Method of generating haematopoietic cells

Abstract

The present invention provides an in vitro method of generating a haematopoietic cell, the method comprising: a) Providing a cell genetically modified to comprise a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tal1) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors; and b) Culturing the genetically modified cell in a differentiation medium which does not comprise the inducer, such that the expression level of the exogenous transcription factors in the cell is reduced to a level whereby the cell differentiates into a haematopoietic cell. Also provided are genetically modified haematopoietic cells, genetically modified cells and therapeutic uses thereof.

Description

[0001] Method of generating haematopoietic cells

[0002] Field of the Invention

[0003] The present invention relates to in vitro methods of generating haematopoietic cells. Also provided are genetically modified haematopoietic cells, genetically modified cells and therapeutic uses thereof.

[0004] Background

[0005] Haematopoiesis is the production of all the cellular components of blood from haematopoietic stem and progenitor cells (HSPCs). There are various stages of haematopoiesis. Developmental haematopoiesis is the generation of the blood-forming system in a developing embryo. In early development, a blastocyst emerges containing epiblast cells, which develop into the three germ layers endoderm, ectoderm and mesoderm. Mesoderm is the germ layer that forms the haematopoietic system. After the emergence of blastocyst, intra-embryonic and extra-embryonic regions are formed and establish the embryo and yolk sac structures. Mesoderm derived cells are found at various anatomical sites and times in development including the extra-embryonic yolk sac, and the embryo proper. The earliest blood is formed within yolk sac blood islands where mesoderm derived haemogenic angioblasts give rise to blood cells. Later within the developing vasculature, haemogenic endothelium (HE) cells emerge, which are rare and transient endothelial cells with haematopoietic potential that undergo endothelial-to-haematopoietic transition. There are different spatiotemporal instances of blood generation from haemogenic angioblasts and HE and their emergence and developmental potential define at least three distinct waves of haematopoiesis. Broadly, the three waves of developmental haematopoiesis are divided into the primitive first wave, definitive second, both of which occurring sequentially within the extra-embryonic yolk sac, and then the definitive third wave occurring within the aorta-gonad-mesonephros (AGM) of the embryo. A summary of the three waves of developmental haematopoiesis is shown in Figure 1 .

[0006] In mice, primitive haematopoiesis is first detectable at embryo day 7.25 (E7.25) as blood islands within the yolk sac vasculature. Blood islands were found to develop from a cell mass termed “haemangioblast”, that develops into both haematopoietic cells and endothelial cells (Murray, 1932). The idea that the haemangioblast was actually a common progenitor cell was later proposed and demonstrated through in vitro differentiation of embryonic stem (ES) cells. The in vitro differentiation of ES cells into embryoid bodies (EBs) encourages differentiation to the three germ layers including mesoderm, which develops into haematopoietic cells.

[0007] Definitive haematopoiesis encompasses the second and third waves of haematopoiesis. Both waves occur through emergence of a haemogenic endothelium (HE) which is an endothelial cell that undergoes endothelial-to-haematopoietic transition (EHT) (Lancrin et al., 2009). HE is a rare and transient cell found only in early development of the haematopoietic system. During the beginning of definitive haematopoiesis (E8.25 in mice) erythro-myeloid progenitors (EMPs) emerge from the yolk sac which include burst forming unit erythroid cells (BFU-Es), and myeloid progenitors including neutrophils and mast cells (reviewed in Frame et al., 2013). This yolk sac wave of definitive haematopoiesis also includes lymphoid progenitors, including T-cells and innate type B-1 B cells, generated from lymphoid primed multi-potential progenitor cells (LMPPs) around E9.5, before the emergence of haematopoietic stem cells (HSCs) (Yoshimoto et al., 2011 , 2012). Primitive and definitive erythroid cells produce different forms of haemoglobin (Brotherton et al., 1979). Detection of embryonic (pH1) and adult (pmaj) haemoglobin can be used to distinguish between primitive and definitive haematopoiesis respectively (Noordermeer and De Laat, 2008) as shown in Figure 2. Although early primitive erythroid cells are nucleated cells this distinction cannot be used to distinguish primitive erythroid cells as they eventually do enucleate (Kingsley et al., 2004).

[0008] The third wave of haematopoiesis generates haematopoietic stem and progenitor cells (HSPCs) which include bone marrow stem cells that can re-populate the blood system of an adult. HSCs emerge from haemogenic endothelium on the wall of the dorsal aorta in the AGM through endothelial-to- haematopoietic transition (EHT) forming intra-aortic haematopoietic clusters (lAHCs) (Muller et al., 1994). HSCs can also be generated from the vitelline and umbilical arteries from E10.5 onward (de Bruijn, 2000). After haematopoietic cells are formed, they migrate to the foetal liver where they proliferate extensively (Medvinsky et al., 1993), then later to the bone marrow where they establish the bone marrow HSC population (Moore and Metcalf, 1970). HSCs can generate both lymphoid and myeloid cells through differentiation and continue to do this throughout the life of an adult.

[0009] HE are the precursor to haematopoietic cells and so are the gateway to haematopoiesis and the production of blood for therapeutic applications. HE is, however, a rare and transient population that has diversity in its origins and developmental potential. HE can be produced by differentiating ES cells or IPSCs through differentiation involving the formation of embryoid bodies. The isolation of Fl k1 + cells from these embryoid bodies enriches for cells that can become HE. The frequency of HE in these cultures is low, making generating HE a bottleneck in studying EHT and producing blood for therapeutic use.

[0010] Due to the limited expansion potential of patient derived blood cells, there is a need to establish scalable platforms to manufacture therapeutically relevant cells for clinical applications. Platforms to generate therapeutically relevant cells have emerged led by companies such as Fate Therapeutics, T-CiRA, and Notch Therapeutics, but the generation of haematopoietic cells in these processes is still a bottleneck. Improving the understanding of how haematopoietic cells are formed, and increasing the efficiency in which they are generated could improve the ability to generate blood cells for therapeutic use.

[0011] Generating mature blood cells and haematopoietic stem cells (HSCs) have been long-standing goals of stem cell biology, developmental biology, and bioengineering due to their clinical opportunities in bone marrow transplantation and cellular immunotherapy. Pluripotent embryonic stem (ES) cells or induced pluripotent stem cells (IPSCs) offer promising starting cell sources to achieve these goals. However, controlled differentiation can be inefficient, and embryonic stem cells require extensive maturation to produce these target cells. Previous work has explored the possibility of establishing HE cell lines as a strategy to generate haematopoietic cells, but the obtained cell lines have been restricted to primitive haematopoiesis with limited cellular progeny (Vereide et al., 2014).

[0012] The present invention has been devised in light of the above considerations. Summary of the Invention

[0013] The present inventors have established a haemogenic endothelium (HE) cell line through ectopic expression of a unique combination of transcription factors. Advantageously, this HE cell line expands extensively and can undergo definitive haematopoiesis when released from ectopic gene expression. The inventors have found that the HE cell line can differentiate into multi-lineage colony-forming cells and mature haematopoietic cells. This obviates the need for extensive differentiation stages from pluripotent stem cells, which can be time-consuming and costly, and instead provides an inexhaustible supply of HE to generate haematopoietic cells.

[0014] According to a first aspect, the present invention provides an in vitro method of generating a haematopoietic cell, the method comprising: a) Providing a cell genetically modified to comprise a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors; b) Culturing the genetically modified cell in a differentiation medium which does not comprise the inducer, such that the expression level of the exogenous transcription factors in the cell is reduced to a level whereby the cell differentiates into a haematopoietic cell.

[0015] In some embodiments, the exogenous transcription factors further comprise a Forkhead box (FOX) family transcription factor. In some embodiments, the FOX family transcription factor comprises Foxc2. In some embodiments, the exogenous transcription factors further comprise MYC. The MYC may comprise c-MYC or n-MYC, preferably c-MYC.

[0016] In some embodiments, the ETS family transcription factor comprises Ets1 or ETV2, preferably ETV2.

[0017] In some embodiments, the GATA family transcription factor comprises GATA1 or GATA2, preferably GATA2.

[0018] In some embodiments, the exogenous transcription factors further comprise LIM-only protein 2 (LMO2).

[0019] In some embodiments, the exogenous transcription factors further comprise a Sry-box (SOX) family transcription factor. The SOX family transcription factor may comprise Sox17 and / or Sox18. In some embodiments, the SOX family transcription factor comprises Sox17.

[0020] The exogenous transcription factors may further comprise Friend leukaemia integration 1 transcription factor (Fli-1) .

[0021] In some embodiments, in step b) the expression level of the exogenous transcription factors in the cell is reduced to an undetectable expression level. In some embodiments, the haematopoietic cell comprises a haematopoietic stem and progenitor cell (HSPC) and / or a blood cell.

[0022] In some embodiments, the inducer comprises Doxycycline.

[0023] Step a) may comprise: i) Providing the genetically modified cell comprising the nucleotide sequence(s) encoding the exogenous transcription factors; and ii) Culturing the genetically modified cell in an inducer medium comprising the inducer to induce a detectable expression level of the exogenous transcription factors in the cell.

[0024] In some embodiments, the cell in step i) comprises a differentiated cell genetically modified to comprise the nucleotide sequence(s). In some embodiments, the cell in step i) comprises a haematopoietic cell previously obtained according to the method of the first aspect.

[0025] In some embodiments, step i) comprises genetically modifying the cell to introduce the nucleotide sequence(s) encoding the exogenous transcription factors into the cell.

[0026] The differentiation medium may comprise a TGF-p inhibitor. Optionally, the TGF-p inhibitor comprises SB431542.

[0027] In some embodiments, the differentiation medium comprises a Wnt activator. Optionally, the Wnt activator comprises CHIR99021 .

[0028] The differentiation medium may comprise bone morphogenetic protein 4 (BMP4). The present inventors have advantageously found that the inclusion of BMP4 in the differentiation medium further increases blood cell output.

[0029] In some embodiments, the differentiation medium comprises a PI3K inhibitor, optionally wherein the PI3K inhibitor comprises LY2940002.

[0030] The cell may be a murine or a human cell. Preferably, the cell is a human cell.

[0031] According to a second aspect, the present invention provides a haematopoietic cell obtainable by the method of the first aspect.

[0032] According to a further aspect, the present invention provides a genetically modified haematopoietic cell comprising a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer.

[0033] In some embodiments, the genetically modified haematopoietic cell comprises no more than a low expression level of the exogenous transcription factors. In some embodiments, the genetically modified haematopoietic cell comprises a haematopoietic stem and progenitor cell (HSPC) and / or a blood cell. Optionally, the genetically modified haematopoietic cell comprises a blood cell. Optionally, the blood cell comprises a myeloid cell and / or a lymphoid cell.

[0034] In some embodiments, the genetically modified haematopoietic cell comprises a macrophage, dendritic cell (DC), granulocyte, T-cell, NK cell, B-cell or erythrocyte.

[0035] In some embodiments, the genetically modified haematopoietic cell further comprises a nucleotide sequence encoding a chimeric receptor or a T-cell receptor (TCR), optionally a chimeric antigen receptor.

[0036] According to another aspect, the present invention provides the genetically modified haematopoietic cell of the above aspect for use in the treatment or prevention of a disease. In some embodiments, the disease comprises cancer or autoimmune disease.

[0037] The present invention also provides a genetically modified cell comprising a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors. Advantageously, such genetically modified cells form a stable HE-like cell line in the presence of the inducer, in contrast to naturally occurring HE cells which are typically rare and transient. The stable nature of the genetically modified cells of the invention makes them particularly amenable to further genetic modification, for example, to express specific chimeric receptors or TCRs, or to have genetically modified MHCI, MHCII and / or CD47 expression such that the cells can advantageously become “invisible” to a host.

[0038] The genetically modified cell may comprise a detectable expression level of Flk1. In some embodiments, the genetically modified cell comprises a detectable expression level of Tie2, and optionally comprises a detectable expression level of cKit.

[0039] According to a further aspect, the present invention provides an in vitro method of generating a hemogenic endothelium (HE) cell, the method comprising: a) Genetically modifying a cell to introduce nucleotide sequence(s) encoding exogenous transcription factors into the cell, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer; b) Culturing the genetically modified cell in a culture medium comprising the inducer to induce expression of the exogenous transcription factors such that the cell forms a HE cell.

[0040] The method may further comprise carrying out a method according to the first aspect on the HE cell generated in step b). Optionally, the HE cell is stored and / or cultured for a period of time prior to carrying out the method according to the first aspect on the HE cell. The HE cell may be cultured for at least about 10 days prior to carrying out the method according to the first aspect on the HE cell, optionally at least about 50 days, further optionally at least about 100 days.

[0041] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0042] Summary of the Figures

[0043] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying Figures in which:

[0044] Figure 1 : Summary of developmental haematopoiesis from mesoderm to mature blood cells. Simplified diagram outlines primitive and definitive haematopoiesis during development. Solid lines represent known differentiation pathways. Dashed lines indicate suspected differentiation pathways.

[0045] Figure 2: Haemoglobin switching between primitive and definitive haematopoiesis.

[0046] Figure 3. Schematic of aspects of the present invention. Doxycycline inducible gene expression in HE cells blocks differentiation and allows for self-renewal. After dox removal, HE cells are released to undergo EHT and generate CD45+ haematopoietic cells.

[0047] Figure 4: Flow cytometry FSC vs SSC profile and GFP histogram for ES cells 24 hours after electroporation with GFP plasmid. 3x10E6 ES cells were re-suspended in 0.5ml electroporation buffer (100mM Na2HPO4, 27mM NaH2PO4, 5mM KCI, 5mM MgCI2, pH=7.2) at room temperature and electroporation was done with a 50pF 600V pulse using a BioRad Gene Pulser Xcell.F

[0048] Figure 5: Genotype analysis by PCR of genetically modified mTmG ES cells. Genomic DNA from Bulk modified ES cells prepared by thermal treatment and proteinase K treatment, then analysed with vector specific primers. PCR products were analysed by gel electrophoresis and imaged on a BioRad gel imager. The vector gene of interest is denoted at the top of the gel.

[0049] Figure 6: Fluorescence and phase contrast overlay of mTmG-ES derived EBs at day 3. Cells were imaged after 3 days of EB culture using both fluorescence imaging and phase-contrast imaging at 20x. (Scale bar denotes 50pm)

[0050] Figure 7: EB time-course in liquid cultures with mTmG ES cells. Cells were harvested at different time points and stained with conjugated antibodies for Flk1 , and then analysed by flow cytometry.

[0051] Figure 8: Overview of derivation of HE Cell lines

[0052] Figure 9: Ectopic expression of Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc in Flk1 + cells cultured in blast media. Genetically modifies Flk1 + enriched cells were plated at 50,000 cells per well in blast media with or without dox. A. Total cell number were measured using a haemocytometer when cells were passaged at day 3 and day 6. B. Microscope images of cells at Day 6 after Flk1 enrichment. Phase contrast images of modified bulk ES cells cultured in blast media on BME2 coated plates with and without dox for 6 days. Figure 10: Surface marker profile of bulk HE Cells cultured in blast media (+Dox) 59 days after Flk1 Isolation. Cells were analysed by flow cytometry and three population gates were defined by Flk1 and Tie2 expression. These populations are represented by different colours and histogram overlays presented for c-Kit, Tie2 and Flk1 .

[0053] Figure 11 : Microscope image of differentiated HE bulk cells 8 days after dox removal and differentiation in blast media (no Dox) containing 4uM SB431542 and 3uM CHIR99021. Cells imaged by phase contrast microscopy with a 20x optic. (Scale bar denotes 50pm)

[0054] Figure 12: A. Flow cytometry plots at day 0 and day 7 to show differentiated cells suppress endothelial markers (Flk1 / Tie2) and express haematopoietic markers (CD41 / CD45). Cells are only the suspension fraction at 7 days after dox removal. Cells were differentiated in blast media with SB431542, CHIR9902 and Bmp4. After 3 days, media was replaced with blast media without VEGF or D4T supernatant, and contained SB431542, CHIR9902, Bmp4, Activin A, Flt3l, FGF, and SCF. After 5 days media was replaced with the same media as the previous step without SB431542, CHIR9902, or Bmp4. B. Differentiated HE cells form multi-potential haematopoietic colonies in methylcellulose. HE cell line differentiated at day 59 after Flk1+ isolation (For 7 Days). Cells were differentiated in blast media with SB431542, CHIR9902 and Bmp4. After 3 days, media was replaced with blast media without VEGF or D4T supernatant, and contained SB431542, CHIR9902, Bmp4, Activin A, Flt3l, FGF, and SCF. After 5 days media was replaced with the same media as the previous step without SB431542, CHIR9902, or Bmp4. HE cells were differentiated in HE cell line compared to wild type mouse bone marrow. Left plot shows images of the HE cells, with right graph showing colony forming potential percentage.

[0055] Figure 13: Example colonies generated from the HE Cell Line. Microscope images demonstrate the appearance of specific colony forming unit (CFU) cells that were scored as CFU - macrophage (CFU-M), CFU - granulocyte (CFU-G), CFU - granulocyte, macrophage (CFU-GM), burst forming unit - erythroid (BFU-E), and CFU - granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM).

[0056] Figure 14: Genotyping CFU-GEMM colony and clonal HE cell to compare ectopic gene signature ‘fingerprint’. Fingerprint is an expression of percentage of ectopic gene detection which assumes equal PCR efficiencies between genes. This is an approximation of relative gene integrations for the purposes of comparative analysis between clones and colonies only. qPCR was performed once for each cell line or colony (n=1). Microscope images of the clonal cell line and colony that was genotyped on shown on the right.

[0057] Figure 15: Clonal HE cell line differentiates into blood after dox removal, measured by hematopoietic marker CD45 and endothelial markers Flk1 and Tie2. Cells were washed in PBS, then cultured in cytokine rich media with 4uM SB431542 and 3uM CHIR99021 . Cells were analysed by flow cytometry at 2 or 3 day intervals.

[0058] Figure 16: A. Dose response toxicity assay for resveratrol, Ly294002, and cycloleucine on clonal HE cells with dox. Cells were seeded in duplicate with doxycycline and the chemical of interest in a limiting dilution. After 2 days of culture cells were incubated with alamarBlue at 37C and analysed at 530nm Excitation, 590 Emission on a SpectraMax Plate Fluorometer. Fluorescent signal was normalised to the average measured signal in the absence of additional chemical and reported as relative normalised proliferation. A chemical concentration before significant loss in proliferation was annotated and used in experiments. B. Impact of Bmp4 addition to differentiation media on generation of CD45+Flk1- cells. (LEFT) HE-B02-C01 cells (pre-treated with Bmp4 or not / CTRL) were differentiated in Big mix base media (With / without Bmp4) and assessed by quantitative flow cytometry after 9 days of culture. Relative number of Flk1-CD45+ cells compared to base media with no pre-treatment. (RIGHT) Phase contrast images (10X) of cells differentiated for 8 days in base media, or base media +Bmp4. Scale bar denotes 400pm.

