) represents BIT-C-P3787PCT

[0505] 76 a seeding density of 150,000 cells / mL and a temperature increase from 37 °C to 39 °C at day 15, (2) represents a seeding density of 300,000 cells / mL and a temperature increase from 37 °C to 39 °C at day 15, (3) represents a seeding density of 450,000 cells / mL and a temperature increase from 37 °C to 39 °C at day 15, (4) represents a seeding density of 150,000 cells / mL, (5) represents a seeding density of 450,000 cells / mL, (6) represents pooled plated donor primary hepatocytes, (7) represents undifferentiated induced pluripotent stem cells, (8) represents reverse transcriptase negative control and (9) represents no template control (negative assay control) NTC. Table 2

[0506] Table 2 shows that the resultant hepatocytes had no expression of Ki67 (a clinically important proliferation marker for grading multiple types of cancers), undesired marker KRT7 and VIM ENTIN or pluripotency markers NANOG and POLI5F1. The hepatocyte showed increased expression of the hepatocyte-specific markers SERPINA, albumin and ASGR1. BIT-C-P3787PCT

[0507] EXAMPLE 2 - Hepatocyte forward programming in bioreactor aggregate culture conditions

[0508] Methods

[0509] Human induced pluripotent stem cells (hiPSCs) were engineered as described in Example 1. The hiPSCs were detached from 2D culture using TrypLE (Gibco 12093745) and seeded into 30 mL ABLE Biott vessels at 300,000 cells / mL for 3 days. The hiPSCs were cultured in Stemscale media (Gibco A4965001) + 10 M ROCKi (Tocris TB1254). ABLE Biotts were incubated at 37 °C with agitation from a magnetic delta-wing shaped impeller (32 mm in diameter) set to 80 rpm (tip speed of 0.134 m / s). A 50% media change was performed every 24 hours and cells were imaged using a Evos Flight microscope.

[0510] On day -1 hiPSCs were passaged for induction into 250 mL Ambr vessels (Sartorius 001- 2A33) by first dissociating into single cell suspension with TrypLE and reseeding into Stemscale + ROCKi at 150,000 cells / mL. 50 or 80% media changes occur every 24 to 48 hours following the schedule listed in table 3 below. 8 x 200 mL Ambr vessels were set up, the conditions of all are listed in table 4.

[0511] Table 3 BIT-C-P3787PCT

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[0513] E6 + DOX- CTS Essential 6 medium (Gibco A4238501) + 1 .g / mL Doxcycline (Sigma D9891) + 1% Poloxamer 188. HPZc + OHD - Hepatozyme SFM (Gibco 17705-021) + 2% Chemically defined lipid concentrate (Gibco 11905-031) + 2% MEM Non-essential amino acid solution (Gibco 11140- 050) + 1% L-Glutamine (Gibco 25030-081) + 14 .g / mL insulin solution (Lonza BE02-033E20) + 30 .g / mL Human Apo-transferrin (R&D Systems 3188-AT) + 20 ng / mL Oncostatin M (ProteinTech HZ-1030) + 50 ng / mL Human Growth Factor (ProteinTech HZ-1084) + 1 .g / mL Doxcycline + 1 % Poloxamer 188.

[0514] HLI+OH - Hepatozyme SFM + 2% Chemically defined lipid concentrate + 2% MEM Non- essential amino acid solution + 1 % L-Glutamine + 2 .g / mL insulin solution + 30 .g / mL Human Apo-transferrin + 20 ng / mL Oncostatin M + 50 ng / mL Human Growth Factor 1 % Poloxamer 188.

[0515] Table 4 BIT-C-P3787PCT

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[0517] The 200 mL Ambr bioreactors had an impeller 30 mm in diameter. At 125 rpm the tip speed was 0.196 m / s. At 175 rpm the tip speed was 0.275 m / s. The tip speed in the ABLE Biott vessel at an rpm of 80 was 0.134 m / s. Note that the impeller speed in the control (ABLE biott) was reduced to 80 rpm due to the much larger impeller generally present in spinner culture vessels such as the ABLE Biott compared with impellers typically used in bioreactors such as the Ambr.

