Haematopoietic stem / progenitor cells and methods for their production

Abstract

The invention relates to differentiating a pluripotent stem cell (PSC) into a definitive haematopoietic stem / progenitor cell which is capable of multilineage engraftment, and to therapeutic uses of such haematopoietic stem / progenitor cells.

Description

HAEMATOPOIETIC STEM / PROGENITOR CELLS AND METHODS FOR THEIR PRODUCTIONField

[0001] The invention relates to differentiating a pluripotent stem cell (PSC) into a definitive haematopoietic stem / progenitor cell which is capable of multilineage engraftment, and to therapeutic uses of such haematopoietic stem / progenitor cells.Background

[0002] Haematopoietic stem cell (HSC) transplantation permits the reconstitution of the blood cell compartment, for example, in patients with haematopoietic disorders or receiving myeloablative therapy. The provision of HSCs from differentiated induced pluripotent stem cells (iPSCs) or embryonic stem cells would be beneficial. For example, HSCs derived from patient induced PSCs would circumvent the donor-host mismatch that leads to graft- versus-host-disease, a major source of morbidity and mortality in recipients of imperfectly matched allogenic transplants.

[0003] The provision of transplantable HSCs via in vitro haematopoietic differentiation has proved challenging, in part, due to difficulties distinguishing between cells resembling derivatives of the yolk-sac (and lacking re-populating activity), and those representing aorta-gonad-mesonephros (AGM)-like haematopoiesis (which possess repopulating activity). Despite considerable efforts demonstrating that induction of HOXA gene expression is associated with an AGM-like differentiation trajectory that may provide transplantable cells, there remains a need to provide transplantable HSCs that possess multilineage engrafting capacity.Summary of Invention

[0004] Through detailed studies, the inventors have established a fully defined culture system involving the timed provision of different cell culture components to differentiate iPSCs in vitro via a HOXA -positive differentiation pathway closely resembling AGM-like definitive haematopoiesis which provides CD34+hematopoietic stem / progenitor cells (HSPCs) that are capable of cryopreservation and subsequent intravenous transplantation, and possess repopulating activity and multilineage engrafting capacity similar to that seen following transplantation with human umbilical cord blood-derived cells.

[0005] Based on the inventors’ findings, a first aspect provides a method for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), the method comprising: a) culturing a population of mesoderm cells obtained from a population of PSCs in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; b) culturing the population of cells from step a) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step a); c) culturing the population of cells from step b) in a medium comprising a FGF, a BMP, an IGF, VEGF, and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b); and d) culturing the population of cells from step c) in a medium comprising a stem cell factor (SCF) and thrombopoietin (TPO), FGF, an IGF, and a retinoic acid signalling agent to produce a population of definitive HSPCs.

[0006] In one embodiment, the method step a) comprises culturing the population of cells for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10, preferably one or more of H0XA5, H0XA7, H0XA9, and HOXA10, even more preferably H0XA5, H0XA7, H0XA9, and HOXA10. In a further embodiment, the method step a) comprises culturing the population of cells for a period of about 2 days.

[0007] In one embodiment, the method step b) comprises culturing the population of cells for a time sufficient for generation of a population of cells expressing CD34 on the cell surface. In a further embodiment, the method step b) comprises culturing the population of cells for a period of about 2 days.

[0008] In one embodiment, the method step c) comprises culturing the population of cells for a time sufficient for generation of a population of cells co-expressing CD34 and CXCR4 on the cellsurface. In a further embodiment, step c) comprises culturing the population of cells for a period of at least about 2 days. In a further embodiment, the concentration of BMP in the medium in step c) is the same as in the medium of step b), or alternatively up to 20 fold lower than the concentration of BMP in the medium of step b).

[0009] In one embodiment, the method step d) comprises culturing the population of cells for a time sufficient for generation of a population of cells co-expressing CD34, CD90 and CD45 on the cell surface. In a further embodiment, step d) comprises culturing the population of cells for a period of about 3 days. In another embodiment, the medium in step d) further comprises, a TGF- beta pathway activator, a BMP, or a combination of one or more thereof.

[0010] In a second aspect, the present invention provides a method for differentiating a population of pluripotent stem cells (PSCs) into a population of definitive haematopoietic stem / progenitor cells (HSPCs), the method comprising: i) culturing the population of PSCs in a basal medium comprising a WNT agonist, a fibroblast growth factor (FGF), and Activin A; ii) culturing the population of cells comprising mesoderm cells from step i) in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; iii) culturing the population of cells from step ii) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step ii); iv) culturing the population of cells from step iii) in a medium comprising a FGF, a BMP, an IGF, VEGF, and a retinoic acid signalling agent wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii), and wherein the concentration of BMP is the same as in the medium of step iii), or alternatively up to 20 fold lower than the concentration of BMP in the medium of step iii); v) culturing the population of cells from step iv) in a medium as recited in step iv) excluding VEGF; andvi) culturing the population of cells from step v) in a medium comprising a stem cell factor (SCF) and thrombopoietin (TPO), FGF, an IGF, and a retinoic acid signalling agent to produce a population of definitive HSPCs.

[0011] In one embodiment, the method step i) comprises culturing the population of cells for a time sufficient for the generation of a population of mesoderm cells expressing CD 13 and CD90 on the cell surface. In a further embodiment, the method step i) comprises culturing the population of cells for a period of about 1 day.

[0012] In one embodiment, the method step ii) comprises culturing the population of cells for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10, preferably one or more of HOXA5, HOXA7, HOXA9, and HOXA10, even more preferably HOXA5, HOXA7, HOXA9, and HOXA10. In a further embodiment, the method step ii) comprises culturing the population of cells for a period of about 2 days.