[0059] Figure 17: A. VeraVec™ co-culture cells (FACS Profile at Day 10 of Differentiation). Flow cytometry of differentiated clonal HE cell line (HE-B02-C01) at day 0, 2 & 10. HE cell line was day 121 post flk1 at time 0. Differentiation involved 2 days in Big mix +SB+CHIR+Bmp4, then 8d VeraVec™ co-culture on StemSpan+KOSR+SB+CHIR+FGF+SCF. The c-Kit+CD45+ cells analysed for LSK are highlighted in purple. B. LSK-SLAM characterisation of haematopoietic cells generated through serum free veravec coculture from HE clonal cell line. Gates selected for LSK-SLAM are highlighted in red.

[0060] Figure 18: Colony forming cell potential of the 3 cell products prepared by 3 different differentiation protocols.

[0061] Figure 19: UMAP projections of differentiating HE cell lines. A. Cell day determined through demultiplexing pooled samples. B. Major cell classifications of cell identities projected onto existing UMAP space. Cell identity alignment determined using the SingleR (Aran et al., 2019) package. Mouse reference data set used for comparison was Heng et al. 2008.

[0062] Figure 20: Heatmap showing cell identity alignment for all cells determined using the SingleR (Aran et al., 2019) package. Mouse reference data set used for comparison was Heng et al. 2008 with minor cell classifications.

[0063] Figure 21 : A. UMAP projections displaying the relative counts for notable genes (Darker purple is higher expression). Additional notation of suspected cell types added onto the UMAPs identifying key cell types based on notable gene expression. B. Haemoglobin genes detected by scRNA-Seq for differentiated HE- B02-C01 cells split by time points and classified as adult or embryonic.

[0064] Figure 22: Flow cytometry and microscope analysis of blood cells generated from HE-B02-C01 . A. Flow cytometry analysis of blood cells split into 3 populations based on expression of Cd11 b, Gr-1 , and Teri 19. Forward and side scatter is shown to demonstrate the size of Teri 19+ cells. B. Haemoglobin visualised by pelleting cells and imaging with a Pixel 3A camera, adjusting for contrast and brightness. C. Microscope image of blood cells generated from HE-B01-C01 demonstrating the presence of small round cells suspected to be RBSs denoted by black arrows.

[0065] Figure 23: May-Grunwald-Giemsa (MGG) stain and cytospin analysis of blood generated from HE-B02- C01 . Cells with distinctive morphology were identified and clustered as macrophages, granulocytes, RBCs, and progenitor cells. Cells were imaged using an Termo Evos XL. Cells of interest were cropped and pooled based on cell identity. Scale bar denotes 100pm. Figure 24: Flow cytometry results for reprogramming (reverse differentiation) of clonal HE cells. Shown are parental clonal HE cells (HE-B02-C01) cells, Day 10 differentiated cells sorted for Gr-1 and CD11 b by MACS, and reprogrammed (reverse differentiated) cells after re-introducing dox.

[0066] Figure 25: Differentiation of reverse differentiated HE cell line measured by flow cytometry on day 0, day 3, and day 7. Cells were stained for Flk1 , Tie2, CD41 , CD45, and c-kit. Resulting cells are presented as smoothened density plots with red being the highest density and blue being lowest.

[0067] Figure 26: Frequency of EHT events determined by extreme limiting dilution analysis (ELDA) software summarising 3 biological replicates, each with 4 technical replicates per condition. Limiting dilution of cells were seeded in differentiation media and scored for the presence of haematopoiesis after 1 week of culture (left, A). Expression of Runxl in cell line expressing dox measured by qRT-PCR in two technical replicates (right, B).

[0068] Figure 27: Chemical screening assay with HE cell line HE-B02-C01-R1 (REV). Limiting dilutions of chemicals performed in differentiation media in BME2 coated 96 well plates. Equal cell number plated in each well and harvested after 4 days of differentiation. Samples stained in 96-well V-bottom plates and equal volumes analysed for CD41 and Tie2 expression on BD high-throughput sampler in high throughput mode. All values normalised to baseline condition with no chemical addition.

[0069] Figure 28: Flow Cytometry analysis for HE cell line (HE-B02-C01-R1) time-course differentiation. Cells differentiated without dox in cytokine rich media with Bmp4, CHIR99021 , SB431542. A. Flow cytometry plots of the bulk culture for each day. B. Quantitative flow cytometry results (achieved through counting beads) for time-course analysis showing total cell number for each FACS population for the culture well.

[0070] Figure 29: Colony forming unit analysis for HE cell line (HE-B02-C01-R1) time-course differentiation. Cells differentiated without dox in cytokine rich media with Bmp4, CHIR99021 , SB431542. A. CFC assay set up for each time point and scored based on colony type and reported as colony number per 10,000 cells. Flow cytometry analysis of Tie2 CD41+(CD45 ) cell percentage for early emerging blood presented on top of CFC data (secondary axis). B. FACS plot of Tie2 CD41+(CD45 ) population from Figure 28 outlining population.

[0071] Figure 30: UMAP analysis of scRNA-Seq data. TOP: Gene expression level for selected genes projected onto the UMAP space. BOTTOM: Subplot of UMAP projection for early time-points from time-course analysis.

[0072] Figure 31 : Pseudotime Analysis of Time-course. A. Slingshot analysis (Street et al., 2018) performed on all cells. Pseudotime is indicated by black line and colour where trajectories start in the centre where cells are designated red, and progress to a blue colour at the endpoints. B. Average gene expression and dot plots for pseudotime generated with plotExpression (show_smooth = TRUE).

[0073] Figure 32: Cell fate trajectory comparative analysis. A. Trajectory bottleneck definitions shown in blue and red. B. Heatmap of DEGs between endothelial and haematopoietic bottleneck populations. Figure 33: Fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter (Sakaue-Sawano et al., 2008) was employed. As the cells were derived from mTmG ES cells and are already dTomato, only the mAG-hGem (green) reporter was used.

[0074] Figure 34: A. Enriched microRNA expression in endothelial trajectory. B. Analysis of Adult vs Embryonic Haemoglobin for cells from HE cell line differentiation time-course expressing haemoglobin.

[0075] Figure 35: Comparative analysis of HE cell line and primary AGM cells. A. UMAP projection and k-means clustering analysis for both AGM and REV HE cell line (HE-B02-C01-REV1) B. Violin plots for Cdh5, Runxl , Gfi 1 , and Gfi 1 b expression in the three k-means clusters for both REV HE and AGM cell datasets.

[0076] Figure 36: A. Venn diagram of shared upregulated and downregulated genes between C1 and C2 for both cell populations. Plots generated using the VennDiagram library B. Dot plot showing selected co- upregulated or co-down regulated genes. Gene expressions was visualised using DotPlotQ function in the Seurat library for select genes. C. GO analysis through WikiPathway _2021_Human for shared up / down regulated presenting adjusted P-values for shared gene pathways.

[0077] Figure 37: A. Validation of genetic engineering at day 6 of human T cells by piggybac transposon and electroporation of plasmid DNA. B. Human T cell derived HE (hT-HE-3-VS1) differentiated for 6 days. Imaged at 20X. C. Flow cytometry analysis of human T-cell derived HE cell line hT-HE-3-VS1 with dox in maintenance media and after 6 days of differentiation culture in cytokine rich media.

[0078] Figure 38: Establishment of human-ES-derived HE cell lines. Flow cytometry analysis of human ES-cell derived HE cell line following incubation with and without dox. Upon doxycycline withdrawal, hHE cells began expressing hematopoietic markers cKIT, CD43, and CD45.

[0079] Figure 39: Multilineage repopulation using mouse HE cell progeny. Tomato-expressing HE cells were cultured without doxycycline for 2, 3, and 4 days, then pooled. Five million cells were injected intraperitoneally into NSG mice following two doses of 100 cGy irradiation, administered 3 hours apart. After 11 weeks, the mice were sacrificed, and the spleen, bone marrow, and thymus were harvested for analysis. Flow cytometry analysis of tomato-positive cells detected in the thymus, spleen, peripheral blood and bone marrow.

[0080] Figure 40: Demonstration of continuous HE cell line amplification in culture. A. Graph to show in vitro expansion of murine HE cells in a long-term culture of 106 days. B. Morphology of HE cells at day 0, day 64 and day 106 of culture.

[0081] Detailed Description of the Invention

[0082] According to a first aspect, the present invention provides an in vitro method of generating haematopoietic cells, the method comprising: a) Providing a cell genetically modified to comprise a nucleotide sequence(s) encoding exogenous transcription factors, comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall , which may otherwise be referred to herein as SCL) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors; b) Culturing the genetically modified cell in a differentiation medium which does not comprise the inducer, such that the expression level of the exogenous transcription factors in the cell is reduced to a level whereby the cell differentiates into a haematopoietic cell.

[0083] The inventors have advantageously found that the genetic modification of a cell to express the exogenous transcription factors programs the cell into a “haemogenic endothelium (HE)”-like state. As the skilled person will appreciate, the haemogenic endothelium is a rare and transient endothelial cell population with haematopoietic potential. The inventors have, crucially, found that maintained expression of the exogenous transcription factors is necessary to maintain the haemogenic endothelial state. Once expression of the exogenous transcription factors is reduced or removed, differentiation of the cells into haematopoietic cells occurs. This enables the controlled and sustained production of a significant quantity of blood cells which have various utilities, including, for example, in therapeutic uses.

[0084] In the context of the present invention, “inducible by culture with an inducer” will be understood to mean that expression of the exogenous transcription factors in the cell is initiated when the cell is cultured in a medium comprising the inducer. When the cell is cultured in a medium which does not comprise the inducer, expression of the exogenous transcription factors in the cell does not occur or is reduced, relative to expression when cultured in the presence of the inducer.

[0085] As used herein, “genetically modified” will be understood to mean that the cell has been modified by the introduction of nucleotide sequence(s) into the cell. The term “genetically modified” may be used interchangeably with the term “genetically engineered”.

[0086] In some embodiments, the transcription factors are derived from murine transcription factors. In other embodiments, the transcription factors are derived from human transcription factors.

[0087] In the context of the present invention, “exogenous transcription factors” will be understood to refer to transcription factors which are expressed from nucleotide sequence(s) introduced into the cell from the genetic modification. It will be appreciated that the nucleotide sequence(s) introduced into the cell are non-naturally occurring. In other words, the nucleotide sequence(s) introduced into the cell are recombinant.

[0088] Typically, the nucleotide sequence(s) comprise an inducer response element nucleotide sequence and nucleotide sequence(s) encoding the exogenous transcription factor(s). The term “inducer response element nucleotide sequence”, as used herein, will be understood to refer to a nucleotide sequence to which an inducing protein can bind and thus initiate transcription of the nucleotide sequence(s) encoding the exogenous transcription factor(s) when in the presence of the inducer. Typically, the combination of the inducer response element nucleotide sequence and a nucleotide sequence(s) encoding the exogenous transcription factor(s) is non-naturally occurring. The inducer response element nucleotide sequence may comprise, for example, a tetracycline response element (TRE) or a promoter which can be bound by the inducing protein.

[0089] Advantageously, the combination of inducer response element nucleotide sequence and the nucleotide sequence(s) encoding the exogenous transcription factor(s) ensures that expression of the exogenous transcription factors is tightly regulated and controllable by an inducer, enabling expression, or, if desired, suppression of expression of the target genes. In the context of the present invention, an inducer will be understood to comprise an extracellular stimuli which, when present, initiates transcription of the target gene, in this instance the exogenous transcription factors. Once the inducer is removed, transcription of the target gene is reduced or stops.

[0090] Various inducible gene expression systems are known to the skilled person and commercially available. These include, for example, tetracycline-controlled gene expression systems, cumate-controlled gene expression systems, doxycycline-controlled gene expression systems, rapamycin-controlled gene expression systems, FKCsA-controlled gene expression system, ER (estrogen receptor) gene expression systems or ABA controlled gene expression systems. Any such inducible gene expression systems are suitable for use in the present invention. For example, the method may utilise a cumate-controlled gene expression system. In such embodiments, it will be appreciated that the inducer response element nucleotide sequence comprises a cumate response element nucleotide sequence and that the inducer comprises cumate.

[0091] In some embodiments, the inducible gene expression system comprises a doxycycline or tetracycline- controlled gene expression system. An exemplary system is the Tet-On system, wherein a reverse tetracycline transactivator (rtTA) protein is capable of binding at specific tetracycline response elements if bound by tetracycline or a derivative thereof, such as doxycycline, thereby initiating the transcription of the target gene. Other systems include, but are not necessarily limited to the Tet-On advanced and Teton 3G inducible gene expression systems. In such embodiments, the inducer comprises tetracycline, or a derivative thereof, such as doxycycline. In some embodiments, the inducer comprises Doxycycline.

[0092] In some embodiments, the inducer response element nucleotide sequence comprises a tetracycline response element (TRE). In some embodiments, the inducer response element comprises a TRE and a promoter.

[0093] In some embodiments, the inducer response element comprises a promoter which can be bound by the inducing protein. This may otherwise be referred to as an inducible promoter. The promoter may, for example, comprise a TetOn inducible promotor, such as a TetOn3G dox inducible promotor. This may include, for example a TRE3G promoter. Various other inducible promoters and systems are commercially available and known to the skilled person.

[0094] In the context of the present invention, a haematopoietic cell will be understood to refer to a cell which is a blood cell precursor and / or a blood cell. By blood cell precursors, this will be understood to refer to cells such as haematopoietic stem and progenitor cells (HSPCs). Thus, in some embodiments, the haematopoietic cell comprises a HSPC and / or a blood cell. In the context of the present invention, a blood cell will be understood to be any type of cell which can be found in the blood.

[0095] In some embodiments, a detectable expression level of the exogenous transcription factors comprises a detectable expression level of exogenous transcription factor proteins. In some embodiments, a detectable expression level of the exogenous transcription factors comprises a detectable expression level of exogenous transcription factor mRNA. Preferably, a detectable expression level of the exogenous transcription factors comprises a detectable amount of exogenous transcription factor mRNA and protein. Methods for measuring the presence of mRNA and / or protein are known in the art and discussed in more detail below. The mRNA and / or protein may be present in any amount in the genetically modified cell. In the context of the present invention, the terms “amount” and “level” are interchangeable.

[0096] Prior to being genetically engineered, expression of the exogenous transcription factors in the cell may be undetectable. In some embodiments, prior to being genetically engineered, expression of the exogenous transcription factors comprises an undetectable amount of exogenous transcription factors mRNA and / or protein in the cell.

[0097] Various methods are known in the art to detect proteins including, for example, western blots, flow cytometry and ELISAs. Normalisation of protein expression may be to a housekeeping protein product. The skilled person will be aware of suitable housekeeping genes and products. The expression and / or detectable amount of protein may comprise a normalised value.

[0098] The expression and / or detectable amount of protein may be detected using flow cytometry. Normalisation of protein expression may be to an isotype control antibody. The expression and / or detectable amount of protein detected using flow cytometry may be quantified by population shift and / or mean fluorescence intensity.

[0099] The expression and / or detectable amount of mRNA can be detected using, for example, PCR or RNA- seq methods. Various RNA-seq methods are commercially available and known to those skilled in the art. RNA-seq methods function by mapping the number of RNA reads aligned to each gene under each biological condition, to obtain a read count. The reads can then be normalised to provide a normalised read count.

[0100] In some embodiments, the exogenous transcription factors further comprise a Forkhead box (FOX) family transcription factor, In some embodiments, the FOX family transcription factor comprises Foxc2.

[0101] In some embodiments, the ETS family transcription factor comprises Ets1 or ETV2, preferably ETV2.

[0102] In some embodiments, the exogenous transcription factors further comprise MYC, optionally wherein MYC comprises c-MYC or n-MYC, preferably c-MYC.

[0103] In some embodiments, the GATA family transcription factor comprises GATA1 or GATA2. In some embodiments, the GATA family transcription factor comprises GATA2. In some embodiments, the exogenous transcription factors further comprise a Sry-box (SOX) family transcription factor. The SOX family transcription factor may comprise Sox17, Sox18 or a combination thereof. In some embodiments, the SOX family transcription factor comprises Sox17.

[0104] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC and a FOX family transcription factor.

[0105] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, c-MYC, and Foxc2.

[0106] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC, a FOX family transcription factor and a SOX family transcription factor.

[0107] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, and c-Myc.

[0108] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, c- Myc, and Lmo2

[0109] In some embodiments, the exogenous transcription factors further comprise LIM-only protein 2 (LMO2).

[0110] In some embodiments, the exogenous transcription factors comprise Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc.

[0111] The exogenous transcription factors may further comprise Friend leukaemia integration 1 transcription factor (Fli-1) .

[0112] In step b), the expression level of the exogenous transcription factors in the cell may be reduced by at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99%. In some embodiments, in step b) the expression level of the exogenous transcription factors in the cell is reduced to an undetectable expression level. Such a reduction in expression level has advantageously been found by the inventors to initiate differentiation of the cell into a haematopoietic cell.

[0113] In some embodiments step a) comprises: i) Providing the genetically modified cell comprising the nucleotide sequence(s) encoding the exogenous transcription factors; and ii) Culturing the genetically modified cell in an inducer medium comprising the inducer to induce a detectable expression level of the exogenous transcription factors in the cell.

[0114] In step a), optionally step ii), the genetically modified cell may comprise a detectable level of Flk1 expression. This may otherwise be referred to as “Flk1+”. In step a), optionally step ii), the genetically modified cell may comprise a detectable level of Tie2 expression. In step a), optionally step ii), the genetically modified cell may be cKit+. In some embodiments, in step a), optionally step ii), the genetically modified cell is Flk1+cKit+. In some embodiments, in step a), optionally step ii), the genetically modified cell is Flk1+cKit+Tie2+In some embodiments, in step a), optionally step ii), the genetically modified cell is cKit+Tie2+. In some embodiments, in step a), optionally step ii), the genetically modified cell is Tie2+.

[0115] In some embodiments in step a), optionally step ii), the genetically modified cell comprises an undetectable expression level of CD41 . This may otherwise be referred to as “CD41 “. In some embodiments in step a), optionally step ii), the genetically modified cell comprises a low level of expression of CD41. This may otherwise be referred to as “CD41 (low)“. A low level of expression can be identified using, for example, flow cytometry. In such embodiments, the level of expression of CD41 in the cell may be plotted against the level of expression of CD41 in other cells. This may be represented, for example, as a dot plot. The dot plot may be divided into quadrants, and CD41 low expression determined to be when the cell is in the bottom left quadrant. In some embodiments, a low level of expression of CD41 could be quantified as a population having a CD41 expression distribution measured by flow cytometry that overlaps between 10% and 80% with the distribution of an isotype negative control, but is higher.

[0116] Advantageously, the present inventors have found that culture of the genetically modified cell in step ii) of step a) can be for a prolonged period without any substantial change in cell morphology. In addition, the inventors have found that the genetically modified cell remains proliferative in long-term culture. This can advantageously provide a long-term and reliable in vitro source of cells from which haematopoietic cells can be generated in large numbers, advantageously from a relatively small number of starting cells. For example, step ii) may comprise culturing the genetically modified cell in an inducer medium comprising the inducer for at least about 10 days. In some embodiments, step ii) comprises culturing the genetically modified cell in an inducer medium comprising the inducer for at least about 20 days, at least about 30 days, at least about 40 days or at least about 50 days. In some embodiments, step ii) comprises culturing the genetically modified cell in an inducer medium comprising the inducer for at least about 60 days. In some embodiments, step ii) comprises culturing the genetically modified cell in an inducer medium comprising the inducer for at least about 70 days, at least about 80 days or at least about 90 days. In some embodiments, step ii) comprises culturing the genetically modified cell in an inducer medium comprising the inducer for at least about 100 days.