[0518] Results hiPSCs formed viable aggregates in both set ups where gas control is provided via overlay and where gas control is provided with sparging (Figures 5 and 6). Viable aggregates formed at a variety of bioreactor impeller speeds (125 rpm (0.196 m / s tip speed) and 150 rpm (0.236 m / s tip speed)) and regardless of whether the media was changed after 24 hours post-seed (Figure 7). Albumin secretion after bioreactor culture was either comparable with the ABLE Biott culture or improved in a variety of culture conditions as set out in table 4 (figure 8). Differences in albumin secretion comparing any of the repeats (i.e. unit 5 compared against unit 1 , unit 6 compared against unit 2, unit 7 compared against unit 3 and unit 8 compared against unit 4) were statistically insignificant, indicating that the culture procedures arrived at repeatable results.

[0519] EXAMPLE 3 - Microglia forward programming in aggregate culture conditions

[0520] Methods

[0521] Human induced pluripotent stem cells (hiPSCs) were engineered using an inducible OPTi-OX system as previously described (WO2018 / 096343). Briefly, two genetic safe harbour (GSH) sites were targeted. The hROSA26 locus was targeted with a constitutively expressed transactivator (rtTA), responsive to doxycycline, and the AAVS1 locus was targeted with the transgenes for SPI1 and CEBPp under a TET-responsive element (TRE).

[0522] The hiPSCs were seeded into either 30 mL or 100 mL ABLE Biott vessels at 150,000 cells / mL. The hiPSC were cultured in Stemscale (Gibco A4965001) + 10 mM ROCKi (Tocris TB1254) or DMEM / F12 media. ABLE Biotts were incubated at 37 °C with agitation from a magnetic delta-wing shaped impeller set to 80 rpm (0.134 m / s tip speed) (30 mL vessel) or 56 rpm (0.135 BIT-C-P3787PCT

[0523] 80 m / s tip speed) (100 mL vessel). Doxycycline was added at day 0. Media exchange was carried out at 50% every 24 hours during expansion. Cells were imaged using a light microscope.

[0524] Optical genome mapping

[0525] DNA of high molecular weight is isolated from cell samples and fluorescently labelled using a DNA dye specific for a 6 base pair motif. Labelled DNA is cleaned of excess dye and DNA backbone is counter stained before loading into flowcells. DNA molecules are linearized in nanochannel arrays on the chip and images. Changes in label spaces and / or orientation when compared to a reference allows detection of all classes of structural variants.

[0526] Phagocytosis

[0527] Cells cultured for 10 days are incubated with 1 pg / 0.33 cm2pHrodo RED labelled E. coli particles for 24 hours + / - cytochalasin D control. Images are acquired every 30 minutes on the Incucyte live imaging platform looking at the red fluorescence and phase contrast. An in house pipeline is then used to assess the images and plot the proportion of cells phagocytosing E. coli particles over the 24 hour period.

[0528] Results

[0529] Culture of the hiPSCs was successfully achieved in the 3D aggregate culture, with more than 95% cell viability and up to 200,000,000 cells cultured in the 100 mL ABLE Biott vessels.

[0530] Optical genome mapping revealed no detected insertions, deletions, inversions, duplications, intra-fusions, inter-translocations, absence-of-heterozygosity regions, loss-of-heterozygosity regions, copy number variation gain segments, copy number variation loss segments, aneuploidy gains or aneuploidy losses compared with 2D culture.

[0531] Table 5 below shows an impressively high percentage retention of pluripotency markers in the hiPSCs after either five passages (30 mL ABLE Biott vessels) or two passages (100 mL ABLE Biott vessels), determined through flow cytometry.