[0013] In one embodiment, the method step iii) comprises culturing the population of cells for a time sufficient for generation of a population of cells expressing CD34 on the cell surface. In a further embodiment, the method step iii) comprises culturing the population of cells for a period of about 2 days.

[0014] In one embodiment, the method step iv) comprises culturing the population of cells for a time sufficient for generation of a population of cells co-expressing CD34 and CXCR4 on the cell surface. In a further embodiment, the method step iv) comprises culturing the population of cells for a period of about 2 days.

[0015] In one embodiment, the method step v) comprises culturing the population of cells for a time sufficient for generation of a population of cells expressing CD34 without CXCR4 on the cell surface. In a further embodiment, the method step v) comprises culturing the population of cells for a period of about 4 days.

[0016] In one embodiment, the method step vi) comprises culturing the population of cells for a time sufficient for generation of a population of definitive HSPCs. In a further embodiment, the method vi) comprises culturing the population of cells for a period of about 3 days. In oneembodiment, the medium in step vi) further comprises, a TGF-beta pathway activator, a BMP, or a combination of one or more thereof.

[0017] In one embodiment of any preceding aspect or embodiment, the WNT agonist is CHIR99021. In a further embodiment, the CHIR99021 is present in said medium at a concentration of about 4 pM.

[0018] In one embodiment of any preceding aspect or embodiment, the concentration of ACTIVIN A in the medium of step i) is about 5 ng / ml to 50 ng / mL. In a further embodiment, the concentration of ACTIVIN A in the medium of step i) is about 30 ng / mL.

[0019] In one embodiment of any preceding aspect or embodiment, the concentration of ACTIVIN A in the medium of step i) is about 5 ng / ml and the medium further comprises BMP at a concentration of about 3 ng / mL.

[0020] In one embodiment of any preceding aspect or embodiment, the ACTIVIN antagonist is SB431542. In a further embodiment, the SB431542 is present in said medium at a concentration of about 3-4 pM.

[0021] In one embodiment of any preceding aspect or embodiment, the retinoic acid signalling agent is a retinoid or retinoic acid analogue, preferably retinol or retinyl acetate (RETA). In a further embodiment, wherein the concentration of the retinoid or retinoic acid analogue in the medium of step a) or step ii) is about 50 nM - 100 nM. In a further embodiment, the retinoid or retinoic acid analogue in the medium of step a) or step ii) is about 50nM of RETA.

[0022] In one embodiment of any preceding aspect or embodiment, the concentration of VEGF in the medium of step a) or step ii) is about 50 ng / ml.

[0023] In one embodiment of any preceding aspect or embodiment, the concentration of VEGF in the medium of step b) or step iii) is about 100 ng / mL - about 200 ng / mL.

[0024] In one embodiment of any preceding aspect or embodiment, the bone morphogenic protein is BMP4. In a further embodiment, the concentration of BMP in the medium of step b) or step iii) is about 20 ng / mL.

[0025] In one embodiment of any preceding aspect or embodiment, the medium of steps a) to c) or steps i) to v) do not comprise SCF. In a further embodiment, the medium of step d) or step vi) comprises SCF at a concentration of about 10 ng / mL and TPO at a concentration of about 10 ng / mL.

[0026] In one embodiment of any preceding aspect or embodiment, the medium of step d) or step vi) comprises StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), and / or erythropoietin (EPO).

[0027] In one embodiment of any preceding aspect or embodiment, the FGF is FGF2, and / or wherein IGF comprises IGF1 and / or IGF2, optionally in equal concentrations when IGF1 and IGF2 are both present.

[0028] In one embodiment of any preceding aspect or embodiment, the medium of step i) comprises a ROCK inhibitor, optionally wherein the ROCK inhibitor is thiazovivin or Y-27632.

[0029] In one embodiment of any preceding aspect or embodiment, the population of cells is cultured as embryoid bodies (EBs). In a further embodiment, the embryoid bodies are cultured with swirling.

[0030] In one embodiment of any preceding aspect or embodiment, the basal medium is SPELS medium.

[0031] In one embodiment of any preceding aspect or embodiment, the PSCs are embryonic stem cell (ESC) or induced PSCs (iPSCs).

[0032] In one embodiment of any preceding aspect or embodiment, the PSC is human, preferably a human induced pluripotent stem cell iPSC.

[0033] In one embodiment of any preceding aspect or embodiment, in any one of the recited steps, the population of cells is cultured under hypoxic conditions, preferably between about 2% and about 10% oxygen.

[0034] In one embodiment of any preceding aspect or embodiment, the population of definitive HSPCs are HLF+and SPINK+.

[0035] In one embodiment of any preceding aspect or embodiment, the population of definitive HSPCs express CD34 and CD45, and one or more of CD90, CD44, KIT, CD201, ITGA3 and ITGA6 on the cell surface.

[0036] In one embodiment of any preceding aspect or embodiment, the method further comprises harvesting cells in suspension following step d) or step vi).

[0037] In one embodiment of any preceding aspect or embodiment, the method further comprises dissociating EBs in suspension following step d) or step vi) and harvesting cells dissociated from the EBs.

[0038] In one embodiment of any preceding aspect or embodiment, the method further comprises enriching, or sorting the harvested cells for CD34+cells.

[0039] In a third aspect, a definitive haematopoietic stem / progenitor cell, or population of cells, differentiated from a PS C by the method of any preceding aspect or embodiment.

[0040] In a fourth aspect, the present invention provides a therapeutic composition comprising the cell or population of cells of any preceding aspect or embodiment.

[0041] In a fifth aspect, the present invention provides an infusion bag comprising the cell or population of cells of any preceding aspect or embodiment.