[0117] In some embodiments, the cell in step i) comprises a differentiated cell genetically modified to comprise the nucleotide sequence(s). In the context of the present invention, a differentiated cell will be understood to refer to a cell which has a specific function and is typically unable to change to a different type of cell in vivo; it is not a precursor cell. Typically, therefore, the function of a differentiated cell is final and does not change substantially. Surprisingly, however, the present inventors have found that the genetic modification of a differentiated cell with nucleotide sequence(s) encoding the exogenous transcription factors of the invention effectively “reprograms” the differentiated cell into a multipotent HE-like state. Even more surprisingly, the present inventors have found that once the reprogrammed genetically modified cell is cultured in a differentiation medium which does not comprise the inducer, differentiation of the cell has improved efficiency relative to non-reprogrammed cells. In some embodiments, the cell in step i) comprises a haematopoietic cell previously obtained according to the method of the first aspect. In some embodiments, the cell in step i) comprises a blood cell previously obtained according to the method of the first aspect. The blood cell may comprise a myeloid or lymphoid cell, for example a T-cell. In some embodiments, the blood cell comprises a PBMC (peripheral blood mononuclear cell). The blood cell may comprise a T-cell, B-cell or NK-cell. The blood cell may comprise a macrophage, granulocyte, T-cell, NK cell, dendritic cell (DC) B-cell or erythrocyte. Without wishing to be bound by theory, the present inventors believe that the reprogramming of a particular blood cell gives the resulting reprogrammed cell a tendency to differentiate with improved efficiency into that particular type of blood cell, for example a T- cell. The method is therefore advantageously capable of providing a sustained supply of substantial numbers of haematopoietic cells, and if desired, haematopoietic cells of a particular type, such as, for example T-cells.

[0118] The cell may be a mammalian cell. For example, the cell may be a human, horse, dog, cat, bovine or murine cell. In some embodiments, the cell is a murine or a human cell. Preferably, the cell is a human cell.

[0119] In some embodiments, the cell is a somatic cell.

[0120] In some embodiments, the cell is an allogeneic cell. In other embodiments, the cell is an autologous cell. As the skilled person will appreciate, autologous cells are cells from the same subject, i.e. cells which have been obtained from a subject which will be administered back to the same subject. Allogeneic cells are cells obtained from a different subject to the subject to which the cells will be administered. The different subjects are typically from the same species. Allogenic cells are thus genetically different to the subject to which they are administered. Advantageously, the method of the present invention enables the generation of a substantial number of allogeneic or autologous blood cells, effectively providing a substantial bank of cells for use, for example, therapeutically.

[0121] In other embodiments, the cell is an embryonic stem cell. In some embodiments, the cell is a murine embryonic stem cell. In some embodiments, the cell is a human embryonic stem cell. In some embodiments, the cell is a human embryonic stem cell previously obtained via parthenogenetically activated human oocytes. In some embodiments, the cell is a human embryonic stem cell previously obtained using a method which did not involve the destruction of human embryos.

[0122] In some embodiments, the cell is an induced pluripotent stem cell. In some embodiments, the cell is a murine induced pluripotent stem cell. In some embodiments, the cell is a human induced pluripotent stem cell.

[0123] In some embodiments, step i) comprises genetically modifying the cell to introduce the nucleotide sequence(s) encoding the exogenous transcription factors into the cell. Genetically modifying the cell may comprise transfecting or transducing the cell with the nucleotide sequence(s) encoding the exogenous transcription factors. In some embodiments, genetically modifying the cell comprises transducing the cell with the nucleotide sequence(s) encoding the exogenous transcription factors. Transduction may comprise transducing the cell with a viral-based vector, for example a lentiviral vector comprising the nucleotide sequence(s) encoding the exogenous transcription factors. Various suitable lentiviral vectors into which the nucleotide sequence(s) can be introduced are known and commercially available to the skilled person. Lentiviral vectors include but are not necessarily limited to self-inactivating lentiviral vectors (so-called SIN vectors). Transduction may comprise transducing the cell with CRISPR / Cas9 genetic engineering. CRISPR / Cas9 genetic engineering will typically include homologous recombination of DNA including the exogenous transcription factors. Various CRISPR / Cas9 gene editing protocols are available and known to the skilled person.

[0124] In other embodiments, genetically modifying the cell comprises transfecting the cell with the nucleotide sequence(s) encoding the exogenous transcription factors. For example, transfection may comprise use of the piggy bac transposon system. As the skilled person will appreciate, the piggybac transposon system comprises co-transfection of transposon plasmids and transposase plasmid into the cell. Advantageously, this achieves stable translocation into a genome. Transposon integrated sequences are less susceptible to gene silencing than lentiviral delivered sequences. The transfection may comprise electroporation.

[0125] A vector may comprise the nucleotide sequence(s) encoding the exogenous transcription factors. In some embodiments, a plurality of vectors comprise the nucleotide sequence(s) encoding the exogenous transcription factors. In some embodiments, one nucleotide sequence encodes the exogenous transcription factors. A vector may comprise the one nucleotide sequence. Alternatively, each exogenous transcription factor may be encoded by a distinct nucleotide sequence. Each distinct nucleotide sequence may be comprised in a separate vector.

[0126] In some embodiments, the genetically modified cell comprises a fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter. The inventors have surprisingly observed that cells comprising this indicator have an increased efficiency and speed of EHT.

[0127] It will be appreciated that the inducer medium comprises the inducer. Any suitable basal culture medium may be used, for example IMDM medium. The inducer medium may further comprise vascular endothelial growth factor (VEGF). The VEGF may be at a concentration of from about 1 to about 50ng / ml, optionally of from about 1 to about 10ng / ml. In some embodiments the VEGF is at a concentration of about 5ng / ml. In some embodiments, the inducer medium further comprises one or more of FBS Pen / Strep, MTG, transferrin, ascorbic acid, D4T supernatant and IL-6. Other formulations of serum free media can be envisaged which may include a base media (ie. StemPro-34) supplemented with L-Glutamine, Pen / Strep, MTG, transferrin, ascorbic acid, VEGF and / or IL-6.

[0128] The inducer may be at a concentration of from about 50ng / ml to about 50pg / ml in the inducer medium. The inducer may be at a concentration of from about 1 pg / ml to about 50pg / ml in the inducer medium. In some embodiments the inducer is at a concentration of no more than 10pg / ml in the inducer medium. In some embodiments, the inducer is at a concentration of about 2pg / ml in the inducer medium.

[0129] The differentiation medium does not comprise the inducer. Therefore, the differentiation medium comprises undetectable levels of the inducer. The differentiation medium may comprise a blast medium. For example, the differentiation medium may comprise IMDM medium. In some embodiments the differentiation medium further comprises one or more of FBS, PFHM-II, L-Glutamine, Pen / Strep, MTG, transferrin, ascorbic acid, SCF, IL-3, GM-CSF, TPO, EPO, mCSF, IL-6 and IL-11. For example, the differentiation medium may further comprise FBS, L-Glutamine, Pen / Strep, MTG, transferrin, ascorbic acid, SCF, IL-3, TPO, IL-6 and IL-11 .

[0130] The differentiation medium may comprise a TGF-p inhibitor. In the context of the present invention, the term “TGF-p inhibitor” will be understood to refer to a modulator which reduces or prevents TGF-p expression. The mechanism of action of the TGF-p inhibitor may comprise interfering with TGF-p bioavailability, TGF-p / receptor interaction or TGF-p kinase function. The TGF-p inhibitor may comprise a TGF-p neutralising antibody, ligand trap or receptor kinase inhibitor. In some embodiments, the TGF-p inhibitor comprises a receptor kinase inhibitor. In some embodiments, the TGF-p inhibitor comprises an activin receptor-like kinase (ALK)5, ALK4 and / or ALK7 inhibitor. For example, the TGF-p inhibitor may include, but not necessarily be limited to SB505124, A83-01 , Galunisertib and / or SB431542.

[0131] In some embodiments, the TGF-p inhibitor comprises SB431542. The differentiation medium may comprise the TGF-p inhibitor at a concentration of at least about 1pM, at least about 2pM, at least about 3pM, at least about 4pM or at least about 5pM. In some embodiments, the differentiation medium comprises the TGF-p inhibitor at a concentration of less than about 15pM, optionally less than about 10pM. In some embodiments, the differentiation medium comprises a TGF-p inhibitor at a concentration of from about 1pM to about 10pM. In some embodiments, the differentiation medium comprises a TGF-p inhibitor at a concentration of from about 1 pM to about 5pM. In some embodiments, the differentiation medium comprises a TGF-p inhibitor at a concentration of about 4 pM.

[0132] The differentiation medium may comprise a Wnt activator. The Wnt activator may comprise CHIR99021 , a frizzled receptor ligand, a GSK-3 inhibitor, BIO(6-bromoindirubin-3'-oxime), LY2090314 and / or Lithium chloride (LiCI). The frizzled receptor ligand may be selected from Wnt1 , Wnt3a and / or Wnt5a. In some embodiments the frizzled receptor ligand comprises Wnt3a.

[0133] In some embodiments, the Wnt activator comprises CHIR99021 . As the skilled person will appreciate, a Wnt activator is a molecule or compound which increases Wnt signalling, either directly or indirectly. In some embodiments, the Wnt activator comprises a GSK-3 inhibitor, known to activate the Wnt / p-catenin signalling pathway.

[0134] In some embodiments the differentiation medium comprises the Wnt activator CHIR99021 at a concentration of at least about 1 pM, at least about 2pM or at least about 3 pM. In some embodiments, the differentiation medium comprises the Wnt activator CHIR99021 at a concentration of less than about 10pM, less than about 7pM or less than about 5pM. In some embodiments the differentiation medium comprises the Wnt activator CHIR99021 at a concentration of from about 1 pM to about 5pM. In some embodiments, the differentiation medium comprises the Wnt activator CHIR99021 at a concentration of about 3pM.

[0135] The differentiation medium may comprise bone morphogenetic protein 4 (BMP4). The differentiation medium may comprise BMP4 at a concentration of at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20ng / ml or at least about 30 ng / ml. In some embodiments, the differentiation medium comprises BMP4 at a concentration of less than about 50ng / ml, less than about 45 ng / ml, less than about 40 ng / ml or less than about 35ng / ml. In some embodiments, the differentiation medium comprises BMP4 at a concentration of from about 10 ng / ml to about 30ng / ml. In some embodiments, the differentiation medium comprises BMP4 at a concentration of about 20ng / ml.

[0136] In some embodiments, the differentiation medium comprises a TGF-p inhibitor, a Wnt activator and BMP4. Optionally, the differentiation medium comprises VEGF.

[0137] In some embodiments, the differentiation medium comprises a PI3K inhibitor. As the skilled person will appreciate, a PI3K inhibitor is a modulator which inhibits expression of one of more of the phosphoinositide 3-kinase enzymes. Exemplary PI3K inhibitors include, but are not necessarily limited to Idelalisib, Copanlisib, Duvelisib, Alpelisib, Umbralisib, Leniolisib, PI828, PI3065, PIK90, CGS15943 and LY2940002. In some embodiments, the PI3K inhibitor comprises LY294002. LY294002 is commercially available from various companies, including, but not necessarily limited to Cell Signaling Technology and Thermo Fisher Scientific.

[0138] The differentiation medium may comprise the PI3K inhibitor at a concentration of at least about 0.5pM or at least about 1 pM. In some embodiments, the differentiation medium comprises the PI3K inhibitor at a concentration of less than about 10pM or less than about 5pM. In some embodiments, the differentiation medium comprises the PI3K inhibitor at a concentration of about 1 pM. In other embodiments, the differentiation medium comprises the PI3K inhibitor at a concentration of about 4pM.

[0139] In some embodiments, the differentiation medium comprises a PI3K inhibitor at a concentration of about 1 pM or 4pM and BMP4 at a concentration of about 20ng / ml.

[0140] In some embodiments, the differentiation medium comprises BMP4 and a PI3K inhibitor, optionally wherein the PI3K inhibitor comprises LY2940002.

[0141] In some embodiments, the differentiation medium further comprises one or more of Activin A, Flt3l, FGF and SCF. For example, the differentiation medium may comprise a TGF-p inhibitor, a Wnt activator, BMP4, Activin A, Flt3l, FGF and SCF.

[0142] In some embodiments, the differentiation medium further comprises Resveratrol. In some embodiments, the differentiation medium further comprises cycloleucine.

[0143] In some embodiments, the differentiation medium does not comprise a KDM1 A / LSD1 inhibitor. In some embodiments, the differentiation medium does not comprise a LATS1 and / or LATS2 inhibitor. For example, in some embodiments the differentiation medium does not comprise the LATS1 and LATS2 inhibitor GA-017 (Hippo Inhibitor).

[0144] The haematopoietic cell may be CD45+. In some embodiments, the haematopoietic cell is CD41+. In some embodiments, the haematopoietic cell is CD45+, CD41+. In some embodiments the haematopoietic cell is c-kit+. In some embodiments the haematopoietic cell comprises an undetectable level of expression of Flk1 , which may otherwise be referred to as FlkT. In some embodiments the haematopoietic cell is CD45+FlkT. In some embodiments the haematopoietic cell comprises an undetectable level of expression of Tie2 and / or CD31 . In some embodiments the haematopoietic cell comprises a detectable expression level of the transcription factor Runxl . This may otherwise be referred to as Runx1+. In some embodiments, the haematopoietic cell is CD43+.

[0145] It will be appreciated that the expression of the exogenous transcription factors in the haematopoietic cell is undetectable or reduced relative to expression in the genetically modified cell in step a) of the method.

[0146] According to a second aspect, the present invention provides a haematopoietic cell obtainable by the method according to the first aspect.

[0147] According to a further aspect, the present invention provides a genetically modified haematopoietic cell comprising a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer.

[0148] Preferably, the genetically modified haematopoietic cell comprises no more than a low expression level of the exogenous transcription factors. For example, the genetically modified haematopoietic cell may comprise a low or undetectable expression level of the exogenous transcription factors. The low expression level may comprise an expression level which is detectable, for example by PCR, but which is at such a low expression level that it does not phenotypically affect the cell. The low expression level may comprise leaky expression of the exogenous transcription factors. The skilled person will be aware of leaky expression, particularly in the context of inducer systems. Leaky expression may comprise no more than about 5% expression, no more than about 4% expression, no more than about 3% expression, 2% expression, no more than about 1% expression, no more than about 0.5% expression or no more than about 0.1% expression. In some embodiments, leaky expression comprises no more than about 0.09% expression, no more than about 0.08% expression, no more than about 0.07% expression, no more than about 0.06% expression, no more than about 0.05% expression, no more than about 0.04% expression, no more than about 0.03% expression, no more than about 0.02% expression or no more than about 0.01% expression. In some embodiments, leaky expression comprises of from about 0.01% expression to about 0.1% expression. In some embodiments, leaky expression comprises of from about 0.05% expression to about 2% expression, optionally of from about 0.1% expression to about 1% expression. In some embodiments, leaky expression comprises about 0.01% expression. When referring to % expression, this may be in the context of a population of cells (i.e. 1% of cells are deemed positive for expression). Alternatively, when referring to % expression, this may be in the context of the percentage intensity of expression per cell, relative to, for example, a cell which is known to be expressing a detectable level of the transcription factors (for example, the genetically modified cell of the aspect below). In some embodiments, the genetically modified haematopoietic cell comprises an undetectable expression level of the exogenous transcription factors. The genetically modified haematopoietic cell may be as defined above in relation to the haematopoietic cell of any of the above aspects. For example, the genetically modified haematopoietic cell may be allogeneic or autologous. In some embodiments, the transcription factors are derived from murine transcription factors. In other embodiments, the transcription factors are derived from human transcription factors.

[0149] In some embodiments, the exogenous transcription factors further comprise a Forkhead box (FOX) family transcription factor, In some embodiments, the FOX family transcription factor comprises Foxc2.

[0150] In some embodiments, the ETS family transcription factor comprises Ets1 or ETV2, preferably ETV2.

[0151] In some embodiments, the exogenous transcription factors further comprise MYC, optionally wherein MYC comprises c-MYC or n-MYC, preferably c-MYC.

[0152] In some embodiments, the GATA family transcription factor comprises GATA1 or GATA2. In some embodiments the GATA family transcription factor comprises GATA2.

[0153] In some embodiments, the exogenous transcription factors further comprise a Sry-box (SOX) family transcription factor. The SOX family transcription factor may comprise Sox17, Sox18 or a combination thereof. In some embodiments, the SOX family transcription factor comprises Sox17.

[0154] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC and a FOX family transcription factor.

[0155] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, c-MYC, and Foxc2.

[0156] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC, a FOX family transcription factor and a SOX family transcription factor.

[0157] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, and c-Myc.

[0158] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, c- Myc, and Lmo2

[0159] In some embodiments, the exogenous transcription factors further comprise LIM-only protein 2 (LMO2).

[0160] In some embodiments, the exogenous transcription factors comprise Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc.

[0161] The exogenous transcription factors may further comprise Friend leukaemia integration 1 transcription factor (Fli-1) .

[0162] In some embodiments, the haematopoietic cell comprises a HSPC and / or blood cell.

[0163] In some embodiments, the genetically modified haematopoietic cell is CD45+. In some embodiments, the haematopoietic cell is CD41 +. In some embodiments, the haematopoietic cell is CD45+, CD41 + . In some embodiments the haematopoietic cell is c-kit+. In some embodiments the haematopoietic cell comprises an undetectable level of expression of Flk1 , which may otherwise be referred to as Flk1 -. In some embodiments the haematopoietic cell is CD45+Flk1-. In some embodiments the haematopoietic cell comprises an undetectable level of expression of Tie2 and / or CD31. In some embodiments the haematopoietic cell comprises a detectable expression level of the transcription factor Runxl .

[0164] In some embodiments, the genetically modified haematopoietic cell comprises a detectable expression level of Spi 1 and / or Myb. In some embodiments, the genetically modified haematopoietic cell is CD43+.

[0165] In some embodiments, the genetically modified haematopoietic cell comprises a detectable level of expression of Gr-1 (which may otherwise be referred to as Gr-1+). In some embodiments, the genetically modified haematopoietic cell comprises a detectable level of expression of CD11 b (which may otherwise be referred to as CD11 b+). In some embodiments, the genetically modified haematopoietic cell is Gr-1+, CD11b+, CD45+. In some embodiments, the genetically modified haematopoietic cell is Teri 19+.

[0166] In some embodiments, the genetically modified haematopoietic cell comprises a detectable expression level of the MuvB complex.

[0167] In some embodiments, the haematopoietic cell comprises a myeloid cell or a lymphoid cell. In some embodiments, the haematopoietic cell comprises a PBMC.

[0168] In some embodiments, the haematopoietic cell comprises a myeloid cell. The myeloid cell may be selected from an erythrocyte, macrophage, granulocyte, dendritic cell, monocyte, neutrophil or combinations thereof. In some embodiments, the haematopoietic cell comprises a neutrophil, granulocyte, macrophage, dendritic cell or monocyte.