[0532] Table 5 BIT-C-P3787PCT

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[0534] The cells also showed a clear overexpression of the microglia forward programming transcription factors SPI1 and CEBPp following exposure to doxycycline. This resulted in the generation of microglia with typical morphology (Figure 9d) and that stain positive for P2RY12 (a microglia-specific marker) and IBA1 (a marker specific to microglia and macrophages) (Figure 10). Flow cytometry analysis revealed that 0.1% of the microglia generated were positive for the pluripotency marker TRA-1-60, 89% were positive for CD45, 71 % were positive for TMEM119, 80% were positive for P2RY12, 90% were positive for CD11b and 86% were positive for CD14 (CD45, TMEM119, P2RY12, CD11 b and CD14 are all markers for microglia). The majority of microglia generated were also able to carry out phagocytosis at a level comparable with microglia generated using 2D methods, showing that the generated microglia were functional. Microglia yields were noticeably increased in 3D aggregate culture conditions compared with equivalent 2D conditions (Figure 11).

[0535] Forward programming of hiPSCs in a 3D aggregate culture was also successfully achieved into GABAergic neurons, glutaminergic neurons and sensory neurons.

[0536] EXAMPLE 4- GABAergic neuron forward programming in aggregate culture conditions

[0537] Methods

[0538] Cell Engineering and Culture Preparation

[0539] Human induced pluripotent stem cells (hiPSCs) were engineered using an inducible dualharbour system as previously described (WO2018 / 096343) that involves the overexpression of the transcription factors ASCL1 and DLX2. This was achieved by targeting the hROSA26 locus with a constitutively expressed transcriptional activator (rtTA) (activable using doxycycline) and the AAVS1 locus with the ASCL1 and DLX2 transgenes under a TET- responsive element (TRE).

[0540] Induction and Aggregation

[0541] The differentiation process is conducted over a period of 12 days, following a specific media change and passage schedule. The protocol begins on Day 1 with an iPSC thaw and seeding in Stemscale media (Gibco A4965001) + 10 pM ROCKi (Tocris TB1254) in a 30 mL Biott BIT-C-P3787PCT

[0542] 82 vessel. A rotational speed of 80 RPM (tip speed 0.134 m / s) is used throughout the entire process. A 50% media change is performed on Day 2 using Stemscale media. On Day 3 and Day 4, a 50% media change and passage is done using Stemscale + ROCKi media. From Day 5 to Day 7, a 50% media change is performed daily using Stemscale media. The induction passage occurs on Day 8 using Stemscale media. From Day 9 to Day 11 , the media change is increased to 80% using GABA Induction media. On Day 12, the cells are frozen using CTS- KOSR + 10% dimethyl sulfoxide (DMSO).

[0543] The GABA Induction Media is composed of the following: 96.5% (v / v) DMEM / F-12, 1 % (v / v) non-essential amino acids 100x, 1 % (v / v) N-2 supplement 100x, 1% (v / v) pluronic acid, and 0.5% (v / v) doxycycline.

[0544] Characterization and Analysis

[0545] Cell morphology and aggregate formation were monitored through brightfield and Incucyte live imaging. Metabolite profiles, including glucose, pH, and lactate levels, were measured throughout the process, showing a metabolic shift from an iPSC-like state to a less metabolically active neuronal aggregate. The total cell yield and viability were assessed before and after freezing.

[0546] Results

[0547] Pre-freeze images (Figure 12) show the transition from small, loosely packed iPSC aggregates (Day -4) to larger, more compact neuronal clusters (Day 0). Post-freeze images (Figure 12) show cells that were cryopreserved as single cells, thawed, and then placed down on Day 1. These single cells further matured into a 2D monolayer of developed GABAergic neurons by Day 12.

[0548] A general trend of increasing cell yield with higher induction seeding densities was observed (Figure 13a), accompanied by a small reduction in pre-freeze viability (Figure 13b).

[0549] A consistent drop in viability following freeze-thaw was seen across all conditions, as expected (Figure 14). Notably, the decreased in viability in 3D cultures was generally smaller than that observed in the 2D WCB (working cell bank) control, suggesting improved cryo-resilience in the 3D system. BIT-C-P3787PCT

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[0551] The successful conversion to GABAergic neurons was confirmed by gene expression analysis at Day 3 and Day 12 post-thaw. As shown in Figure 15, the pluripotency markers POLI5F1 and NANOG were highly expressed in iPSCs but were rapidly downregulated upon differentiation. In contrast, the pan-neuronal marker TLIBB3, the early GABAergic lineage transcription factors ASCL1 , DLX1 , and DLX2, and the GABAergic markers GAD1 , GAD2, and VGAT all showed increased expression over time, confirming neuronal and GABAergic specification.