[0042] In a sixth aspect, the present invention provides a method for treating a condition, disease or disorder requiring HSC transplantation, the method comprising administering to a subject the cell or population of cells of the third aspect or the therapeutic composition of the fourth aspect. In one embodiment, the condition, disease or disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), Non-Hodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, other solid tumor, thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.

[0043] In a seventh aspect, the present invention provides a use of the cell or population of cells of the third aspect or the therapeutic composition of the fourth aspect in the manufacture of amedicament for treating a condition, disease or disorder requiring HSC transplantation. In one embodiment, the condition, disease or disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), Non-Hodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, other solid tumor, thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.

[0044] In an eighth aspect, the present invention provides a cell or population of cells of the third aspect for use in treating a condition, disease or disorder requiring HSC transplantation. In one embodiment, the condition, disease or disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), Non-Hodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, other solid tumor, thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.

[0045] In an ninth aspect, the present invention provides a kit for use in generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF) and thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor.

[0046] In a tenth aspect, the present invention provides a kit when used for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF) and thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor.Brief Description of Drawings

[0047] Figure 1 shows In vitro hematopoietic differentiation of iPSCs. (a) Swirling embryoid body (EB) differentiation protocol indicating differentiation stages transitioning from undifferentiated iPSCs to hematopoietic, endothelial and stromal cells. Growth factors for each stage are shown in Figure 7. EHT, endothelial to hematopoietic transition, (b) 60 mm dish at day 7 showing hundreds of swirling EBs. (c) Overlaid bright field (BF) and TOMATO (TOM) fluorescence images of developing swirling EB cultures. Scale bar, 200 pm. (d) Flow cytometry of day 14 suspension hematopoietic cells showing expression of surface CD45, CD34, KIT, CD44 and CD90. (e) Dissociated day 14 swirling EB cells were typically enriched to >90% CD34+endothelium and blood using magnetic bead separation (MACS). These cells comprised CD45+blood cells (profiles with red borders) and CD45’ endothelium (profiles with blue borders). Endothelium was categorized as arterial, venous or hemogenic based on expression of CD34, CD44, CXCR4 and CD73. Flow cytometry panels in (d) and (e) are from one representative experiment of greater than 20 experiments performed.

[0048] Figure 2 shows Multilineage engraftment depends on CHIR and retinoids during iPSC differentiation, (a) Swirling embryoid body (EB) differentiation protocol indicating mesoderm induction factors provided during the first day of differentiation and retinoids during endothelium formation from day 3 to day 5 to generate the 12 differentiation conditions, outlined as screening protocol #1 in Figure 7. Numbers indicate concentration of CHIR (CH) in pM, and concentrations of BMP4 (B) and ACTIVIN A (A) in ng / ml. EHT, endothelial to hematopoietic transition; ROL, retinol; RETA, retinyl acetate, (b) Scatter dot plot correlating % BM human cells with differentiation conditions in cohort #1. Error bars, mean+SEM. Number of mice receiving cells subjected to each mesoderm induction (n) is shown. Number of unengrafted (NEG) mice indicated for each condition, (c) Scatter dot plot correlating concentration of CHIR during mesoderm induction with phenotype of engrafted human cells in the BM (colored circles). Number of mice displaying a MLE phenotype differed between those receiving cells treated with 4CH and 1CH, * P = 0.03, two-sided Fisher's exact test. Error bars, mean+SEM. Data from 4CH 3B5A and 4CH 30A mesoderm inductions was pooled, (d) Scatter dot plot correlating inclusion of retinoid (ROL or RETA) during iPSC differentiation with phenotype of engrafted human cells in the BM (colored circles). Number of mice displaying a MLE phenotype differed between those receiving cells treated with or without retinoid. ROL or RETA vs NIL (no retinoid), * P = 0.03, two-sided Fisher's exact test. Error bars, mean+SEM. Data from 4CH 3B5A and 4CH 30A mesoderm inductions was pooled, (e) Engraftment phenotypes in 42 / 51 mice transplanted with cells treated with thecombination of 4 |jM CHIR and retinoid (RET) that showed engraftment. 9 / 51 (17.6%) transplanted mice showed multilineage engraftment. Error bars, mean+SEM.

[0049] Figure 3 shows transcriptional profiling of in vitro differentiated iPSCs. (a) Swirling embryoid body (EB) differentiation protocol showing the mesoderm induction and retinoid combinations used to differentiate RM TOM and PB1.1 BFP iPSCs. Each cell line was subject to two mesoderm induction conditions, with 4 pM CHIR, 3 ng / ml BMP4 and 5 ng / ml ACTIVIN A (4CH 3BA5) or 4 pM CHIR, 30 ng / ml ACTIVIN A (4CH 30A), and three or four retinyl acetate (RETA) exposure patterns. Samples were harvested from swirling EB and suspension hematopoietic cell fractions at day 14 of differentiation, leading to 28 samples subjected to scRNA seq. (b and c) UMAP of integrated samples for individual lines (b) and following pooling of samples (c), showing annotation of cell clusters, allocated based on cluster specific gene expression, (d and e) Feature plots depicting selected genes identifying cell lineages in integrated samples, (f) Feature plots depicting expression of six human HSC signature genes in arterial (Artl), endothelial / stromal (En / Str), hemogenic (HE) and HLP+SP!NK2+cells from stem / progenitor clusters 1-3 (HSPC) in integrated samples, (g) Violin plots showing expression of selected stem cell genes in CS14 and 15 embryos and HLP+SP1NK2+cells from HSPC clusterl in PB 1.1 BFP and RM TOM cells. Cell numbers: CS14, 51; CS15, 70; PB 1.1 BFP, 2983; RM TOM, 1112. (h) Comparison of the expression profiles of HLP+SP1NK2+cells from HSPC clusters from PB 1.1 BFP and RM TOM cells to reference data from human embryonic and CB derived endothelial and hematopoietic cell populations, using the ACTINN machine learning algorithm to determine the percentage of the iPSC-derived hematopoietic cells displaying the greatest similarity to each reference data set. Data stratified by retinoid treatment is shown for each cell line. The bar height represents the percentage of HLP+SP1NK2+putative iHSCs that map most closely to each reference sample. EC, endothelial cell; VE, venous endothelium; AE, arterial endothelium; preHE, pre-hemogenic endothelium (representing aortic endothelium); HE, hemogenic endothelium; HSPC, hematopoietic stem or progenitor cell; CS, Carnegie stage; W, week; CB, cord blood; YS, yolk sac; Plac, placenta; Ery, erythroid; Prog, progenitor; Meg, megakaryocyte; Mast, mast cell; Mono, monocyte; Mac, macrophage; Gran, granulocyte.