[0169] In some embodiments, the haematopoietic cell comprises a lymphoid cell. The lymphoid cell may be selected from a B cell, NK cell, NKT cell or T cell, for example a CD4+or CD8+T-cell. In some embodiments the T cell comprises a CD4+CD8+T-cell.

[0170] In some embodiments, the genetically modified haematopoietic cell comprises a macrophage, granulocyte, T-cell, NK cell, B-cell or erythrocyte.

[0171] In some embodiments, the genetically modified haematopoietic cell comprises a macrophage, T-cell, B- cell or erythrocyte.

[0172] In some embodiments, the genetically modified haematopoietic cell comprises a T-cell, NK cell or B cell. Optionally, the genetically modified haematopoietic cell comprises a T-cell or NK cell. The genetically modified haematopoietic cell may comprise a T-cell.

[0173] In some embodiments, the haematopoietic cell comprises a population of haematopoietic cells. In embodiments comprising a population of haematopoietic cells, the population may comprise a mixture of autologous and allogeneic haematopoietic cells.

[0174] In some embodiments, the genetically modified haematopoietic cell further comprises a nucleotide sequence encoding a chimeric receptor or a T-cell receptor (TCR). In some embodiments, the genetically modified haematopoietic cell comprises a nucleotide sequence encoding a chimeric receptor, optionally a chimeric antigen receptor (CAR). The term “chimeric receptor”, as used herein, will be understood to refer to a genetically engineered receptor capable of specifically binding to a target molecule. By chimeric antigen receptor (CAR), this will be understood to refer to a genetically engineered receptor capable of specifically binding with a target antigen. The CAR may comprise a first, second, third or fourth generation CAR. First-generation CARs typically comprise a binding domain that is capable of specifically binding to an epitope on a target antigen, a transmembrane domain, and one or more intracellular signalling domains. Typically, first-generation CARs comprise a binding domain that is capable of specifically binding to an epitope on a target antigen, a transmembrane domain, and one intracellular signalling domain. The extracellular binding domain may comprise a single-chain variable fragment (scFv) from a monoclonal antibody. A first-generation CAR may comprise a CD3 chain domain or a variant thereof as the intracellular signalling domain. In addition to the components specified for first-generation CARs, second-generation CARs also comprise a co-stimulatory domain, such as CD28 and / or 4-1 BB. Typically, second generation CARs contain one co-stimulatory domain, such as CD28 or 4-1 BB. The inclusion of an intracellular co-stimulatory domain improves T-cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence. The co-stimulatory domain of a second- generation CAR is typically in cis with and upstream of the one or more intracellular signalling domains. Generally, the co- stimulatory domain of a second-generation CAR is typically in cis with and upstream of the one intracellular signalling domain. Third-generation CARs combine multiple co-stimulatory domains in cis with one or more intracellular signalling domains, to augment T-cell activity. Typically, third generation CARs combine two co-stimulatory domains in cis with an intracellular signalling domain. For example, a third-generation CAR may comprise co-stimulatory domains derived from CD28 and 41 BB, together with an intracellular signalling domain derived from CD3 zeta. Fourth-generation CARs (also known as TRUCKS or armoured CARs), combine the features of a second-generation CAR with further factors to enhance anti-tumour activity (e.g., cytokines, co-stimulatory ligands, chemokines receptors or further chimeric receptors of immune regulatory or cytokine receptors). The factors may be in trans or in cis with the CAR, typically in trans with the CAR. In some embodiments, the CAR is specific for a cancer antigen.

[0175] In some embodiments, the genetically modified haematopoietic cell is an HSPC cell and exhibits an improved rate of differentiation and / or differentiation efficiency relative to a non-genetically modified HSPC cell. In the context of the present invention, the rate of differentiation will be understood to refer to the speed at which differentiation into a specific differentiated cell type, for example a myeloid or lymphoid cell, occurs. For example, the genetically modified HSPC cell may differentiate at a rate at least about 1 .5 fold, 2 fold, 3 fold, 4 fold or five fold faster than a non-genetically modified HSPC cell. By “differentiation efficiency”, this may be understood to refer to the proportion of cells which differentiate into the desired differentiated cell type. Thus, in some embodiments, the cell is an HSPC cell and exhibits an improved differentiation efficiency relative to a non-genetically modified HSPC cell.

[0176] In some embodiments, the genetically modified haematopoietic cell exhibits an increased proliferative rate relative to a non-genetically modified HSPC cell. For example, the genetically modified haematopoietic cell may exhibit a proliferative rate at least about 25%, at least about 50% or at least about 100% greater than a non-genetically modified haematopoietic cell. The genetically modified haematopoietic cell may exhibit other functional improvements, relative to a non- genetically modified HSPC cell. For example, in embodiments where the genetically modified haematopoietic cell comprises a CD8+ T-cell, the cell may exhibit improved cytotoxic activity.

[0177] In some embodiments, the genetically modified haematopoietic cell has an increased expression level of Runxl compared to a non-genetically modified haematopoietic cell. For example, the genetically modified haematopoietic cell may have at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, or at least about 200% increased expression of Runxl compared to a non- genetically modified haematopoietic cell. The genetically modified haematopoietic cell may have at least two fold, at least three fold, at least four fold or at least five fold expression level of Runxl compared to a non-genetically modified haematopoietic cell.

[0178] Also provided is a method for treating or preventing a disease in a subject, wherein the method comprises administering to the subject the haematopoietic cell of the second or third aspects of the invention.

[0179] The method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the haematopoietic cell of the invention. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease and / or abolishes one or more symptoms, such as all the symptoms, of the disease. The therapeutically effective amount preferably cures the disease. A prophylactically effective amount is an amount which prevents the onset of the disease and / or prevents the onset of one or more symptoms, such as all the symptoms, of the disease. The prophylactically effective amount preferably prevents the subject from developing the disease. Suitable amounts are discussed in more detail below.

[0180] The haematopoietic cell of the invention may be administered to a subject that displays symptoms of disease. The haematopoietic cell of the invention may be administered to a subject that is asymptomatic, i.e. does not display symptoms of disease. The haematopoietic cell of the invention may be administered when the subject’s disease status is unknown or the subject is expected not to have a disease. The haematopoietic cell of the invention may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease.

[0181] The subject may be a mammal. Optionally, the subject is a human, horse, dog or cat. In some embodiments, the subject is human. Alternatively, the subject may be a horse.

[0182] In some embodiments, the disease comprises cancer or autoimmune disease.

[0183] In some embodiments, the disease comprises autoimmune disease. Exemplary autoimmune diseases may include, but not necessarily be limited to rheumatoid arthritis (RA), Crohn’s disease, multiple sclerosis (MS), psoriasis, diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Addison’s disease, Graves’ disease, Sjogren’s disease, Hashimoto’s thyroiditis, Myasthenia gravis, Coeliac disease and autoimmune vasculitis.

[0184] In some embodiments, the disease comprises cancer. For example, the cancer may comprise breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, pancreatic cancer, colon cancer, gastric cancer, bladder cancer, skin cancer, myeloma, non-Hodgkin lymphoma, prostate cancer, oesophageal cancer, leukaemia, head and neck cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, or renal cell carcinoma. Exemplary cancers may include, but not necessarily be limited to breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, pancreatic cancer, colon cancer, gastric cancer, bladder cancer, skin cancer, myeloma, non-Hodgkin lymphoma, prostate cancer, oesophageal cancer, head and neck cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, or renal cell carcinoma. In some embodiments, the cancer comprises colon, breast, ovarian, lung, skin head and neck or pancreatic cancer.

[0185] The subject may have been pre-treated with a chemotherapeutic agent.

[0186] The administration of the haematopoietic cell of the invention to the subject may result in a decrease in tumour size of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated tumour.

[0187] Any suitable form of administration may be used for the haematopoietic cell. For example, the haematopoietic cell may be administered subcutaneously, intranasally, orally, topically, intraperitoneally or intravenously. In some embodiments, the haematopoietic cell may be intraperitoneally administered.

[0188] In embodiments wherein the haematopoietic cell comprises a population of haematopoietic cells, the number of haematopoietic cells administered to the subject should take into account the route of administration, the disease (for example, cancer) being treated, the weight of the subject and / or the age of the subject. In general, from about 1 x 106to about 1 x 1011haematopoietic cells are administered to the subject. In some embodiments, from about 1 x 107to about 1 x 1010haematopoietic cells, or from about 1 x 108to about 1 x 109haematopoietic cells are administered to the subject.

[0189] The invention also provides the haematopoietic cell of the invention for use in any of the therapeutic methods described above. Thus, also provided is the haematopoietic cell of the invention for use in the treatment or prevention of a disease. In particular, the invention provides the haematopoietic cell of the invention for use in the treatment or prevention of cancer or autoimmune disease, preferably cancer.

[0190] Also provided is the use of the haematopoietic cell of the invention for the manufacture of a medicament for the treatment or prevention of a disease. Optionally, the disease is cancer or autoimmune disease. Further provided is use of the haematopoietic cell of the invention for the treatment or prevention of cancer or autoimmune disease, optionally cancer.

[0191] According to a further aspect, the present invention provides a genetically modified cell, the cell comprising a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors. Advantageously, as described herein, the ectopic expression of the exogenous transcription factors reprograms cells into a hemogenic endothelium (HE) cell. The inventors have found that such HE like cells are able, upon reduction or removal of expression of the exogenous transcription factors, to differentiate into blood cells. The inventors have found that, advantageously, the HE cells can be maintained for substantial periods of time in vitro, providing a long-lasting, regulatable and efficient source of blood cells.

[0192] In some embodiments, the transcription factors are derived from murine transcription factors. In other embodiments, the transcription factors are derived from human transcription factors.

[0193] In some embodiments, the exogenous transcription factors further comprise a Forkhead box (FOX) family transcription factor, In some embodiments, the FOX family transcription factor comprises Foxc2.

[0194] In some embodiments, the ETS family transcription factor comprises Ets1 or ETV2, preferably ETV2.

[0195] In some embodiments, the exogenous transcription factors further comprise MYC, optionally wherein MYC comprises c-MYC or n-MYC, preferably c-MYC.

[0196] In some embodiments, the GATA family transcription factor comprises GATA1 or GATA2. In some embodiments, the GATA family transcription factor comprises GATA2.

[0197] In some embodiments, the exogenous transcription factors further comprise a Sry-box (SOX) family transcription factor. The SOX family transcription factor may comprise Sox17, Sox18 or a combination thereof. In some embodiments, the SOX family transcription factor comprises Sox17.

[0198] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC and a FOX family transcription factor.

[0199] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, c-MYC, and Foxc2.

[0200] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC, a FOX family transcription factor and a SOX family transcription factor.

[0201] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, and c-Myc.

[0202] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, c- Myc, and Lmo2

[0203] In some embodiments, the exogenous transcription factors further comprise LIM-only protein 2 (LMO2).

[0204] In some embodiments, the exogenous transcription factors comprise Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc.

[0205] The exogenous transcription factors may further comprise Friend leukaemia integration 1 transcription factor (Fli-1) .

[0206] The genetically modified cell may have an in vitro doubling rate of from 0.5 to 1 .5 days. In some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.7 to 1 day. In some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.8 to 0.9 days. Advantageously, the doubling rate of the genetically modified cell can be maintained for a prolonged period of culture. Thus, in some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.5 to 1 .5 days for at least about 50 days. In some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.5 to 1 .5 days for at least about 100 days. In some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.7 to 1 day for at least about 50 days. In some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.7 to 1 day for at least about 100 days. The genetically modified cell may have an in vitro doubling rate of from 0.8 to 0.9 days for at least about 50 days. In some embodiments, the genetically modified cell has an in vitro doubling rate of from 0.8 to 0.9 days for at least about 100 days.

[0207] The genetically modified cell may be as defined in relation to the genetically modified cell of the first aspect of the invention. For example, the genetically modified cell may comprise a detectable expression level of Flk1. In some embodiments, the cell comprises a detectable expression level of Tie2, and optionally comprises a detectable expression level of cKit.

[0208] The cell may be a mammalian cell. For example, the cell may be a human, horse, dog, cat, bovine or murine cell. In some embodiments, the cell is a murine or a human cell. Preferably, the cell is a human cell.

[0209] In some embodiments, the cell is a somatic cell. In other embodiments, the cell is derived from an embryonic stem cell. For example, the cell may be derived from a murine embryonic stem cell. In other embodiments, the cell is derived from a human embryonic stem cell. In some embodiments, the cell is derived from a human embryonic stem cell previously obtained via parthenogenetically activated human oocytes. In some embodiments, the cell is derived from a human embryonic stem cell previously obtained using a method which did not involve the destruction of human embryos. In some embodiments, the cell is derived from an induced pluripotent stem cell.

[0210] According to a further aspect of the invention, there is provided an in vitro method of generating a hemogenic endothelium (HE) cell, the method comprising: a) Genetically modifying a cell to introduce nucleotide sequence(s) encoding exogenous transcription factors into the cell, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer; b) Culturing the genetically modified cell in a culture medium comprising the inducer to induce expression of the exogenous transcription factors such that the cell forms a HE cell.

[0211] In some embodiments, the culturing in step b) is for a time period of at least about 48 hours, at least about 72 hours or at least about 96 hours.

[0212] As described herein, the present inventors have found that culture of the genetically modified cell can be for a prolonged period of time without loss of proliferation or morphology. Thus, in some embodiments, the culturing in step b) is for a time period of at least about 10 days, at least about 20 days, at least about 30 days, or at least about 40 days. In some embodiments, the culturing in step b) is for a time period of at least about 50 days. In some embodiments, the culturing in step b) is for a time period of at least about 60 days, at least about 70 days, at least about 80 days or at least about 90 days. In some embodiments, the culturing in step b) is for a time period of at least about 100 days.

[0213] In some embodiments, the transcription factors are derived from murine transcription factors. In other embodiments, the transcription factors are derived from human transcription factors.

[0214] In some embodiments, the exogenous transcription factors further comprise a Forkhead box (FOX) family transcription factor, In some embodiments, the FOX family transcription factor comprises Foxc2.

[0215] In some embodiments, the ETS family transcription factor comprises Ets1 or ETV2, preferably ETV2.

[0216] In some embodiments, the exogenous transcription factors further comprise MYC, optionally wherein MYC comprises c-MYC or n-MYC, preferably c-MYC.

[0217] In some embodiments, the GATA family transcription factor comprises GATA1 or GATA2, preferably GATA2.

[0218] In some embodiments, the exogenous transcription factors further comprise a Sry-box (SOX) family transcription factor. The SOX family transcription factor may comprise Sox17, Sox18 or a combination thereof. In some embodiments, the SOX family transcription factor comprises Sox17.

[0219] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC and a FOX family transcription factor.

[0220] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, c-MYC, and Foxc2.

[0221] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , an ETS family transcription factor, MYC, a FOX family transcription factor and a SOX family transcription factor.

[0222] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, and c-Myc.

[0223] In some embodiments, the exogenous transcription factors comprise Gata2, Tall , Etv2, Foxc2, Sox17, c- Myc, and Lmo2

[0224] In some embodiments, the exogenous transcription factors further comprise LIM-only protein 2 (LMO2).

[0225] In some embodiments, the exogenous transcription factors comprise Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc.

[0226] The exogenous transcription factors may further comprise Friend leukaemia integration 1 transcription factor (Fli-1) .

[0227] In some embodiments, the method further comprises carrying out the method of the first aspect on the HE cell generated in step b). For example, the genetic modification may be as described in relation to the first aspect. The inducer may be as defined for the first aspect. In some embodiments, the HE cell is stored for a period of time prior to carrying out the method of the first aspect on the HE cell. In some embodiments, the HE cell is cultured for a period of time prior to carrying out the method of the first aspect on the HE cell. In some embodiments, the HE cell is stored and cultured for a period of time prior to carrying out the method of the first aspect on the HE cell. Storage may comprise cryogenic storage. Culture may be in an inducer medium as defined above for the first aspect. The period of time may comprise a period of at least about three days, at least about four days, at least about five days, at least about one week, at least about two weeks, at least about one month, at least about three months, at least about six months or at least about 12 months. In some embodiments, the period of time comprises at least about 50 days or at least about 100 days.

[0228] Also provided is a pharmaceutical composition comprising any of the cells as defined above and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0229] The carrier and / or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0230] Suitable further agents for inclusion in the pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogen-sulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0231] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd Edition).

[0232] A pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoural, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoural, intrapleural and intra-sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intratumourally. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake.

[0233] Alternatively, the pharmaceutical composition of the invention can be administered by a nonparenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0234] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents. Alternatively, the pharmaceutical composition may be for intravenous administration.

[0235] The invention also provides a kit comprising any of the cells of the above aspects of the invention, for example, the haematopoietic cells of the second or third aspects of the invention. The kit may further comprise instructions for use. In some embodiments, the cells are provided in an aqueous solution, optionally buffered solution and / or at a temperature of at least -20°C.

[0236] Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0237] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting.

[0238] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0239] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0240] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0241] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0242] Examples

[0243] MATERIALS AND METHODS

[0244] Murine ES Culture and Differentiation

[0245] Murine Embryonic Stem Cells (mESCs) were cultured and differentiated as embryoid bodies (EBs) following a published protocol (Sroczynska et al., 2009) described briefly as follows. ES cells were thawed and passaged in DMEM-ES media (DMEM (Gibco, Prod: 11965092, with 15% serum, 2% LIF (Conditioned Media), 150 pM MTG (Merck, Prod: M6145), 2mM L-Glutamine (Gibco, Prod: 25030081), and 100 U / ml Penn / Strep (Gibco, Prod: 15140122)). Cultures were maintained on feeder mouse embryonic fibroblasts (MEFs) cells plated onto 6-well plates (CytoOne, Prod: CC7682-7506) pre-coated with 0.1% gelatin (Sigma, Prod: G1890) solution, and used within one week of plating. Typical passage includes washing cells with PBS, incubating with TrypLE (Gibco, Cat: 12605010) to detach cells, washing cells with media including centrifugation of 300g for 5min, then seeding at 2.5-3.0x10E5 cells per MEF coated well in a 6 well plate. For differentiation of ESC to EBs, cells were prepared by two 24 hour passages on gelatin in IMDM-ES (DMEM-ES replacing DMEM with IMDM) incubating on a tissue culture plastic for 30min during each passage to remove MEF cells before seeding. For EB generation, cells were washed in IMDM (Gibco, Prod: 12440053) with 15% serum, then seeded at 2.5x10E4 cells / ml in EB Media (15% serum, 0.5ng / ml ascorbic acid (Sigma, Prod: A-4544), 180 pg / ml transferrin (Roche, Prod: 652-202), 450 pM MTG, 2mM L-Glutamine, 100 U / ml Penn / Strep in IMDM) on polyHEMA (Sigma, Prod: P3932) coated plastic plates to prevent attachment.