[0552] Enhanced neurite branching in 3D cultures in a seeding density-dependent manner was seen (Figures 16 and 17). Distinct morphological differences were observed between 3D-derived and 2D neuron cultures (Figure 16).

Claims

BIT-C-P3787PCT84CLAIMS1 . An ex vivo method for culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions.

2. The method according to claim 1 , wherein the forward programming comprises 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 the stem cells.

3. The method according to claim 1 or claim 2, wherein the stem cells are pluripotent stem cells, more preferably induced pluripotent stem cells.

4. The method according to any one of claims 1 to 3, wherein the cells are mammalian cells, preferably human cells.

5. The method according to any one of claims 1 to 4, wherein the lineage-restricted cells are selected from the list consisting of hepatocytes, microglia, glutamatergic neurons, GABAergic neurons, sensory neurons and pancreatic beta cells, preferably hepatocytes, microglia and GABAergic neurons.

6. The method according to any one of claims 1 to 5, wherein the expression of the transcription factors is increased by contacting the cell population with one or more exogenous expression 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.

7. The method according to claim 6, wherein the expression of the transcription factors is increased by contacting the cell population with a single exogenous expression cassette encoding genes for all of the polypeptides having transcription factor activity and / or the transcription factors.

8. The method according to any one of claims 1 to 7, which comprises culturing under suitable conditions for less than 40, 35, 30 or 25 days.BIT-C-P3787PCT859. The method according to any one of claims 1 to 8, wherein the aggregates are less than 1000, 900, 800, 700, 600 or 500 pm in diameter and / or more than 30, 40, 50, 60, 70 or 80 pm in diameter.

10. The method according to any one of claims 1 to 9, wherein the culturing takes place at an initial cell seeding density of between 50,000 cells / mL and 400,000 cells / mL, between 100,000 cells / mL and 350,000 cells / mL and between 125,000 cells / mL and 325,000 cells / mL.11 . The method according to any one of claims 1 to 10, wherein the culture takes place in a culture vessel and:(i) where the culture vessel is a bioreactor, the tip speed of the impeller within the bioreactor is between 0.10 m / s and 0.40 m / s, between 0.12 m / s and 0.36 m / s, between 0.14 m / s and 0.32 m / s, between 0.16 m / s and 0.30 m / s or between 0.19 m / s and 0.28 m / s; and(ii) where the culture vessel is not bioreactor, the tip speed of an impeller present within the culture vessel is between 0.05 m / s and 0.20 m / s, between 0.07 m / s and 0.18 m / s, between 0.09 m / s and 0.17 m / s or between 0.10 m / s and 0.16 m / s.

12. The method according to any one of claims 1 to 11 , wherein a sequence encoding one or more polypeptides having transcription factor activity and / or the transcription factors is introduced into the stem cell using a method comprising:- insertion, preferably targeted insertion, of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of the stem cell; and- insertion, preferably targeted insertion, of an inducible cassette into a second genomic safe harbour site of the stem cell, wherein said inducible cassette comprises said sequence, wherein the transcription of said sequence is regulated by the transcriptional regulator protein.

13. The method according to any one of claims 1 to 12, wherein the stem cells are present in the form of a single cell suspension before forward programming takes place.

14. A method of preparing a cell or tissue suitable for therapeutic or in vivo diagnostic purposes, comprising: (i) culturing stem cells in conditions that allow for aggregates to form, wherein the cells undergo forward programming into cells that are lineage-restricted in such culturing conditions; (ii) collecting a lineage-restricted cell or lineage restricted cell aggregate; and (iii) preparing a therapy or diagnostic comprising the lineage-restricted cell or lineage- restricted cell aggregate.BIT-C-P3787PCT8615. A cell or an aggregate of cells made by the method of any one of claims 1 to 13.

16. A cell or aggregate of cells according to any one of claim 15, for use in therapy, in vitro diagnostics or drug screening.

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