[0050] Figure 4 shows hematopoietic cells exposed to retinoid throughout differentiation possess multilineage engraftment potential, (a) Swirling embryoid body (EB) differentiation protocol showing the mesoderm induction and retinoid combinations used to differentiate RM TOM iPSCs. Cells were subjected to two mesoderm induction conditions, and six retinoid exposure patterns,prior to harvesting and cryopreservation at day 14 - 16. (b) Scatter dot plot correlating human cells in the bone marrow (BM) with the interval of retinoid (R) treatment during differentiation (shown as days) in cohort#2. Each circle represents one animal, color coded to represent myeloid (M), myelo-lymphoid (ML), ery thro -myeloid (EM) and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment. Number of mice receiving each duration of retinyl acetate (n) is shown. Number of unengrafted (NEG) mice indicated. Data from 4CH 3B5A and 4CH 30A mesoderm inductions was pooled. Error bars, mean+SEM. (c) Confocal images of bone marrow cells from an engrafted (mouse (m)536) and unengrafted (m534) recipient. Scale bar, 50 pm. BF, bright field; TOM, TOMATO fluorescence, (d - g) Flow cytometry profiles from (d) bone marrow (BM), (e) peripheral blood (PB), (f) spleen (SPL) and (g) thymus (THY) of a multilineage repopulated recipient (m490). (d) Erythroid cells (CD43+GYPA+) were enriched in the TOM low (lo) BM fraction. The TOM high (hi) BM cells comprised CD19+B cells, CD33+and CD13+myeloid cells, and CD45+CD34+CD38lo / _HSC-like cells (HSC) (boxed in red), (f) The spleen contained CD45+CD19+sIGM+B cells, (g) The thymus contained immature CD45+CD3“ thymocytes including CD4 CD8’ cells, transitioning through immature CD4+to CD4+CD8+double positive cell states, whilst CD45+CD3+thymocytes included CD4+CD8+double positive and CD4+and CD8+single positive cells.

[0051] Figure 5 shows robust hematopoietic engraftment with cells differentiated using protocol #3. (a - d) Engraftment of bone marrow (BM) and spleen (S) in transplant recipients of RM TOM (a), PB1.1 BFP (b), PB5.1 (c) and PB 10.5 (d) cells showing the phenotype of engrafting cells and the level of engraftment. Error bars, mean+SEM. (e - h) Tissue distribution of engrafting cells in MLE recipients of RM TOM (e). PB1.1 BFP (f), PB5.1 (g) and PB10.5 (h) cells in bone marrow (BM), spleen (S), thymus (THY) and peripheral blood at 12 (PB 12) and 16 (PB 16) weeks. Error bars, mean+SEM. (i) Flow cytometry analysis of bone marrow in engrafted mice for each cell line showing GYPA+erythroid lineage and CD45+lymphoid and myeloid cells, (j) Bone marrow, spleen and thymus / mediastinal lymph node tissue of RM TOM engrafted mouse m574, showing GYPA+erythroid, CD45+CD19+B cell, CD45+CD3+T cell, CD45+CD33+ / CD13+myeloid and CD45+CD34+CD3810stem cell populations in the bone marrow, CD45+sIgM+B cells and CD45+CD3+T cells in the spleen, and thymus / mediastinal lymph node tissue containing CD45+CD3+CD4+and CD45+CD3+CD8+T cells and a population of CD45+CD19+B cells.

[0052] Figure 6 shows engraftment patterns of MLE engrafted iHSC and CB transplanted mice, (a) Upper panel bar graphs show the level of human engraftment in BM of MLE mice receivingthe indicated cell lines (individual recipients identified on X-axis). Lower panels depict stacked column graphs showing the lineage distribution of human cells in the BM of iHSC engrafted recipients. UN, unclassified cells include myeloid, dendritic and natural killer cells not detected by the antibodies used, (b - h) Characteristics of engrafted CB cells, (b) Scatter plot correlating calculated dose of injected CD34+CB cells with phenotype and level of human engraftment in the bone marrow. Each circle represents one animal, color coded to represent myeloid (M), myelo- lymphoid (ML), myelo-erythroid (ME) and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment. Error bars, mean+SEM. Total of 39 animals transplanted, (c) Flow cytometry plot showing GYPA+erythroid cells and CD45+lymphoid and myeloid cells, (d) Tissue distribution of engrafting cells in MLE recipients of CB cells in bone marrow (BM), spleen (S), thymus (THY) and peripheral blood at 12 (PB 12) and 16 (PB 16) weeks, (e) Analysis of paired samples of peripheral blood showing increased levels of human cells in 6 / 8 recipients between 12 and 16 weeks, (f, g) Lineage distribution in the bone marrow (f) and spleen (g) in CB recipients, (h) Upper panel bar graphs show the level of human engraftment in BM of MLE mice receiving CB cells (individual recipients identified on X-axis). Lower panels depict stacked column graphs showing the lineage distribution of human cells in the BM of CB engrafted recipients. UN, unclassified cells include myeloid, dendritic and natural killer cells not detected by the antibodies used.