[0246] Human ESC Culture and Differentiation

[0247] Man5 (University of Manchester) human Embryonic stem cells (hESCs) were cultured and differentiated as embryoid bodies (EBs) following a published protocol (Garcia-Alegria et al, 2021) described briefly as follows. Human ESCs were maintained on mitomycin C inactivated DR4 mouse embryonic fibroblasts in KO-DMEM (Gibco, Thermo Fisher Scientific) supplemented with 20% KO Serum replacement (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (50 mM, Gibco, Thermo Fisher Scientific), 1 % minimum essential medium (MEM) non-essential amino acids solution (100*, Gibco, Thermo Fisher Scientific), 1 % L-glutamine, 0.5% penicillin / streptomycin, and 8 ng / mL of human basic fibroblast growth factor (bFGF) (PeproTech). Four days before differentiation, hESCs were plated on Geltrex LDEV-Free, hES-Qualified, reduced growth factor basement membrane matrix (Thermo Fisher Scientific) and maintained in TeSR-E8 (STEMCELL Technologies). Differentiation of hESCs was driven by EB formation, using 10 million hESCs, in serum-free StemPro-34 SFM (Thermo Fisher Scientific) supplemented with the sequential addition of human cytokines (all from PeproTech) during 6 days as follows: 10 ng / mL BMP4 at day 0, 5 ng / mL of bFGF at day 1 , and 0.9 ng / mL activin A at day 2. From days 4 to 6 the medium was supplemented only with 5 ng / mL of bFGF and 12 ng / mL of VEGF. At day 6, EB were dissociated to single cells and stained with CD31 , CDH5 / CD144 and CD43.

[0248] Genetic engineering was achieved using the piggybac transposon system where co-transfection of transposon plasmids and transposase plasmid achieve stable translocation into a genome. The PB- TRE3G-MYCN dox inducible piggyback transposon backbone plasmid (Addgene, Plasmid #104542) was used as a backbone to generate the PB-TRE3G plasmids (Vereide et al., 2014). PB-TRE3G plasmids encoding transcription factor cDNA were derived from this backbone by amplifying cDNA for the transcription factors of interest with overhang PCR primers and ligating inserts into the AsiSI and Aflll sites of the backbone plasmid by standard molecular genetics protocols. These transposon plasmids require a transposase plasmid and the one used contains a constitutively expressed hyperactive piggyback transposon (HyBase) which was obtained from the Welcome Trust Sanger Institute (Yusa et al., 201 1). A piggybac transposon vector including a constitutively expressed dox inducible reverse tetracycline trans-activator (rtTA) within plasmid PB-EF1 a-TetOn3G (Addgene, Plasmid #104543) was used to achieve dox inducible expression (Loew et al., 2010; Vereide et al., 2014). DNA was amplified in E. Coli and purified using NucleoBond Xtra Midi kits (Macherey-Nagel, Prod: 740410.50). For genetic engineering of ES 3x10E6 cells were re-suspended in 0.5ml of electroporation buffer (1 OOmM Na2HPO4, 27mM NaH2PO4, 5mM KCI, 5mM MgCI2, pH=7.2). 49pg of plasmid DNA was added to the cells including 5pg of each of the 8 transposon plasmids (TRE3G: mEtv2, mFoxc2, hSox17, hcMyc, mGata2, mTall , mLmo2, EF1 :TetOn3G), 7pg of hyPBASE (Transposase), and 2 pg of a GFP spike plasmid. The mixture was pipetted into a 0.4cm BioRad cuvette and electroporated with a 50pF 600V pulse using a BioRad Gene Pulser Xcell. Immediately afterward the cells were diluted to 2ml in DMEM-ES, washed by centrifugation and plated at 5x10E5 cells per well in 6 wells of a 6-well plate. Two days later GFP+ cells were sorted on a FACS using a BD FACS Aria II to enrich for modified ES cells.

[0249] Genetic engineering was achieved using the piggybac transposon system where co-transfection of transposon plasmids and transposase plasmid achieve stable translocation into a genome. The PB- TRE3G-MYCN dox inducible piggyback transposon backbone plasmid (Addgene, Plasmid #104542) was used as a backbone to generate the PB-TRE3G plasmids. PB-TRE3G plasmids encoding transcription factor cDNA were derived from this backbone by amplifying cDNA for the transcription factors of interest with overhang PCR primers and ligating inserts into the AsiSI and Aflll sites of the backbone plasmid by standard molecular genetics protocols. These transposon plasmids require a transposase plasmid and the one used contains a constitutively expressed hyperactive piggyback transposon (HyBase) which was obtained from the Welcome Trust Sanger Institute (Yusa et al., 201 1). A piggybac transposon vector including a constitutively expressed dox inducible reverse tetracycline trans-activator (rtTA) within plasmid PB-EF1 a-TetOn3G (Addgene, Plasmid #104543) was used to achieve dox inducible expression. DNA was amplified in E. Coli and purified using NucleoBond Xtra Midi kits (Macherey-Nagel, Prod: 740410.50).

[0250] For genetic engineering of hESCs the Neon™ Transfection System (Invitrogen, Fisher Scientific) was used. Per electroporation, 2x10E6 cells were re-suspended in 0.1 ml of Neon™ electroporation buffer. 10pg of plasmid DNA was added to the cells including 0.75pg of each of the 8 transposon plasmids (TRE3G: hEtv2, hFoxc2, hSox17, hcMyc, hGata2, hTall , hLmo2, EF1 :TetOn3G), 2.5pg of hyPBASE (Transposase), and 1.5pg of a GFP spike-in plasmid. The mixture was electroporated using a 30pF 1 100V pulse and a 100 pL Neon™ Tip. 6x10E6 electroporated hESCs were plated in a Geltrex (LDEV-Free, hES-Qualified, reduced growth factor basement membrane matrix (Thermo Fisher Scientific)) coated 10 cm tissue culture dish and maintained in TeSR-E8 (STEMCELL Technologies). Two days later GFP+ cells were sorted on a FACS using a BD FACS Aria II to enrich for modified hESCs.

[0251] Mouse HE Cell line Derivation, and Maintenance

[0252] EBs were generated with modified ES cells. Day 3.5 EBs were harvested and washed with PBS using a 40pm cell strainer where EBs are collected on top of the strainer. Cells were then treated with TrypLE to disassemble EBs, washed with 10% FBS PBS. A single cell suspension was generated by straining the dissociated EBs with a 40pm cell strainer, collecting the flow-through. Flk1+ cells were isolated using magnetic-activated cell sorting (MACS) using biotin labelled anti-mouse Flk1 antibody (Miltenyi Biotec, Prod: 130-101-915), anti-biotin microbeads (Miltenyi Biotec, Prod: 130-090-485) and a Miltenyi Biotec LS column (Miltenyi Biotec, Prod: 130-042-401). Eluted Flk1+ cells were seeded on a basement membrane extract 2 (BME2) (Bio-Techne Part: 3533-010-02) pre coated (1 :100 for >1 hr) 24-well plate (CytoOne, Prod: CC7682-7524) at 5.0x10E4 cells per well in blast media (10% FBS in IMDM with 2mM L-Glutamine, 100 U / ml Penn / Strep, 450 pM MTG, 30 pg / ml transferrin, 0.25ng / ml ascorbic acid, 7.5% D4T supernatant, 5ng / ml VEGF (Peprotech, Prod: 450-32), 10ng / ml II-6 (Peprotech, Prod: 216-16) with 2pg / ml doxycycline (Dox) (Sigma, Prod: D5207). Cells were passaged every 2-3 days when about 80% confluent and split at a 1 :5 split ratio.

[0253] Human HE culture

[0254] Sorted CD31+CDH5 / CD144+ cells from EBs at day 6 were cultured on gelatin-coated plates in serum-free StemSpan medium (STEMCELL Technologies) supplemented with human cytokines (25 ng / mL insulin growth factor 1 (IGF-1), 25 ng / mL IGF-2, 50 ng / mL stem cell factor (SCF), 50 ng / mL thrombopoietin, 20 ng / mL Fms-like tyrosine kinase-3 ligand (FLT3L), 5 ng / mL interleukin-11 (IL-11), 5 ng / mL VEGF-A, and 5 ng / mL fibroblast growth factor 2 (all from PeproTech). At this stage, Doxycline was added to induce the expression of the seven transgenes.

[0255] Differentiation of Mouse HE Cell Lines

[0256] Once HE cell lines were established, differentiation assays were performed as follows. Cells were washed with PBS and then re-suspended in differentiation media without dox, and plated with 5.0x10E4 cells per well in a BME2 pre-treated 24 well plate. Differentiation media included blast media without dox with the addition of 3uM CHIR99021 (Sigma, Prod: SML1046) and 4uM SB431542 (Tocris, Prod: 1614). Differentiation media also included big mix media (10% FBS in IMDM with 10% PFHM-II, 2mM L- Glutamine, 100 U / ml Penn / Strep, 450 pM MTG, 180 pg / ml transferrin, 0.25ng / ml ascorbic acid, 1% SCF (supernatant), 1% IL-3 (supernatant), 1% GM-CSF (supernatant), 1% TPO (supernatant), 4U / ml EPO (Janssen, Prod: Eprex®, Epoetin alfa), 10ng / ml mCSF (Peprotech), 10ng / ml IL-6 (Peprotech, Prod: 216- 16), and 10 ng / ml IL-11 (Peprotech, Prod: 220-11) (including 10ng / ml Bmp4 (Peprotech, Prod: 315-27), 3uM CHIR99021 and 4uM SB431542 in some instances). In some instances differentiations were done in a minimum (Min) media (10% FBS in IMDM with 2mM L-Glutamine, 100 U / ml Penn / Strep, 450 pM MTG, 180 pg / ml transferrin, 0.25ng / ml ascorbic acid, 1% SCF, 1% IL-3, 1% TPO, 10ng / ml IL-6, and 10 ng / ml IL- 11). Cells were incubated at 21% 02 at 37°C with 5% CO2, and 70% relative humidity (RH). A 2% Methylcellulose (4000 cPs) (Alfa Aesar Prod: 36718) solution was prepared in IMDM with 0.3% sodium bicarbonate and frozen into aliquots. After thaw, a 1% methycellulose media was prepared with a final composition of 10% FBS, 10% PFHM-II (Gibco, Prod: 12040077), 2mM L-Glutamine, 100 U / ml Penn / Strep, 450 pM MTG, 180 pg / ml transferrin, 0.25ng / ml ascorbic acid, 1% SCF, 1% IL-3, 1% GM- CSF, 1% TPO, 4U / ml EPO, 10ng / ml mCSF, 10ng / ml IL-6, and 10 ng / ml IL-11. Cells were re-suspended in this final mixture at 10,000 cells / ml and 1.0ml was plated in a 35mm petri dish (Falcon, Prod: 351008) with surrounding open dishes with water and incubated at 37C and 5% CO2, with 70% RH. Colonies were scored after at least one week of culture to allow for differentiation and scoring by morphology.

[0257] Cells were re-suspended in 10Oul of PBS and loaded into a cytofunnel assembled with a glass cytoslide and absorptive filter guard. Samples were spun at 900g for 10 minutes and left to air dry overnight. Slides were prepared for staining by applying a circular hydrophobic barrier around the sample area using a PAP pen to reduce staining reagent use. Samples were stained in May-Grunwald stain (Sigma, Prod: MG500) for 5min, rinsed in PBS, and then stained in 1 :20 dilute Giemsa solution for 20min before rinsing in dH2O and then air drying. A cover slide was fixed onto the sample to allow for cells to be imaged facing down on an inverted microscope with a 40x optic and images were taken with an Evos XL Imaging System (Thermo, Prod: AME3300).

[0258] PBMCs were purchased from Research Donors UK with full consent to establish cell lines. Cells were thawed (96% viability) and re-suspended in RPMI containing 10% FBS, 2mM L-Glutamine, 100 U / ml Penn / Strep, 50uM p-mercaptoethanol (Gibco, Prod: 31350010), and 10ng / ml II-2 (Peprotech, Prod: 200- 02). Cells were mixed with activating human CD3 / CD28 Dynabeads (Thermo, Prod: 11161 D) at a cell to bead ratio of 1 :1 and seeded at 5.0 x10E5 cells / ml in 6 well plates with 3ml per well. Cells were cultured in activating conditions for 3 days, before washing in PBS and introducing plasmid DNA with a ThermoFisher Neon electroporator. Conditions were one 20ms pulse at 1700V administered to 2.0M cells in 10Oul of buffer R re-suspended with 10ug of purified plasmid DNA. Plasmid DNA included 2.5pg of hyPBASE, and 0.94pg of each of the 8 transposon plasmids (TRE3G:hEtv2, hFoxc2, hLmo2, hGata2, hcMyc, hSox17, EF1 :TetOn3G). Cells were added to warm media without antibiotics (Penn / Strep) to recover following the equipment supplier’s recommendation. Cells were then cultured for three days to allow for gene integration by piggybac transposase, and expression of the TetOn3G dox inducible promotor. Three days after electroporation, cells were re-suspended in blast media (+Dox) and transferred to BME2 coated plates and incubated for one week to allow for reprogramming. Cells were maintained by routine passage using trypsinisation with TrypLE to harvest cells and plating onto BME2 coated plates in blast media (+Dox). Differentiation of Human T Cell Derived HE Cell line

[0259] Human T cell derived HE cell lines were washed in PBS to remove residual dox and plated on BME2 coated tissue culture plastic in cytokine rich media (IMDM with 20% FBS, 2mM L-Glutamine, 100 U / ml Penn / Strep, 450 pM MTG, 180 pg / ml transferrin, 0.25ng / ml ascorbic acid,1 % IL-3 (Supernatant), 1% SCF (Supernatant), 5ng / ml mlL-6, 10ng / ml Bmp4, 5ng / ml VEGF, 10ng / ml hlL-2, 20ng / ml IGF-1 (Peprotech, Prod: 100-11), 1 ng / ml FGFb (Peprotech, Prod: 100-18B), 5ng / ml Flt3L (Peprotech Prod: 300-19), 5ng / ml IL-7 (Peprotech, Prod: 200-07), 3uM CHIR99021 , and 4uM SB431542). Samples were harvested by collecting supernatant, washing in PBS, treating with TrypLE and collecting adherent cell fraction.

[0260] Reverse Transcription Quantitative PCR (RT-qPCR)

[0261] RNA from 0.5-2 million cells was extracted from samples using an RNeasy Mini Kit (Qiagen, Prod: 74134) which includes a genomic DNA removal step. Samples were eluted in 20pl. An additional DNA removal step was performed using DNA-free DNA Removal Kit (Invitrogen, Prod: AM1906). 4ul of RNA was used for reverse transcription with MultiScribe Reverse Transcriptase (Thermo, Prod: 4311235) to generate cDNA in a 10pl volume. Quantitative PCR (qPCR) was done with FastStart Universal SYBR Green Master Mix (ROX) (Roche, Prod: 4913850001) in 10ul volumes. 1 ul of cDNA was used per reaction with primer sets at a final concentration of 0.3uM. RT-qPCR was performed on a Roche LightCycler® 96 instrument with a pre-incubation of 10 minutes at 95°C, then 2 step amplification of 95°C for 15s and 60°C for 60s. Additional melt curves were done with a 95°C 15, 60°C for 30s and then increasing slowly to 95°C. Calculated Ct scores were used to determine relative gene expression normalised to a housekeeping gene.

[0262] EHT Limiting Dilution Analysis

[0263] Cells were prepared by serial dilution in differentiation media and then plated in replicate in a BME2 precoated 96-well plate (CytoOne, Prod: CC7682-7596). Cells were serially diluted down to achieve approximately 1 cell per well at the lowest dilution. After 7 days of culture at 37°C and 5% CO2 wells were scored for morphological evidence of haematopoiesis by microscopic inspection. To determine the frequency of EHT events, the fractions of positive wells for each dilutions was scored and processed using software for extreme limiting dilution analysis (ELDA) (Hu and Smyth, 2009).

[0264] Flow Cytometry and FACS

[0265] Cells were washed with 10% FBS PBS, and 2.0x10E6 cells or less were re-suspended in 20ul volumes of conjugated antibodies at 1 :100- 1 :200 dilutions (1-2pg / ml final concentration). The antibodies used to analyse HE and EHT include PE / Cy7-anti-mFlk1 (Biolegend, 136413), APC anti-mTie2 (Biolegend, 124010), SuperBright 436-anti-mCKit (Invitrogen, 62-1171-82), FITC-anti-mCD41 (Invitrogen, 11-0411- 82), PerCP-Cy5.5- anti-mCD45 (eBiosciences, 45-0451-82) PerCP-Cy5.5-anti-hCD34 (Biolegend, Prod: 343611), APC-anti-hCD45 (Biolegend, Prod: 304011). Cells were incubated at 4°C for 15 minutes before washing with 10% FBS PBS and re-suspended in the same buffer with Hoechst 33258 (Invitrogen, Prod: H3569) included for viability analysis. The cells were then analysed by flow cytometry (BD Fortessa), or re-suspended in culture media for sorting. Single stain compensation bead controls were generated for each fluorophore used, as well as an mTmG (mTomato) unstained control. Channel voltages were set for each experiment series to optimise signal. Compensation was performed using the compensation tool in FlowJo v10 to correct for channel spill-over, although this was minimal. A general gating strategy included excluding small granular dead cells and debris with a restrictive FSC-SSC gate, excluding dead cells that are Hoechst positive, then selection of mTomato+ cells to ensure measurements were cells. Cell surface markers were plotted against each other as either dot or contour plots.

[0266] Genotyping DNA with PCR

[0267] Genotyping analysis was done with between 105-106 cells per sample, which were washed in PBS and then re-suspended in 20ul of PBS and frozen at -20°C. To prepare genomic DNA, cells were burst by thermal treatment of 95°C for 10 min, and then cooled to 20°C. 1 pl of 20mg / ml proteinase K was added to each sample, mixed by pipetting, then incubated at 55°C for 30 minutes, treated at 95°C for 8 minutes to deactivate proteinase K, then cooled to 12°C. Samples were then tested by PCR using previously published primer sets containing a common reverse primer and unique forward primer inside the cDNA sequence. PCR of DNA was done in 10p I volumes using 0.5pl of template DNA, 0.25pM for each primer, and 1X PCR master mix. PCR conditions were an initial 5 minute hot start at 95°C, then 35 cycles with 94°C for 20s separation, 60°C for 15s annealing, and 72°C for 60s elongation, followed by a final elongation of 72°C for 5 minutes . PCR products were run on a 1 .5% agarose gel with SybrSAFE at 100V for 30-60 minutes, then visualised on a Biorad UV Imager.

[0268] High-Throughput Screening of Factors that Influence EHT

[0269] Cells were washed and re-suspended with the factors to be tested in differentiation media (10% FBS in IMDM with 10% PFHM-II, 2mM L-Glutamine, 100 U / ml Penn / Strep, 450 pM MTG, 180 pg / ml transferrin, 0.25ng / ml ascorbic acid, 1 % SCF (supernatant), 1 % IL-3 (supernatant), 1 % GM-CSF (supernatant), 1 % TPO (supernatant), 4U / ml EPO, 10ng / ml mCSF, 10ng / ml IL-6, and 10 ng / ml IL-11 , 10ng / ml Bmp4, 3uM CHIR99021 and 4uM SB431542) with 8,400 cells seeded per well in 10Oul in a 96-well plate. After 4 days of culture, cell supernatant was harvested, adherent cells were trypsinised and pooled in a 96-well v- bottom plate. Cells were stained in 20ul volumes with 1 :200 diluted conjugated antibodies, washed and re-suspended in 40pl 2% PBS containing Hoechst 33258 for viability, and CountBright™ counting beads (Thermo, Prod: C36950) for quantitative flow cytometry. 96-well plates were analysed using the high- throughput sampler (HTS) on a BD Fortessa flow cytometer run in high-throughput mode analysing 10pl per sample. Flow cytometry data was analysed in FlowJo V10. Cell RNA 1OX Genomics Platform

[0270] Multiplexing of cells by sample day was achieved by barcoding each sample with distinct oligos attached to LMO anchor and co-anchor following a protocol developed by Chris McGinnis (Mcginnis et al., 2019). A barcode table was generated using the deMULTIplex library (Mcginnis et al., 2019). The reverse transcribed library generated with the 10x Genomics platform was sequenced to obtain 400M reads. The reads were aligned to the mouse genome using the 10X Cell Ranger alignment pipeline to obtain a count matrix. The Seurat library (Butler et al., 2018; Hao et al., 2021 ; Satija et al., 2015; Stuart et al., 2019) was used to create a Seurat assay object with the counts matrix. The LMO barcode data was added by intersecting the barcode table with the distinct cell IDs in Seurat. De-multiplexing was achieved by normalising the barcode table using a centre log-ratio (CLR) transformation (when applicable), and applying the HTODemux method with a positive quantile of 0.99999 to ensure lower error rate. Doublets and negative barcodes were removed. In addition, the data was cleaned by removing poor quality cells (nFeature_RNA > 500, nCount_RNA > 5000, percent.mt < 10, and nCount_RNA < 90000).

[0271] The resulting cleaned demultiplexed Seurat object was analysed using FindVariableFeatures (vst method, and 2000 features). Principle component analysis reduction was performed on the original object, and then again on the resulting subset after removing day 0 before RunllMAP was performed. Gene expressions were visualised using FeaturePlot, VlnPlot, DotPlot, and DoHeatmap functions within the Seurat library. For trajectory analysis the Seurat object was converted to a SingleCellExperiment object, Kmeans clustering was performed, and the slingshot method was applied to the UMAP projection specifying a start and two end point kmeans clusters. Independent trajectories were assigned a metadata variable for pseudotime in the original Seurat object, and trajectory bottlenecks clusters were defined by trial and error before performing differentially expressed genes (DEG) and gene set enrichment analysis (GSEA) analysis. DEG analysis was performed using FindAIIMarkers with a min. pct = 0.1 and a minimum change in expression threshold of 5% (logfc.threshold=0.07). Average expression for each trajectory group were calculated using AverageExpressionQ for DEGs with a p_val_adj<0.05, and then ranked for uploading into GSEA from the Broad Institute (Mootha et al., 2003; Subramanian et al., 2005). GSEA analysis was performed using MH: Hallmark Gene Sets (Mh.all.v2022.1 .Mm.symbols.gmt), and M2: Curated Genes (m2.all.v2022.1 .Mm.symbols.gmt) databases with 1000 permutations. The Top 10 up and down-regulated genes were reported.

[0272] For comparative EHT analysis, both HE cell line and AGM cell reads (Fadlullah et al., 2022) were log- normalised and scaled by a factor of 10,000. PCA analysis was performed and Kmeans clustering was done on the HE data (resolution = 0.3), to select an EHT subset. To identify DEGS, FindAIIMarkers (min. pct = 0.1 , logfc.threshold = 1) was performed. Venn Diagrams were created by comparing both up and down regulated genes using the VennDiagram library. For pathway analysis marker genes with p_val_adj <0.05 were analysed using the enrichR library and the WikiPathway_2021_Human database. Pathways with adjusted p-values <0.05 were reported. Gene expressions were visualised using FeaturePlotQ, VlnPlotQ, DotPlotQ, and DoHeatmapQ functions. EXAMPLE 1: a Clonal HE-Like Cell Line from Mouse ES Cells

[0273] Introduction

[0274] To establish an HE cell line with inducible gene expression we employed a tight tetracycline responsive element (TRE3G) dox inducible promotor (Loew et al., 2010) within a genetic engineering piggybac (PB) transposon vector. The coding DNA sequences for the transcription factors Sox17, Etv2, Foxc2, Tall , Lmo2, Gata2, and cMyc were cloned into this PB-TRE3G vector. The TRE3G promotor and gene of interest in this vector is flanked by piggybac transposon inverted terminal repeat (ITR) sequences allowing for transposition of the sequence into recipient cells, through what can be described as a “cut- and-paste” mechanism. Transposon integrated sequences are typically less susceptible to gene silencing than lentivirus delivered sequences making them a better option to establish a stable cell line.

[0275] In vivo, HE are the progeny of haemangioblasts (HB) which are typically enriched in ES derived embryoid bodies (EBs) at the onset of Flk1 expression by sorting Flk1+ cells (Fehling et al., 2003). A strategy was developed to enrich HBs and then attempt to block the differentiation of the arising HE cells by inducing ectopic gene expression. To enrich HB, we first characterised the timing for emergence of Flk1 expression during EB differentiation for the particular ES cell line used in this work. Getting good HB yields from ES was important to ensure enough cells were available to overcome the likely low frequency of emerging HE cells that were successfully carrying all the introduced genes.

[0276] Design and Cloning of TRE3G Piggybac Transposon Gene Engineering Plasmids

[0277] The TRE3G promotor requires the reverse tetracycline trans-activator (rtTA) to allow dox dependent transcription at TRE3G promotors. The rtTA was cloned under a constitutive expression (EF1a) on a piggybac transposon vector (Miller et al., 2018). Integration into genomic DNA of the different piggyback plasmids was achieved through the activity of the transposase enzyme. For this, a DNA plasmid containing the hyperactive transposase (HyPBase) under a constitutive promotor (Yusa et al., 2011) was co-transfected to achieve transient transposase activity.

[0278] Modification of ES with Inducible Transcription Factors

[0279] Plasmid DNA was introduced into the ES cells by electroporation. An efficient electroporation protocol was developed involving a custom electroporation buffer (100mM Na2HPO4, 27mM NaH2PO4, 5mM KCI, 5mM MgCI2, pH=7.2) and electroporation with a 50pF 600V pulse using a BioRad Gene Pulser Xcell. Successful delivery of plasmid encoding for GFP into ES cells can be seen in Figure 4 which shows that 24 hours after electroporation, more than 60% of the cells were GFP positive measured by flow cytometry. The FSC vs SSC plot demonstrates that 26.5% of all events were small and low in granularity indicating a large fraction of healthy cells after electroporation.

[0280] Having validated electroporation conditions, we then established ES cells with the dox inducible transcription factors. We used mT / mG ES cells (Muzumdar et al., 2007) in our experiments which have a membrane tagged dimer tomato (dTomato) fluorescent marker. Expressing dTomato makes these cells easy to distinguish from control cells by flow cytometry. These were chosen to enable clear identification of ES derived cells for any potential future in vivo studies.

[0281] Mouse mT / mG ES were modified with the piggybac transposon plasmids to establish dox inducible expression of transcription factors. As there is no reporter on the PB-TRE3G plasmids, a basic GFP spike-in plasmid was included in the plasmid mixture at 10% by mass during electroporation. Using fluorescence activated cell sorting (FACS) two days after the electroporation, GFP positive cells were sorted to enrich for cells into which DNA plasmids were successfully introduced. One week after sorting, genomic DNA was analysed to confirm the integration of piggybac transposon vectors. The primer sets shared a common primer in the common piggybac transposon backbone, and had a unique primer within the cDNA sequence for the transcription factor of interest. As shown in Figure 5, bands were observed for all the specific transposon vectors indicating successful integration in the pool of ESCs. This bulk population of genetically modified cells is expected to contain a variety of unique clones with varying vector integration profiles. Instead of attempting to isolate an ES cell clone containing all the vectors, experiments were performed on the pool and cells with desired phenotype were enriched based on their ability to establishing a cell line. This forced evolution by enriching for cells which could establish cell lines in the conditions tested allowed for the fast selection of clones with desirable properties. These modified heterogeneous ESCs were bulked up, saved by cryogenically storing samples, and used in the following experiments.

[0282] Generation of Expanding HE-Like Cell Line

[0283] To express the transcription factors in HE, haemangioblasts (HBs), which readily form HE cells in vitro, were generated, enriched and cultured. Starting with HBs allowed some time for dox inducible expression to occur while HE were formed in vitro. This overcame the challenge of delayed gene expression when adding dox to in vitro cultures. HBs can be generated from ES cells through EB differentiation in which Brachyury+positive mesoderm first emerges, followed by Brachyury+Flk1+cells which contain HBs. EB differentiations from ES cells can be achieved in liquid culture following a previously published protocol (Sroczynska et al., 2009). EB differentiation from ES cells in liquid culture forms tightly packed cluster of cells (Figure 6).

[0284] As HBs emerge at the onset of Flk1 expression, we characterised Flk1 expression emergence in our differentiation conditions to determine the optimal time to harvest EBs and enrich for Flk1+ cells. An EB time-course experiment was performed and EBs were harvested and analysed for the expression of Flk1 at 0.5-day intervals. The results of this time-course (shown in Figure 7) indicate the emergence of a substantial population of Flk1+cells (20.6% of total cells) at 3.5 days of culture. This time point was therefore selected to harvest HBs giving rise to HE.

[0285] To establish an HE cell line, the schematic shown in Figure 8 was followed. In the start of the experiment, EB differentiation cultures were set up with the genetically modified mTmG ES cells containing the TRE3G vectors. After 3.5 days, Flk1+ cells were isolated from EBs using MACS. These Flk1+ cells were cultured in blast media. Cells were cultured with or without dox, and we investigated the impact of ectopic expression of Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc.

[0286] Six days after Flk1+enrichment, cells cultured in the presence of dox were substantially more abundant than cells cultured without dox. With dox inducible expression a 14-fold increase in total cell number was achieved, compared to a total reduction of viable cells without dox (Figure 9a).

[0287] With ectopic expression of Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc, cells appear more abundant, with observable rounded and adherent tight cell structures (Figure 9b).

[0288] Continuous culture of Flk1+cells in blast media with or without dox revealed a strong proliferative advantage in cells expressing ectopic genes, establishing a cell line which proliferated for more than 150 days. To characterise the phenotype of these cells, we employed flow cytometry to investigate the presence of surface markers typically found on HE. The surface marker profile of the HE cell line two months (59 days) after Flk1 isolation is shown Figure 10. Three sub-populations can be distinguished based on their expression of Tie2 and Flk1 . Gates were drawn for three populations and histogram overlays showing expression of Flk1 , Tie2, and c-Kit are shown. The Flk1+Tie2+cKit+population (red) most closely resembled phenotypic HE, which are Flk1+Tie2+. Within the cell population there was also a Flk1+Tie2 cKit population (blue) which was less frequent. There was also a small Flk1 Tie2 cKit+(yellow) population. In conclusion, flow cytometry analysis demonstrates the existence of a promising and persistent Flk1+Tie2+cKit+population which corresponds to a HE population. The existence of a phenotypic HE subpopulation after two months of culture was surprising as HE are transient cells, existing for several days at most in normal in vitro culture.

[0289] Characterisation of the Developmental Potential of Novel HE Cell line

[0290] The developmental potential of the HEs generated above was then assessed. The HE cell line was differentiated in the absence of dox, releasing the HE cells from ectopic gene expression. Specifically,

[0291] HE cells were washed in PBS to remove dox, and then cultured in blast media comprising the TGF-p inhibitor SB431542 and the Wnt activator CHIR99021 , without dox. Differentiated cells were observed due to formation of budding cell clusters and cells with rounded morphology as shown in Figure 11 . Rounded cell clusters emerging from an adherent cell layer appeared to be haematopoietic cells emerging from endothelial cells through EHT.

[0292] A separate differentiation was then performed with more elaborate media composition steps. Initially after dox removal, cells were differentiated in blast media with SB431542, CHIR9902 and Bmp4. After 3 days, media was replaced with blast media without VEGF or D4T supernatant, and additionally containing SB431542, CHIR9902, Bmp4, Activin A, Flt3l, FGF, and SCF. After 5 days the media was replaced with the same media as the previous step but without SB431542, CHIR9902, or Bmp4. Analysis of the suspension fraction of differentiated cells after 7 days demonstrated the presence of both endothelial cells expressing the surface markers Flk1 and Tie2, and haematopoietic cells expressing the early blood marker CD41 and pan-haematopoietic marker CD45 (Figure 12A). The parental HE cell line cultured in the presence of dox does not express CD41 or CD45 demonstrating the emergence of haematopoietic cells after the removal of dox. Within the differentiated cells, both single positive CD41+CD45- and double positive CD45+CD41+ are present, which correspond to consecutive developmental stages of blood generation. Differentiated haematopoietic cells also expressed the common haematopoietic progenitor marker c-Kit. These results demonstrate the successful establishment of an expanding HE cell line that maintains its developmental potential to undergo EHT and generate blood cells.

[0293] The haematopoietic developmental potential of the HE cell line was further characterised. Colony forming cell (CFC) assays were used to evaluate multi-lineage potential. Cells were re-plated after 7 days of differentiation culture in cytokine rich semi-solid media for one week and colony forming cell type contribution were scored by morphology. CFC assays performed on the haematopoietic cell progeny from the HE cell line formed a variety of colony types including multi-potential CFU-GEMM. The HE cell line derived colonies were compared to colonies derived from mouse bone marrow as a control. Morphology of a CFU-GEMM scored colony derived from the HE cell line and one from mouse bone marrow is shown in Figure 12B, left images. These colonies have a variety of cell types based on size and morphology, contain a large number of cells, and have a dark red colour in the middle, in line with the CFU-GEMM identity. The presence of multi-lineage colonies demonstrate the multipotent potential of the HE cell line. The frequency of colonies identified is similar between HE cell line and bone marrow. A variety of single cell type colonies and multi-potential colonies were also identified. The frequency of colonies generated from the HE cell line after 7 days of differentiation was 116 per 100,000 cells, which is in the same order of magnitude as wild-type bone marrow (296 per 100,000 cells), suggesting HE derived haematopoietic progeny have substantial multi-potential CFC potential (Figure 12B, right graph). Demonstrative examples of all CFC colony types scored are presented in Figure 13. The morphology of the CFU-GEMM, CFU-GM, CFU-G, and CFU-M colonies are in agreement with expectations, however the BFU-Es appear more tightly packed and resemble human BFU-Es instead of mouse BFU-Es. The erythroid attributes of these BFU-Es was confirmed by visual observation of a rich maroon red colour, similar to CFU-GEMMs, giving confidence that these are indeed BFU-Es. Generating multi-lineage colonies after dox removal indicates that expanded HE cells can re-enter developmental haematopoiesis, even after expansion as an HE cell line for several months.

[0294] EXAMPLE 2: Isolation and differentiation of clonal HE-like cell lines

[0295] Having demonstrated that the bulk HE cell line had the potential to generate haematopoietic cells, efforts were directed to isolate and characterise a clonal HE cell line with the same potential. The bulk HE cell line was cloned by limiting dilution, seeding several 96 well plates with an average seeding density of 0.5 cells per well. After about one week, wells were screened by microscopy to identity wells with only one clone and these were taken forward by serial passaging. Some instances of multiple clones in one well were observed, identified by two distinct adherent cell clusters in one well, and these were not carried forward. After expansion, 41 clonal cell lines were banked and genotyped for ectopic gene integration. A ‘fingerprint’ metric was developed for comparative analysis by calculating the relative percentage of gene integration for a specific gene by measuring each gene by qPCR and assuming equal PCR efficiency between all genes. Although the assumption of equal PCR efficiency is not completely correct, it allows for a useful comparative metric to compare clones as the PCR efficiency for a given gene is expected to be consistent between PCR reactions.

[0296] Five unique fingerprint identities were detected with unique integration profiles. The similarity between fingerprints identified to share the same unique fingerprint identity suggested these clones came from a common parental cell or share an integration pattern compatible with expansion. One unique fingerprint (clones: 1 , 2, 3, 4, 5, 6, 7, 37, 38, & 40) carried all the transcription factors introduced, confirming the presence of cells with the complete transcription factor combination. Analysis of the gene fingerprint genes of all the clones tested demonstrated consistent integration of Gata2, Etv2, c-Myc, Tall , and Foxc2.

[0297] It was decided to identify the gene fingerprint that was responsible for the multi-lineage haematopoietic colonies previously observed. Genotype analysis of gene integration was performed on multi-lineage CFC colonies (generated in semi solid media and therefore clonal) to identify the HE cell line clone that has the ability to form multi-lineage haematopoietic progenitor cells. The fingerprint signatures for the cell lines were matched to the genotype fingerprints of genotyped colonies. As shown in Figure 14 the clonal cell line 1 , named HE-B02-C01 , had a similar genotype fingerprint to a multi-lineage CFU-GEMM colony. Comparative genotype analysis suggested that the multi-potential colony and this clonal cell line share a common parental cell, and the clonal cell line should have equivalent colony forming developmental potential. This multi-lineage CFC and isolated clonal HE line contained the dox inducible vectors expressing Tall , Lmo2, Gata2, Etv2, Foxc2, Sox17, and c-Myc. A phase contrast image of the clonal cell line indicated an adherent and tightly packed phenotype (Figure 14). Clonal HE cell line 1 (HE-B02-C01) was the first clone identified of many with equivalent gene integration fingerprints and was used in further experiments as a representative clone. Only one clonal cell line among equivalent integration fingerprints was characterised further to reduce experimental work.

[0298] This clonal HE cell was differentiated to confirm its capacity to undergo EHT and generate haematopoietic cells. Culture of the clonal HE cell line in cytokine rich media (Big Mix) with 4uM SB431542 and 3uM CHIR99021 after dox removal resulted in expression of endothelial markers Flk1 and Tie2. The emergence of haematopoietic cells was confirmed by the presence of cells expressing the common haematopoietic marker CD45 and not the endothelial marker Flk1 (Figure 15). The presence of CD45+FlkT which represent 38% of the cell population after 12 days of differentiation confirms the haematopoietic developmental potential of the clonal HE line. At day 12 there is also the sustained presence of Tie2+Flk1+cells, which are likely endothelial cells (Figure 15). This demonstrates that the HE cell line can differentiate into either haematopoietic cells or endothelial cells after dox removal. EXAMPLE 3: Characterisation of clonal mouse HE cell line

[0299] Improving Endothelial to Haematopoietic Transition (EHT) from Clonal HE-Like Cells

[0300] It was next decided to explore media components that could impact haematopoietic yield using a 96-well plate based screening assay leveraging high-throughput flow cytometry.

[0301] A high-throughput differentiation screening assay was developed in 96-well plate format where conditions could be tested and then analysed by flow cytometry using a high-throughput sampler (HTS) on a BD Fortessa flow cytometer. This assay was used to screen many pre-treatments and differentiation conditions with a variety of molecules in an initial attempt to optimise the differentiation of the HE cell line. Ly294002 (Ly29) was included, which is a PI3K inhibitor that has been shown to promote lymphoid development (Park et al., 2018). Resveratrol was included as it has been characterised to promote Notch signalling activity (Pinchot et al., 2011). Bmp4, Activin A, and FGF2 were included as they have been shown to be important for establishing blood generating HE from ESCs (Pearson et al., 2015). MAT inhibitor Cycloleucine and SAHH inhibitor D-Eritadenine (DE) were also investigated which are both involved in histone methylation (Lee et al., 1992; Zhou et al., 2015) which is important for epigenetic changes involved in differentiation. Initial experiments with resveratrol, Ly294002, and cycloleucine showed cell toxicity at published concentrations so dose response toxicity assays were performed that determined optimal working concentration of 5pM Resveratrol, 1 pM Ly294002, and 5pM Cycloleucine (Figure 16A). These working concentrations were determined to be the highest tolerable concentration without resulting in a decrease in proliferation.