[0053] Figure 7 shows schematic outline of the growth factors used for iPSC differentiation in screening protocols #1 and #2 and in protocol #3. Cohorts of mice transplanted with each protocol are indicated. Concentrations of growth factors used are provided below.

[0054] Figure 8 shows flow cytometry live cell gating strategy and negative control samples. Data collected in the indicated fluorochrome channels for unstained suspension hematopoietic cells and disaggregated swirling embryoid body (EB) cells. See also Figure Id and le. The same strategy was used for evaluation of iPSC engraftment in mouse hematopoietic tissues shown in Figures 4d- g, 5i-j, 6c and Figures 15b, c and 18a - d.

[0055] Figure 9 shows (a) Combinations of mesoderm induction factors provided during the first day of differentiation, and retinoids during endothelium formation from day 3 to day 5, generated 12 differentiation conditions transplanted into cohort#l mice. See also Figure 2a. Concentration of CHIR (CH) is in pM, and concentrations of BMP4 (B) and ACTIVIN A (A) are in ng / ml. (b) Bone marrow (BM) and spleen (SPL) engraftment in 134 transplant recipients. Each circle represents one animal, color coded to indicate myeloid (M), myelo-lymphoid (ML), lympho-myeloid (LM) and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment. Total number of mice is shown, as is number of unengrafted (NEG) mice. Error bars, mean+SEM. (c) Engrafted recipients categorized by engraftment phenotype demonstrate higher levels of human cells in the BM and SPL of lympho-myeloid and multilineage engrafted animals. Number of mice with each phenotype is shown. BM: M vs LM, * P = 0.0175; M vs MLE, **** P < 0.0001, oneway ANOVA (Kruskal-Wallis) test with Dunn's multiple comparisons test. SPL: ML SPL vs LM SPL, * P = 0.0447; ML SPL vs MLE SPL, **** P < 0.0001, one-way ANOVA (Kruskal- Wallis) test with Dunn's multiple comparisons test. Error bars, mean+SEM.

[0056] Figure 10 shows transcriptional profiling of human embryo and iPSC arterial and hematopoietic stem / progenitor cell (HSPC) populations, (a) Left panel depicts UMAP of 11,877 single cells from CS14 and CS15 embryos, with endothelium, stroma, HSPC and non-HSPC populations indicated. Middle panel shows subset analysis of 4,532 endothelial cells and HSPCs, selected on expression of either CD34 and CD31 or RUNX1 and CDH5. In the right panel, 634 cells from artery, aorta and HSPCs were reclustered, (b) Feature plots of the reclustered artery, aorta and HSPCs showing expression of human embryonic HSC signature genes, (c) Bar graphs displaying the expression of HSC signature genes in cells differentiated from RM TOM and PB 1.1 BFP iPSC lines, under three retinyl acetate (RETA) conditions and two mesoderm induction protocols, no R, no RETA; R3 - 5, RETA day 3-5; R3 - 11+, RETA day 3-11, -13, or -14. Both the percentage of positive cells and the average expression of each gene is shown, (d) Bar graphs displaying the percentage of cells expressing HOXA genes in arterial, hemogenic endothelium and hematopoietic stem and progenitor (HSPC) cells differentiated from RM TOM and PB 1.1 BFP iPSC lines under three retinyl acetate (RETA) conditions.

[0057] Figure 11 shows differentially expressed genes in cultures exposed to different durations of retinyl acetate (RETA), (a) UMAP showing arterial (Artl), hemogenic (HE) and stem / progenitor clusters (HSPC1) differentiated from RM TOM and PB 1.1 BFP iPSC lines. Integrated data from all samples was used. Differential gene expression was determined by comparing (b) genes expressed following a pulse of RETA from day 3 - 5 or for a prolonged period (day 3 - day 11, day 13 and day 14) with cells not treated with RETA. (c) Number of differentially expressed genes in each cluster with a false discovery rate (FDR) <0.05 and the subset of these genes up- or down-regulated by a fold change (FC) > 2 are indicated, (d, e) Venn diagrams demonstrating the overlap in genes (d) up- and (e) down-regulated in response to retinoidexposure in each cluster, (f) Heatmaps displaying the 30 most highly up- and down-regulated genes in each cluster in response to RETA.

[0058] Figure 12 shows effects of retinoids on expression of stem cell genes during the endothelial to hematopoietic transition in vitro and expression of retinoid dependent genes in iPSC -differentiated cells and CS10 - CS17 human embryos, (a) Bar graphs displaying the expression of HSC signature genes in cells differentiated from RM TOM and PB 1.1 BFP iPSC lines, shown for each cluster (see Figure 3f) under three retinyl acetate (RETA) conditions, no R, no RETA; R3 - 5, RETA day 3-5; R3 - 11+, RETA day 3-11, -13, or -14. Both the percentage of positive cells and the average expression of each gene is shown, (b) Feature plots showing expression of selected retinoid responsive genes in day 14 differentiated human iPSCs correlated with RETA exposure. Integrated data from 4CH 3B5A and 4CH 30A mesoderm inductions was pooled. Endothelial (endo), hematopoietic (hem) and stromal (stroma) populations indicated, (c) Feature plots of CD34 and selected retinoid responsive genes throughout human embryogenesis from CS10 - CS17. Key endothelial, arterial and HSC containing populations are circled. Note overlap of these cell clusters expressing CD34, marking endothelium and HSPCs, with retinoid responsive genes.