[0302] For the screening experiment, cells were pre-treated in a variety of different pre-treatment maintenance media formulations (with dox) and the seeded in a variety of differentiation media formulations (without dox) in a 96 well plate. After 9 days of culture, cells were harvested and stained for an endothelial marker (Flk1) and a haematopoietic marker (CD45) in a 96-well v-bottom plate. Cells were then analysed by quantitative flow cytometry using counting beads to determine the total number of blood cells generated in each well. Each measurement of total CD45+FlkT cells for a given condition was normalised against the total number of CD45+FlkT cells measured for the control which was maintenance in blast media and differentiation in big mix cytokine rich media (with 4uM SB431542 and 3uM CHIR99021). The relative number of blood cells (CD45+FlkT) measured for a given condition are presented in Table 1 below.

[0303]

[0304] Table 1 : Relative haematopoietic cell output from high-throughput screening of differentiation of clonal HE cells. The addition of Bmp4 to the standard big mix media used during differentiation increased the output number of haematopoietic cells by roughly 100 fold. Another clear improvement was the pre-treatment of cells with either 4pM Ly294002, or 1 pM Ly294002 in combination with 20ng / ml Bmp4. These conditions improved the total number of blood cells generated by 36 fold and 59 fold respectively when differentiated in normal big mix differentiation media. This improvement was not additive with Bmp4 during differentiation as pre-treatment could not improve the total blood cell generation beyond no pre-treatment when differentiated with Bmp4. Another observation is the impact of cell density on blood generation; reducing the seeding cell number from 50,526 to 33,684 cells in a 96-well plate reduced the total number of blood cells back down to blood yields observed without the addition of Bmp4.

[0305] A summary of the impact of Bmp4 as a pre-treatment and during differentiation is presented in Figure 16B. The addition of Bmp4 to the differentiation media improved the measured blood cells by several orders of magnitude (-100 fold, Figure 16B). Pre-treatment with Bmp4 did not appear to influence this efficiency when differentiated under normal conditions. The combination of pre-treatment and differentiation with Bmp4 resulted in a similar fold increase in blood cells. This suggests the impact of Bmp4 may be restricted to the differentiation step. This suggests an important role for Bmp4 signalling to establish the EHT trajectory from the HE cell line, and also that Bmp4 addition prior to differentiation does not significantly affect the magnitude of blood generation. Visual inspection of differentiating cells with and without Bmp4 suggests that EHT events are more frequent with increased frequency of haematopoietic clusters (Figure 16B). This increase in emerging haematopoietic clusters with Bmp4 addition was reproducible in separate biological differentiation experiments giving confidence to its impact on EHT propensity and not proliferation after emergence. Having shown that Bmp4 could increase haematopoietic cell generation, it was included in differentiation media as standard when differentiating the HE cell line.

[0306] Generation of LSK-SLAM Cells

[0307] The extensive blood forming potential of the HE cell line encouraged exploration of the haematopoietic stem cell (HSC) potential of these cells. For this, we attempted to generate HSCs from the HE cell line. We employed a VeraVec™ co-culture strategy. Specifically, differentiating HE cultures were moved to VeraVec™ co-cultures after 2 days of differentiation, and then cultured in the absence of serum with cytokines. The differentiation protocol included initial culture on basement membrane extract 2 (BME2) coated tissue culture plastic (TCP) in big mix media with Bmp4, SB431542, and CHIR99021 for 2 days. Then cells were moved to a VeraVec™ co-culture in serum free Stemspan media with knock-out serum replacement (KOSR), FGF, SCF, SB431542, and CHIR99021.

[0308] Haematopoietic stem cells are commonly characterised by flow cytometry based on a lack of lineage commitment markers (Lin-), and expression of both Sca-1 and c-Kit, commonly referred to as LSK. At day 10, cells contained a significant proportion of CD45+ blood cells with some having a LSK phenotype (Figure 17A). This warranted a more comprehensive analysis of phenotypic HSCs employing additional markers such as SLAM family of receptors with HSCs further enriched in CD150+CD48_. In a separate experiment, cells were differentiated following a similar protocol with the exception of a minimum (Min) media used instead of Big Mix that had fewer cytokines to prevent progenitor specification. The differentiated cells were analysed for LSK markers, as well as the addition of SLAM markers CD150 and CD48 and compared with wild-type bone marrow. As shown in Figure 17B, LSK-SLAM blood cells were generated from the clonal HE cell line that resemble LSK-SLAM HSCs from mouse bone marrow. A comparison of total frequency between cell line derived cells and mouse bone marrow shows comparability with 0.0074% Lin-Sca1+cKit+CD48-CD150+ in differentiated HE cell line derived cells compared to 0.0039% Lin-Sca1+cKit+CD48-CD150+ in mouse bone marrow. This measured frequency in bone marrow is similar to 0.0066% reported previously (Kiel et al., 2007).

[0309] HE Cell Line Differentiation

[0310] It was then decided to investigate the transcriptome of the cell line at various stages of differentiation. To do this, multiplexed single RNA sequence (scRNA-Seq) was employed using the 10X genomics platform and LMO barcode multiplexing. The EHT trajectory was also characterised by assigning cells from the differentiation time-course along a developmental trajectory based on a global characterisation of their transcriptome.

[0311] The HE cell line and its progeny from a differentiation time-course were analysed concurrently by 10X. Cells from day 0, day 1 , day 3, and day 9 of differentiation obtained with three differentiation protocols were harvested for analysis (Table 2, below).

[0312] Table 2: In vitro differentiation experiment for analysis by multiplexes scRNA-Seq

[0313] Three different differentiation conditions were included in the dataset to increase our understanding of the developmental potential of the cell line with alternate differentiation strategies. To investigate the impact of days between passage under routine cell maintenance, samples seeded with dox at either 1 , 3, or 5 days prior to harvest were also included. The final day 9 differentiation cultures were seeded into CFC assays to confirm the presence of multi-lineage colony forming cells. The differentiation protocols yielded cell populations with varying colony forming capacity with the addition of Bmp4 increasing colony forming output from 0.2% to 4.5% of cells, and co-culture with Veravec cells increasing it further to 11% of the final culture population (Figure 18). These cell products were used in the multiplexed scRNA-Seq experiment.

[0314] Multiplexed scRNA-Seq of Differentiation Experiment

[0315] Single cell RNA sequencing (scRNA-Seq) was performed using the 10X microfluidic platform. This involves the generation of single cell droplets, in which reverse transcription of RNA is performed that introduces a unique cell barcode, and then the cDNA is read by next generation sequencing (NGS). Multiplexing of the scRNA-Seq run was achieved by introducing an additional sample LMO barcode for different cell samples allowing for determination of a sample identity by de-multiplexing LMO barcodes. Unlabelled cells were identified and removed from the analysis. Doublets were also identified as cells containing more than one barcode. Doublet removal through de-multiplexing is an advantage of the technique, as doublets are normally identified solely by read count number, but here the detection of multiple barcodes confirms the presence of two cells in a 10X emulsion droplet.

[0316] All the cells from Day 0, 1 , 3, and 9 were pooled and clustered using dimensional reduction to achieve a uniform manifold approximation and projection (UMAP) shown in Figure 19. The cells formed distinct clusters, which correlate to some extent with differentiation time. Cells from day 0 formed a unique cluster, whereas cells from day 1 , 3 and 9 appeared to form a continuum of development that bifurcates into two distinct directions. There also appeared to be another unique cluster of cells which were made up of predominantly day 9 cells. 10X RNA sequencing by NGS allows for the non-biased discovery of cell types present in a sample as the technique indiscriminately analyses RNA present in the cell. Utilising the library SingleR (Aran et al., 2019) the RNA transcriptome of all the cells were compared to a mouse cell reference data set (Heng et al., 2008). This allowed for the non-biased classification of each cell by its most closely related cell type based on correlative analysis. The tool classified cells into mostly endothelial, stem and progenitor cells, macrophage and neutrophil cells. These identities were projected onto the UMAP space which allows interpretation within the context of differentiation time. Early time points day 1 and day 3 were mostly classified as endothelial cells, which is in alignment with expression of endothelial markers Tie2 and Flk1 at that stage by flow cytometry. The cells at day 9 were mostly identified as endothelial cells, stem and progenitor cells, macrophages, and neutrophils (Figure 20).

[0317] More discriminate cell type correlations were performed with SingleR and the same mouse reference dataset to reveal subtypes within the hierarchical classifications already presented based on correlation scoring (Figure 20). It is important to consider that the alignment to different cell types depends on the reference library used and these classifications serve as a guide within broader analysis. The majority of cells identified as stem and progenitor cells were classified as granulocyte-macrophage progenitors (GMPs), multi-lymphoid progenitors (MLPs), or monocyte-dendritic cell progenitors (MDPs), as well as at least some cells identified as long-term haematopoietic stem cells (LTHSCs).

[0318] The broad cell clusters identified by SingleR were used to identify differentially expressed genes (DEGs) which were ranked. These identified genes were used to explore notable genes for each cell type. A summary of some key genes identified by DEGs analysis or manually selected based on genes expected in emerging haematopoietic cells are shown on the UMAP projection in Figure 21A. Endothelial cells can be visualised by expression of VE-Cadherin. Haematopoietic cells express Runxl which is critical for definitive haematopoiesis during EHT and Spi1 which is involved in HSC maintenance as well as GMP and B-cell lineage commitment. Cells also express CD41 which is a marker for early haematopoietic commitment and mature megakaryocytes. Gene expression analysis also demonstrates expression of the macrophage marker Maf and Lcn2 which is a Neutrophil associated gene. The presence of these genes suggests that both endothelial and haematopoietic progenitor cells are present and generated from the HE cell line. The progeny of the cell line appear to be preferentially biased to GMP, which could be an inherent bias of the cells or due to the culture media, which may support these progenitors preferentially.

[0319] Analysis of haemoglobin gene expression (Figure 21 B) showed that erythroid genes were expressed. The haemoglobin was mostly adult haemoglobin, which is restricted to definitive haematopoiesis.

[0320] Orthogonal cell characterisation was done to reaffirm the findings of scRNA-Seq. The haematopoietic progeny of the clonal HE cell line were characterised by flow cytometry and cytospin analysis. For flow cytometry analysis, cells were stained with mature blood markers CD11 b, which stains monocytes, macrophages, natural killer cells and granulocytes, Gr-1 , which stains monocytes and neutrophils, and Ter119, which stains maturing erythroid cells. As shown in Figure 22A, these markers are present on differentiated progeny suggesting the presence of maturing blood progenitors. A large population of CD11b was observed including both Gr-1 positive and negative cells. Regarding the presence of red blood cells, erythroid cells decrease in size as they mature and express the erythroid marker Teri 19. A population of small non-granular cells (low FSC & SSC signal) was detected with expression of Ter119+ and not CD11 b, Gr-1 , or CD45, which indicates these cells are indeed maturing erythroid cells or red blood cells. Further, the cell pellets from these cultures were red indicating the presence of haemoglobin (Figure 22B). The microscope image in Figure 22C confirms the expected morphology of the various detected cells. Macrophages are distinctly visible due to their large and granular nature. RBC are also clearly visible as very small and round cells as detected by flow cytometry.

[0321] To further confirm the morphological identity of the cells, May-Grunwald-Giemsa (MGG) stain and cytospin was performed (Figure 23) on the differentiated cells. Large granular macrophage cells were clearly identifiable. Neutrophil progenitors, a type of granulocyte are also distinguishable based on their distinctive multi-lobe nuclei and lightly stained cytoplasm. The visible detection of neutrophils confirms their detection by scRNA-Seq analysis. RBCs were also distinguishable based on the lack of nucleus, as RBCs lose their nucleus and have only small residual RNA that can be detected during the final stages of erythropoiesis. Collectively, we conclude that the HE cell line can undergo EHT and generate macrophages, granulocytes, and RBCs with adult haemoglobin. These are the progeny of definitive haematopoiesis, which confirms the HE cell line undergoes definitive haematopoiesis.

[0322] HE Reprogramming / Reverse Differentiation

[0323] It was next decided to investigate if the transcription factors identified to block cells in a HE state could reprogram cells to HE. To test the potential to derive HE cells from mature cells, differentiated blood cells derived from the clonal HE cell line were assessed for their ability to re-establish a HE cell line.

[0324] Mature haematopoietic cells were enriched for expression of Gr-1 or CD11 b (confirmed to be CD45+) from day 10 differentiated blood cells derived from the clonal HE cell line using MACS (Figure 24). These Gr-1+CD11 b+CD45+cells were then re-introduced into the blast media with dox used for routine maintenance of the HE cell line. Culture of these mature blood cells in normal proliferation blast media with dox established a reprogrammed (reverse differentiated) HE cell line designated HE-B02-C01-REV. These reprogrammed cells re-established their parental surface marker profile, expressing low levels or Tie2 and losing haematopoietic markers Gr-1 , CD11 b and CD45 as shown in Figure 24.

[0325] The reprogrammed (reverse differentiated) cells were then re-differentiated and surprisingly the cells underwent differentiation faster and more efficiently (Figure 25). Contrasting with the differentiation of the parental cell line (Figure 15) a significant population of haematopoietic cells (CD41+FlkT) is detectable in the reprogrammed cell differentiation cultures by day 3. By day 7, the roughly 60% of cells were haematopoietic (CD41+FlkT).

[0326] To define this improvement in EHT efficiency further, limiting dilution assay (LDA) differentiation experiments were performed with the reverse differentiated (REV) line and its parental control (CTRL). These experiments tested the lowest number of cells in a well that can establish EHT and haematopoiesis. Serial dilutions of cells were plated in technical replicates in a 96-well plate in differentiation media. After one week the cells were manually scored for the presence of differentiated rounded blood cells and the fraction of nonresponding cells was used to determine the frequency of EHT events following extreme limiting dilution analysis (ELDA). The results are a summary of 3 biological replicates, each with 4 technical replicates per condition (Figure 26). The reverse differentiation of cells endowed a 200-fold increase in EHT frequency. The estimated frequency of EHT went from 1 :3438 cells to 1 :16 cells. This increase in frequency was clear as wells with just 16 cells plated had detectable haematopoiesis. Gene expression analysis on the control and reverse cell line was performed by qRT- PCR and revealed a 30-fold increase in bulk Runxl mRNA expression between REV and CTRL cells. The higher level of base-line Runxl expression reflects the higher proportion of cells that undergo EHT. This may suggest that the increase in EHT efficiency is a result of higher base line Runxl expression.

[0327] Chemical Screening Analysis with REV line

[0328] Due to the ease of generating large quantities of REV HE that undergo EHT efficiently, large chemical dilution experiments can be done with large libraries of molecules. To explore this potential, differentiation experiments were performed for serial dilutions of various chemicals in 96-well plate format, and the cells were analysed at Day 4 by FACS with the BD high throughput sampler (HTS) system. Factors which influence the percentage of blood cells are shown in Figure 27.

[0329] CHIR99021 improved both the total blood cell number and percentage of blood in the cultures. CHIR99021 is a GSK-3 inhibitor, which increases the stability of p-catenin and therefore emulates Wnt signalling. In contrast, both FGFb and VEGF reduced the percentage of blood cells after 4 days. However, analysis of total cell number indicates this may be a dilution effect from the supportive effect of the chemical on endothelial (Tie2+) cell proliferation. KDM1A / LSD1 inhibitor blocked the formation of blood, while not affecting the total endothelial cell number. The LATS1 and LATS2 inhibitor GA-017 (Hippo Inhibitor) dramatically decreased the ability to generate blood, while increasing endothelial cell proliferation. This demonstrates that sustained YAP activity, through inhibiting hippo signalling, can block EHT.

[0330] Flow Cytometry Analysis of Time-Course Differentiation

[0331] The dynamics of EHT was further explored through a time-course analysis. A 7-day differentiation timecourse experiment was performed analysing cells concurrently by flow cytometry, CFC assays, and multiplexed scRNA-Seq 10X analysis. A differentiation time-course was set up by staggering differentiation cultures so that 7 days of differentiation could be harvested at the same time.

[0332] Representative flow cytometry plots are shown in Figure 28A for the 7-day time-course. The development of blood through EHT transition can be observed clearly in the flow cytometry data. The endothelial marker Tie2 is present on the majority of cells after one day of dox removal, representing the starting endothelial population. Tie2+ HE cells then upregulate CD41 expression then lose the expression of Tie2 to establish CD41+Tie2- early blood progenitor cells. These blood cells then acquire the expression of the more mature blood marker CD45, and then some cells become positive for the myeloid marker CD11 b by day 7. Quantitative analysis of the flow cytometry measurements (Figure 28B) achieved by including counting beads, reveal a transient Tie2+CD41+ population established between days 2-3, and another transient Tie2-CD41 + population which increased rapidly at day 5. The acquisition of mature blood marker CD11 b is observed at day 6 and represents a highly proliferative blood progenitor cell. The dynamics of surface marker expression is in agreement with expectations based on normal HE development from ES cells. The flow cytometry data provides a reference for the other experiments performed concurrently.

[0333] Colony forming cell assays were performed on differentiated cells throughout the time-course to evaluate the emergence of colony forming cell potential. The peak of colony frequency was at day five with 293 / 10,000 cells plated (2.9%) (Figure 29). The timeline of this emergence correlates with emerging blood cell population Tie2-CD41+CD45- (shown on the secondary axis) which emerges rapidly at day 5 of differentiation. This correlation between early blood emergence and colony forming potential aligns with the multi-potential status of the earliest forming blood cells.

[0334] Multiplexed scRNA-Sequencing of Time-Course Differentiation

[0335] The flow cytometry results for the time-course differentiation demonstrate the presence of both endothelial cells and haematopoietic cells at various stages of development. To confirm the endothelial origin of early haematopoietic cells, single cell RNA sequencing was performed. Using this technique, cells are indiscriminately correlated to similar cells and can be used to construct a developmental trajectory. Further, scRNA-Seq investigation of our trajectory analysis allowed for the exploration of EHT dynamics and determinants of the blood forming process.

[0336] Multiplexed single cell RNA-Sequencing was performed for all samples in the 7-day differentiation timecourse using HTO barcoding and the 10X scRNA-Seq platform. Once cleaned, the dataset was analysed by principle component analysis (PCA), sub-plotting the cells by day for early time-point samples. Cells from the day 0 time-point formed a distinct population of cells independent of the day 1 cells, indicating that removal of dox quickly changes the transcriptome profile of the cells.

[0337] A uniform manifold approximation and projection (UMAP) for cells between Day 1 and Day 7 demonstrated a clear transition between either endothelial or haematopoietic endpoints in development (Figure 30). Sub-plotting of cells by differentiation day showed a clear starting endothelial cluster with Cdh5 (VE-Cadherin) expression. Cells appeared to develop either to haematopoietic lineage with upregulation of Runxl and CD41 (Itga2b), or toward an endothelial phenotype with higher expression of Cdh5 (VE-Cadherin). This dimensional reduction analysis indicates a connected trajectory between endothelial and haematopoietic cell identities, which aligns with the early expression of blood markers occurring at day 2 and day 3 of the time-course analysis by flow cytometry.

[0338] Trajectory Analysis of HE Cell Line and Cell Fate Decisions

[0339] Trajectory analysis was performed using the slingshot package (Street et al., 2018) within the UMAP projection from the clonal HE differentiation time-course and is shown in Figure 31 A. To generate this pseudotime result a starting population and two terminal populations were confidently defined utilising differentiation time information available from our multiplexed approach as shown previously in Figure 30. The ability to confidently define the start and end points in trajectory analysis is an advantage utilising a multiplexed time-course dataset generated from a cell line. The trajectory analysis demonstrates a lineage commitment step and subsequent development into either endothelial or haematopoietic lineage. Plotting the average expression throughout the haematopoietic trajectory pseudotime demonstrates EHT clearly with a down-regulation of endothelial genes VE-Cad and Sox17, and the upregulation of Runxl and CD41 , with expression of CD45 following (Figure 31 B). The cell cycle gene Cdk1 is transiently expressed during this transition between cell fates.

[0340] This pseudotime analysis allowed for the selection of cells which appeared to be in a transition zone toward either cell fate (Figure 32A). Comparing cells in these two “bottlenecks” using differentially expressed genes (DEGs) allowed for comparative analysis between genes upregulated in either trajectory. A summary of the top 10 DEGs between the bottleneck clusters identified Sox17 and Edn1 in the endothelial cluster and Birc5, and H2afz in the haematopoietic cluster (Figure 32B).

[0341] To explore the large dataset of DEGs, gene set enrichment analysis (GSEA) was performed on cluster averaged expression using software available from the Broad Institute and UC San Diego (Mootha et al., 2003; Subramanian et al., 2005). The top 10 Hallmark (MH), and Curated (M2) gene sets upregulated in the haematopoietic or endothelial bottlenecks are shown in Tables 3 and 4, respectively.

[0342]

[0343] Table 3: GSEA analysis: Genes upregulated in haematopoietic trajectory bottleneck cluster.

[0344]

[0345] Table 4: GSEA analysis: Genes upregulated in endothelial trajectory bottleneck cluster.

[0346] Enrichments in the haematopoietic transition bottleneck included genes in G2M checkpoint, mitotic spindle, and mitosis Lin 9 targets, which are all involved in the G2M phase of the cell cycle. For the endothelial bottleneck, genes enriched include TGFp and TNFa signalling targets.

[0347] To explore the role of cell cycle in EHT, a fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter (Sakaue-Sawano et al., 2008) was employed. The FUCCI system has two separate components, which can be introduced into cells to either fluorescence red when the cells are in G1 phase, or green when they are in S / G2 / M phase. As the HE cells were derived from mT / mG ES cells, which are already dTomato, only the green vector was introduced. The green vector (mAG-hGem) includes a constitutively expressed monomeric Azami Green (mAG) protein fused to a truncated human geminin (hGem) protein with an intact destruction box and nuclear localisation signal, but without the Cdt1 binding domain limiting its activity (Sakaue-Sawano et al., 2008) (Figure 33A). This allows the fluorescent fusion protein to be present during the S / G2 / M phase of the cell cycle but reduces its ability to influence the cell cycle through Cdt1 . The mAG-hGem vector has a selection marker, and cells integrating the vector were selected with puromycin. As haematopoietic cells emerge from the HE cell line early in differentiation cultures, cells were analysed at an early time-point of differentiation. Comparison of cells at day 1 of differentiation demonstrates that CD41+cells are 5-times more likely to be in the S / G2 / M phase of the cell cycle than CD4T cells (Figure 33B). This suggests a correlation between cell cycle and the early stages of EHT. Expression of the mature blood marker CD45 was also measured in CD41+cells suggesting that cells maintain the ability to undergo haematopoiesis (Figure 33C). We also, surprisingly, observed that cells with the reporter have an increased efficiency and speed of EHT compared to the parental line. Cells expressing the reporter have a 10-fold increase in blood after 2 days (46% vs 4%), and by day 3 more than half the cultures had become CD41+Tie2_suggesting a much higher proportion than 1 :16 cells in the culture undergo EHT (Figure 33C).

[0348] The differential gene expression of the two trajectories of the HE cells was then analysed to identify potential determinants of cell fate decisions. Highly up-regulated in the endothelial trajectory was the microRNA Mir22hg (Figure 34A). Haemoglobin analysis was also performed on all the cells expressing haemoglobin from the time-course analysis (Figure 34B). The majority of haemoglobin detected was beta adult s chain haemoglobin (hbb-bs).

[0349] Comparing HE Cell Line with HE from murine AGM

[0350] \Ne next compared the HE cell line with HE cells from murine aorta-gonad-mesonephros (AGM) and contrasted the EHT they both undergo. To do that, a recently published scRNA-Seq dataset generated from primary cells obtained from mouse AGM (Fadlullah et al., 2022) was utilised. To obtain a comparable subset of cells for analysis we performed kmeans clustering analysis on the HE cell line dataset and identified a subset of clusters identified to be involved in EHT. Four clusters were identified (boxed) as cells undergoing development toward blood. These clusters were pooled into one subset for comparative analysis.

[0351] Both the HE cell line subset and the primary AGM cell subset independently underwent new k-means clustering and dimensional reduction to a UMAP space as presented in Figure 35A. Three k-means populations were identified for each subset as “C1 ”, “C2”, and “C3”. Expression of VE-Cadherin, Runxl , Gfi 1 , and Gfi 1 b in these populations was characterised to confirm the developmental maturity of each population and similarity between equivalent populations in REV HE and AGM cells (Figure 35B). Preliminary analysis indicated similarities in expression of these four markers between subsets and “C1”, “C2”, and “C3” represent successive cell populations that progress through EHT.

[0352] Differentially expressed gene analysis was performed between C1 and C2 populations for both subsets to characterize early stages of EHT. Comparison of genes up-regulated and down-regulated during the first stage of EHT for both AGM and the REV HE cell line identified 92 shared downregulated genes and 54 shared up-regulated genes (Figure 36A). Dot plots of some of these genes are visualized in Figure 36B where a loss of endothelial genes (Cdh5, Pecaml , Sox17, Ece1), and increase in haematopoietic genes (Runxl , Spi1 , Myb) is apparent. Gene pathway enrichment was performed with these shared differentially expressed genes indicating that both HE shared the downregulation of focal adhesion, Notchl regulation of endothelial cells, and VEGF signalling, as well as upregulation of haematopoietic stem cell differentiation (Figure 36C).

[0353] EXAMPLE 6: Reprogramming Human T-Cells to Create an HE Line

[0354] Culture and Genetic Engineering of Primary Human T-Cells

[0355] It was decided to examine if human T cells could be reprogrammed into a human HE cell line with definitive haematopoietic potential.

[0356] Primary peripheral blood mononuclear cells (PBMCs) were thawed and cultured in the presence of activating CD3 / CD28 beads with IL-2 for 3 / 4 days to activate and enrich for T cells. This simple expansion procedure enriches T cells to >95% purity. The T cell populations present were 66% CD4+, 28% CD8+, and 3% CD4+CD8+T cells.

[0357] Cells were then electroporated using a Neon electroporation system after 4 days of activation. To confirm successful gene editing with our protocol a constitutive GFP transposon vector was used as a control in a separate experiment and the GFP expression was measured 6 days after electroporation, at which point GFP expression is likely from integrated DNA and not transient expression from the plasmid introduced into the cells. Electroporated cells were stained for T-cell markers CD4 and CD8 to understand the composition of cells successfully genetically modified. As shown in Figure 37A, 23% of all cells were successfully genetically modified to express GFP. Comparing the representation of T cell populations between all cells and GFP+ cells suggested that CD8+ T-cells were preferentially modified where 91% of all modified cells are CD8+, compared to 78% of all cells, however all cell types appeared to have been modified and are present.

[0358] Establishment of Human T-Cell Derived HE Cell Lines and Preliminary Characterisation

[0359] After confirming successful genetic engineering in a control GFP experiment, we used electroporation to introduce dox inducible (TRE3G) piggybac transposon vectors containing human cDNA for Sox17, Etv2, Foxc2, Tall , Lmo2, Gata2, and cMyc into primary human T cells activated for 3 days. We also included a constitutively expressed rtTA piggybac plasmid to achieve dox inducible gene expression, and an expression plasmid for HyBase transposase to achieve transposition of the target genes stably into the genome. After allowing 2 days for the cells to recover and express the rtTA, cells were cultured in the same blast media used for mouse derived HE with and without dox.

[0360] Addition of dox yielded cultures with a semi-adherent appearance compared to no cell growth without dox addition. Cells lines were derived in multiple wells of a 6-well plate and kept separate as different bulk cell lines. These bulk cell lines were tested for their ability to generate haematopoietic cells by culture in cytokine rich media without dox. After 6 days of differentiation one cell line generated colony like structures (Figure 37B), and flow cytometry analysis indicated that after 6 days cells expressed both CD34 and CD45 (Figure 37C), indicating that these cells have blood forming potential. EXAMPLE 7: Establishment of Human ES-derived HE Cell Lines

[0361] Human ES cells were electroporated to introduce dox inducible (TRE3G) piggybac transposon vectors containing human cDNA for Sox17, Etv2, Foxc2, Tall , Lmo2, Gata2, and cMyc. We also included a constitutively expressed rtTA piggybac plasmid to achieve dox inducible gene expression, and an expression plasmid for HyBase transposase to achieve transposition of the target genes stably into the genome. The electroporated cells were differentiated as embryoid bodies according to the protocol described in Garcia-Alegria et al. in the absence of dox. CD317Cdh5+cells were isolated from day six embryoid bodies by flow cytometry and cultured in HE media, where the seven factors were activated by doxycycline induction, generating the human HE (hHE) cell line. The HE media comprised StemSpan media supplemented with 5 ng / mL FGF2, 25 ng / mL IGF1 , 25 ng / mL IGF2, 50 ng / mL SCF, 50 ng / mL TPO, 5 ng / mL IL11 and 20 ng / mL FLT3-L. These cells were expanded over 15 passages over a total of three months before dox removal. Upon doxycycline withdrawal, hHE cells began expressing the hematopoietic markers cKIT, CD43, and CD45 (Figure 38), indicating successful differentiation toward hematopoietic lineages.

[0362] Tomato-expressing murine HE cells containing dox inducible nucleotide sequences encoding Sox17, Etv2, Foxc2, Tall , Lmo2, Gata2, and cMyc were cultured without doxycycline for 2, 3, and 4 days, then pooled. Five million cells were injected intraperitoneally into NSGS mice following two doses of 100 cGy irradiation, administered 3 hours apart. After 1 1 weeks, the mice were sacrificed, and the spleen, bone marrow, and thymus were harvested for analysis.

[0363] Figure 39 is a representative sample from a mouse transplanted with these Tomato HE cells. This figure demonstrates multilineage, long-term repopulation. Tomato-positive T cells (CD4 and / or CD8 expression), erythroid cells (Ter119 expression), and myeloid / B cells (MAC1 and B220 expression, respectively) were detected in the thymus, spleen, peripheral blood, and bone marrow, respectively.

[0364] EXAMPLE 9: Demonstration of Continuous HE Cell Line Amplification in Culture

[0365] Murine HE cells were cultured in blast media containing IMDM, FCS, D4T supernatant, MTG, glutamine, penicillin-streptomycin, ascorbic acid, transferrin, VEGF, IL-6, and doxycycline. Cells were maintained in 12-well plates coated with basement membrane extract (BME2) and passaged twice weekly using trypsin, with 80,000 cells re-seeded at each passage.

[0366] Over 106 days and 25 passages, the HE cells showed continuous expansion, achieving a 6.13 x 10E35- fold amplification with an estimated doubling time of 0.8 to 0.9 days (Figure 40A). The HE cells maintained a constant morphology over that time (Figure 40B). In addition, the potential of the HE cells to undergo endothelial-to-haematopoietic transition (EHT) to give rise to blood cells remained consistent across these different time points. This was measured through extreme limiting dilution analysis (ELDA) (Hu et al., 2009) and is shown in Table 5, below.

[0367] Table 5: Confidence intervals for capacity of HE cells to form blood cells (1 / undifferentiated cell frequency) Specifically, at day 0, one in 5.7 cells (confidence interval: 1 in 3.5-9.3), at day 64, one in 3.7 cells (confidence interval: 1 in 2.2-6.1), and at day 106, one in 4.3 cells (confidence interval: 1 in 2.6-7.1) had the capacity to form blood cells (Table 5). Furthermore, statistical analysis indicated no significant differences in blood-forming potential across these time points, demonstrating that blood potential is retained upon long-term culture (Table 6).

[0368] Table 6: Pairwise statistical tests of differences between the different cell groups

[0369] References

[0370] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

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Claims

Claims:1 . An in vitro method of generating a haematopoietic cell, the method comprising: a) Providing a cell genetically modified to comprise a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors; b) Culturing the genetically modified cell in a differentiation medium which does not comprise the inducer, such that the expression level of the exogenous transcription factors in the cell is reduced to a level whereby the cell differentiates into a haematopoietic cell.

2. The method of claim 1 , wherein the exogenous transcription factors further comprise MYC, optionally wherein MYC comprises c-MYC or n-MYC, preferably c-MYC.

3. The method of claim 1 or claim 2, wherein the exogenous transcription factors further comprise a Forkhead box (FOX) family transcription factor, optionally wherein the FOX family transcription factor comprises Foxc2.

4. The method of any one of the preceding claims, wherein the ETS family transcription factor comprises Ets1 or ETV2, preferably ETV2.

5. The method of any one of the preceding claims, wherein the GATA family transcription factor comprises GATA1 or GATA2, preferably GATA2.

6. The method of any one of the preceding claims, wherein the exogenous transcription factors further comprise LIM-only protein 2 (LMO2).

7. The method of any one of the preceding claims, wherein the exogenous transcription factors further comprise a Sry-box (SOX) family transcription factor, optionally wherein the SOX family transcription factor comprises Sox17 and / or Sox18, further optionally wherein the SOX family transcription factor comprises Sox17.

8. The method of any one of the preceding claims, wherein the exogenous transcription factors further comprise Friend leukaemia integration 1 transcription factor (Fli-1) .

9. The method of any one of the preceding claims, wherein in step b) the expression level of the exogenous transcription factors in the cell is reduced to an undetectable expression level.

10. The method of any one of the preceding claims, wherein the haematopoietic cell comprises a haematopoietic stem and progenitor cell (HSPC) and / or a blood cell.

11. The method of any one of the preceding claims, wherein the inducer comprises Doxycycline.

12. The method of any one of the preceding claims, wherein step a) comprises: i) Providing the genetically modified cell comprising the nucleotide sequence(s) encoding the exogenous transcription factors; and ii) Culturing the genetically modified cell in an inducer medium comprising the inducer to induce a detectable expression level of the exogenous transcription factors in the cell.

13. The method of claim 12, wherein the cell in step i) comprises a differentiated cell genetically modified to comprise the nucleotide sequence(s).

14. The method of claim 13, wherein the cell in step i) comprises a haematopoietic cell previously obtained according to the method of any one of the preceding claims.

15. The method of claim 12 or claim 13, wherein step i) comprises genetically modifying the cell to introduce the nucleotide sequence(s) encoding the exogenous transcription factors into the cell.

16. The method of any one of the preceding claims, wherein the differentiation medium comprises a TGF-p inhibitor, optionally wherein the TGF-p inhibitor comprises SB431542.

17. The method of any one of the preceding claims, wherein the differentiation medium comprises a Wnt activator, optionally wherein the Wnt activator comprises CHIR99021 .

18. The method of any one of the preceding claims, wherein the differentiation medium comprises bone morphogenetic protein 4 (BMP4).

19. The method of any one of the preceding claims, wherein the differentiation medium comprises a PI3K inhibitor, optionally wherein the PI3K inhibitor comprises LY2940002.

20. The method of any one of the preceding claims, wherein the cell is a murine or a human cell, preferably wherein the cell is a human cell.21 . A haematopoietic cell obtainable by the method of any one of claims 1 to 20.

22. A genetically modified haematopoietic cell, the haematopoietic cell comprising a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA familytranscription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer.

23. The genetically modified haematopoietic cell of claim 22, wherein the haematopoietic cell comprises no more than a low expression level of the exogenous transcription factors.

24. The genetically modified haematopoietic cell of claim 22 or claim 23, wherein the haematopoietic cell comprises a haematopoietic stem and progenitor cell (HSPC) and / or a blood cell, optionally wherein the haematopoietic cell comprises a blood cell, further optionally wherein the blood cell comprises a myeloid cell and / or a lymphoid cell.

25. The genetically modified haematopoietic cell of any one of claims 22 to 24, wherein the genetically modified haematopoietic cell comprises a macrophage, dendritic cell (DC), granulocyte, T-cell, NK cell, B-cell or erythrocyte.

26. The genetically modified haematopoietic cell of any one of claims 22 to 25, wherein the genetically modified haematopoietic cell further comprises a nucleotide sequence encoding a chimeric receptor or a T-cell receptor (TCR), optionally a chimeric antigen receptor.

27. The haematopoietic cell of any one of claims 21 to 26 for use in the treatment or prevention of a disease.

28. The haematopoietic cell for use of claim 27, wherein the disease comprises cancer or autoimmune disease.

29. A genetically modified cell, the cell comprising a nucleotide sequence(s) encoding exogenous transcription factors, the exogenous transcription factors comprising an ETS family transcription factor, T- cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer, and wherein the cell comprises a detectable expression level of the exogenous transcription factors.

30. The genetically modified cell of claim 29, wherein the cell comprises a detectable expression level of Flk1 .31 . The genetically modified cell of claim 29 or claim 30, wherein the cell comprises a detectable expression level of Tie2, and optionally comprises a detectable expression level of cKit.

32. An in vitro method of generating a hemogenic endothelium (HE) cell, the method comprising: a) Genetically modifying a cell to introduce nucleotide sequence(s) encoding exogenous transcription factors into the cell, the exogenous transcription factors comprising an ETS familytranscription factor, T-cell acute lymphocytic leukaemia protein 1 (Tall) and a GATA family transcription factor, wherein expression of the exogenous transcription factors from the nucleotide sequence(s) is inducible by culture with an inducer; b) Culturing the genetically modified cell in a culture medium comprising the inducer to induce expression of the exogenous transcription factors such that the cell forms a HE cell.

33. The method of claim 32, further comprising carrying out the method of any one of claims 1 to 20 on the HE cell generated in step b), optionally wherein the HE cell is stored and / or cultured for a period of time prior to carrying out the method of any one of claims 1 to 20 on the HE cell.

34. The method of claim 33, wherein the HE cell is cultured for at least about 10 days prior to carrying out the method of any one of claims 1 to 20 on the HE cell, optionally at least about 50 days, further optionally at least about 100 days.

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