[0059] Figure 13 shows comparison of the transcriptomes of iPSC-derived cells from the HSPC clusters that co-expressed HLF and SP1NK2, with those of HLF+SP!NK2+cells from CS14 and CS15 embryos. The scorecards developed by the Mikkola laboratory were used as templates, (a) Nascent HSC. (b) HSC transcription factors, (c) HSC maturation, (d) HSPC waves, (e) Hematopoietic cell identity. Cell numbers: CS14, 51; CS15, 70; PB noR, 489; PB R3-5; 880; PB R3-11+, 1614; RM noR, 424; RM R3-5, 400; RM R3-14, 288. Abbreviations: PB, PB1.1 BFP; RM, RM TOM.

[0060] Figure 14 shows comparison of the transcriptomes of iPSC-derived cells from the HSPC clusters that co-expressed HLF and SP1NK2, with those of HLF+SP!NK2+cells from CS14 and CS15 embryos. The scorecards developed by the Mikkola laboratory were used as templates, (a) Liver SPINK2+genes, (b) Proliferation and metabolic activity, (c) Signaling, (d - f) Endothelial to hematopoietic transition. Samples for panels (e) and (f) are the clusters shown in Figure 3f. Cell numbers for panels (a) - (d): CS14, 51; CS15, 70; PB noR, 489; PB R3-5; 880; PB R3-11+, 1614; RM noR, 424; RM R3-5, 400; RM R3-14, 288. Abbreviations: PB, PB1.1 BFP; RM, RM TOM.

[0061] Figure 15 shows engraftment of blood cells from PB 1.1 BFP iPSCs in cohort#3. (a) Scatter dot plot correlating human cells in the bone marrow (BM) with the interval of retinoid (R) treatment during differentiation (shown as days). Each circle represents one animal, color coded to represent myeloid, myelo-lymphoid, and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment. Number of mice receiving each duration of retinoid during differentiation (n) is shown. Number of unengrafted (NEG) mice indicated. Error bars, mean+SEM. Data from 4CH 3B5A and 4CH 30A mesoderm inductions was pooled. Flow cytometry profiles from (b) bone marrow (BM) and (c) spleen (SPL) of a multilineage repopulated recipient (mouse (m)410). (b) Erythroid cells (GYPA+CD43+) were enriched in the BFP low (lo) BM fraction. BFP high (hi) cells included erythroid cells (GYPA+CD45 ), CD19+B cells, CD33+and CD13+myeloid cells, and CD45+CD34+CD38lo / _HSCs. (c) The spleen also contained erythroid cells (GYPA+CD45 ) and CD19+CD45+B cells.

[0062] Figure 16 shows tissue distribution and lineages in cohort #1 - #3 multilineage engrafted recipients of iHSCs. (a) Left panel, tissue engraftment showing human cells in bone marrow (BM), spleen (SPL), thymus (THY) and peripheral blood at 12 weeks (PB 12). Middle and right panels, lineage distribution in the BM and SPL of reconstituted mice. ERY, erythroid; B, B cell; T, T cell; MYE, myeloid; STEM, hematopoietic stem and progenitor cells. Error bars, mean+SEM. (b) Left panel, major thymic T cell subset distribution. Right panel, distribution of T cell subsets in CD3+and CD3“ thymocytes. Statistics, CD8+CD3+vs CD8+CD3“, ** P = 0.0039, two-tailed Wilcoxon matched-pairs signed rank test. CD4 CD8 CD3+vs CD4 CD8 CD3’, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. Error bars, mean+SEM.

[0063] Figure 17 shows removal of VEGF at day 7 of differentiation accelerates the loss of arterial endothelial markers, (a) Swirling embryoid body (EB) differentiation protocol outlining the VEGF (V) titration. Numbers represent VEGF concentration in ng / ml. (b) CD34 expression in RM TOM cells by flow cytometry correlated with VEGF concentration. Error bars, mean+SEM, n=3. One-way ANOVA test for CD34 linear trend, P<0.0001. (c) Percentage of RM TOM CXCR4+CD73lo / +arterial cells, subsetted from CD34+cells, by flow cytometry correlated with VEGF concentration. Error bars, mean+SEM, n=3. One-way ANOVA test for CXCR4+CD73lo / +linear trend, P<0.0001. Comparison of d8 and d9 samples continuing VEGF with d3-7 VEGF, both P<0.0001, one-way ANOVA with Sidak's multiple comparisons test, (d) CD34 expression in PB5.1 cells by flow cytometry correlated with VEGF concentration. Error bars, range, n=2 independent experiments. One-way ANOVA test for CD34 linear trend, P=0.0018. (e) Percentageof PB5.1 CXCR4+CD73lo / +arterial cells, subsetted fromCD34+cells, by flow cytometry correlated with VEGF concentration. Error bars, range, n=2. One-way ANOVA test for CXCR4+CD73lo / +linear trend, P=0.0011. Comparison of d9 and dl2 samples continuing VEGF with d3-7 VEGF, both P<0.0001, one-way ANOVA with Sidak's multiple comparisons test.

[0064] Figure 18 shows removal of VEGF at day 7 of differentiation increases expression of aortic pre-hemogenic endothelial genes, (a) Flow cytometry analysis of differentiating PB5.1 cells showing the increase in CD34+CXCR4+CD73lo / +arterial cells in response to VEGF at 150ng / ml (V150) from d3 - 7. (b) Continuing VEGF maintains CXCR4 expression, (c) VEGF removal leads to CXCR4 downregulation, (d) Negative control samples unstained for CD34 or for CXCR4 and CD73. (e) Real time PCR analysis of differentiated samples of PB5.1 analysed from d5 - 11 for the indicated arterial endothelium, retinoid signaling and hematopoietic genes. Samples with continued VEGF signaling are compared to samples where the VEGF was removed after d7. Error bars, mean+SEM, n=3 independent experiments. V150 vs V150 d3-7 at dl l: AGTR2, P=0.0125; IL33, P=O.O3O7 RUNXI, P=0.0009; HLF, P=0.0100; mixed-effects analysis (two-way ANOVA) with Sidak's multiple comparisons test.

[0065] Figure 19 shows contribution and lineage distribution of human cells in the bone marrow, spleen, thymus and peripheral blood of cohort #4 - #7 mice receiving cells differentiated under protocol#3. (a) Paired samples of peripheral blood analysed at 12 (PB12) and 16 weeks (PB 16). (b) Bone marrow engraftment in transplanted mice stratified by recipient sex. RM TOM cells, Female vs Male mice, P=0.0012; Female MLE vs Male MLE, P= 0.0427; PB5.1, Female vs Male mice, P=0.0253; PB1.1 BFP and PB10.5, no significant gender differences. One-way ANOVA (Kruskal-Wallis test) with Dunn's multiple comparisons test, (c) Bone marrow and (d) spleen lineage distribution in cohort #4 - #7 mice, (e) T cell subsets and B cells in mediastinal lymphoid tissue, comprising thymus and lymph node tissue.

[0066] Figure 20 shows gender differences in MLE mice transplanted with RM TOM cells, (a, b) Tissue distribution in (a) cohort#l-3 and (b) cohort#4 multilineage engrafted female and male mice. Female vs male cohort#l-3 mice, bone marrow engraftment, P=0.0041; peripheral blood 12 week engraftment, P=0.0357. Female vs male cohort#4 mice, bone marrow engraftment, P=0.0003; spleen, P=0.0044; thymus, P=O.O133; peripheral blood 12 week engraftment, P<0.0001; peripheral blood 16 week engraftment, P=0.0004. Comparing cohort#l-3 with cohort#4 female mice, engraftment was greater in cohort#4 female mice in the bone marrow(P=0.0030), spleen (P=0.0006) and peripheral blood at 12 weeks (P=0.0266). Engraftment comparisons between male mice were not statistically significant. Mann- Whitney t- tests, (c, d) Distribution of lineages in bone marrow, spleen, and thymus / lymph node in (c) cohort#l-3 and (d) cohort#4 multilineage engrafted female and male mice, (c) In cohort# 1-3 mice, the proportions of erythroid, myeloid, B cell and stem cells were similar in male and female recipients, (d) In cohort #4 mice, T cell engraftment was greater in female mice in the bone marrow (P=0.0049) and spleen (P=0.0065) and thymic CD4+CD8+cells were more abundant in male mice (P= 0.0058). Myeloid cells were more abundant in the BM of female mice (P=0.0378). Thymic CD8+cells were more abundant in female mice (P=0.0352). Mann-Whitney t-tests.

[0067] Figure 21 shows engraftment patterns of MLE engrafted iHSC and CB transplanted mice.(a) Upper panel bar graphs show the level of human engraftment in BM of MLE mice receiving RM TOM and PB 1.1 BFP lines in cohort#l-3 transplants (individual recipients identified on X- axis). Lower panels depict stacked column graphs showing the lineage distribution of human cells in the BM of iHSC engrafted recipients. UN, unclassified cells include myeloid, dendritic and natural killer cells not detected by the antibodies used, (b, c) Characteristics of engrafted CB cells.(b) Scatter plot correlating calculated dose of injected CD34+CB cells with phenotype and level of human engraftment in the bone marrow, with results stratified by recipient gender. Each circle represents one animal, color coded to represent myeloid, myelo-lymphoid, myelo- erythroid and erythro-myelo-lymphoid multilineage (MLE) patterns of engraftment. Error bars, mean+SEM. Total of 39 animals transplanted. Stem cell frequency was estimated by limit dilution assay, (c) Tissue distribution, bone marrow and spleen lineages of engrafting cells in MLE recipients of CB cells stratified by recipient gender. Female recipients displayed higher levels of bone marrow (P=0.0117), thymus (P=0.0357), and peripheral blood engraftment at 12 weeks (P=0.0340). Mann-Whitney t-tests.

[0068] Figure 22 shows correlation between time of tissue analysis post-transplantation and human cell engraftment in bone marrow (n = 78) and spleen (n = 78) in multilineage engrafted recipients of iHSCs, stratified for sex of the recipient. Panels show a trend of increasing human cell contribution in recipients engrafted for longer periods. The plateau appears earlier for female (18-19 w) than for male (at least 22-24 w) recipients. For mice analyzed after 20 w, the level of engraftment was higher for female mice in the bone marrow (P=0.0002) and the spleen (P=0.0374). Mann- Whitney t-tests.

[0069] Figure 23 shows effect of VEGF on bone marrow engraftment. (a) Bone marrow engraftment by cells from RM TOM and PB 1.1 BFP iPSC lines differentiated with 50 ng / ml VEGF throughout the differentiation (+VEGF) or with 150 ng / ml VEGF limited to days 3-7 (-VEGF d7). Mean+SEM, number of unengrafted mice (NEG) and total number of transplanted mice (n) indicated. Red dots, multilineage engrafted mice. P, Student’s t-test. (b) Contingency analyses indicating increased frequency of multilineage engrafted (MLE) mice in recipients of cells differentiated with VEGF limited to days 3 - 7. P, Fisher’s exact test.Description of Embodiments

[0070] Through detailed studies, and using newly developed methods, the inventors have functionally and transcriptionally characterized human iPSC derived HSPCs and demonstrated that the HSPCs generated using these methods display characteristics of HSCs or MPPs, evidenced by erythroid, myeloid, and lymphoid engraftment of immune deficient mice and establishment of bone marrow hematopoietic stem cell-like cells. Transcriptional profiling of haematopoietically differentiated iPSCs prior to transplantation revealed a HLF+SPINK2+population whose transcriptional profile was similar to HSPCs in the AGM region at day 32 of gestation (CS14), when the first human HSCs emerged from the embryo.

[0071] As disclosed herein the inventors have surprisingly determined that the generation of multilineage engrafting cells is dependent on an initial higher concentration of CHIR during mesoderm induction, followed by exposure to a retinoic acid precursor. Moreover, an increased concentration of VEGF during the generation of arterially patterned hemogenic endothelium, followed by its complete removal led to enhanced endothelial to hematopoietic transition and the robustness of engraftment.

[0072] Accordingly, provided herein is a fully defined culture system to differentiate human iPSCs in vitro to HSPCs that closely resemble the earliest HSCs in the human embryo. These cells are capable of cryopreservation prior to transplantation by injection into the tail vein of immune deficient mice, resulting in long-term, multilineage engraftment similar to that seen following transplantation with human CB. Importantly, this workflow recapitulates clinical hematopoietic stem cell transplantation, in which donor hematopoietic cells harvested from the bone marrow or peripheral blood are cryopreserved prior to intravenous transplantation into the recipient.

[0073] The invention generally relates to methods and compositions for differentiating stem cells toward a definitive hematopoietic cell fate. More particularly, the invention provides a multi-stage, fully defined culture system wherein PSCs (e.g. iPSCs) or PSC-derived cells at various stages of development can be induced to assume a definitive hematopoietic phenotype, including definitive hemogenic endothelium, to hematopoietic stem / progenitor cells (HSPCs) and their progeny, wherein the HSPCs have multiple lineage engraftment capacity. That is, the invention provides methods, and cells obtained by such methods, involving the coordinated temporal provision of cell culture components (e.g. WNT- agonists, retinoic acid precursors and growth factors) that permits a definitive HSPC fate to be assumed (e.g. a CD34+definitive hematopoietic population) by PSC- derived cells wherein the HSPCs are capable of multilineage engraftment.Definitions

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

[0075] Definitions of common terms in cellular and molecular biology, and biochemistry can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 9780911910421, 0911910425); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 2008 (ISBN 3527305424, 9783527305421); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1- 56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2016 (ISBN 9780815345510, 0815345518); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al , Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Laboratory Methods in Enzymology: RNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN: 9780124200371, 0124200370); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; andCurrent Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), Immunological Methods, Ivan Lefkovits, Benvenuto Pemis, (eds.) Elsevier Science, 2014 (ISBN: 9781483269993, 148326999X), the contents of which are all incorporated by reference herein in their entireties.

[0076] As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, optionally include plural referents unless the content clearly dictates otherwise. For example, "a" cell includes one cell, one or more cells and a plurality of cells.

[0077] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0078] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0079] While reference may be made in this disclosure to the invention comprising a combination of a plurality of elements, it is also understood that this invention is regarded to comprise combinations which omit or exclude one or more of such elements, even if this omission or exclusion of an element or elements is not expressly stated herein, unless it is expressly stated herein that an element is essential to the applicant' s combination and cannot be omitted. It is further understood that the related prior art may include elements from which this invention may be distinguished by negative claim limitations, even without any express statement of such negative limitations herein. It is to be understood, between the positive statements of applicant's invention expressly stated herein, and the prior art and knowledge of the prior art by those of ordinary skill which is incorporated herein even if not expressly reproduced here for reasons of economy, that any and all such negative claim limitations supported by the prior art are also considered to be within the scope of this disclosure and its associated claims, even absent any express statement herein about any particular negative claim limitations.

[0080] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also bepreferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0081] The term “about” as used herein contemplates a range of values for a given number of ±25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of ±30%, ±20%, ±15%, ±10%, or ±5% the magnitude of that number. For example, in one embodiment, “about 3 pM” indicates a value of 2.7 to 3.3 pM (i.e. 3 pM ±10%), and the like.

[0082] Similarly, while differentiation processes include ordered, sequential events, the timing of the events may be varied by at least 25%. For example, while a particular step may be disclosed in one embodiment as lasting one day, the event may last for more or less than one day. For example, “one day” may include a period of about 18 to about 30 hours. In other embodiments, periods of time may vary by ±20%, ±15%, ±10%, or ±5% of that period of time. Periods of time indicated that are multiple day periods may be multiples of “one day,” such as, for example, about two days may span a period of about 36 to about 60 hours, and the like. In another embodiment, time variation may be lessened, for example, where 1 day is 24±3 hours; 3 days is 72±3 hours; 4 days is 96±3 hours; 5 days is 120±3 hours; 6 days is 144±3 hours; 7 days is 168±3 hours; 11 days is 264±3. As used herein, about 3 days may be 2.5, 3 or 3.5 days, about 4 days may be 3.5, 4 or 4.5 days, about 5 days may be 4.5, 5 or 5.5 days, about 6 days may be 5.5, 6 or 6.5 days, about 7 days may be 6.5, 7 or 7.5 days, about 11 days may be 10, 10.5, 11, 11.5, or 12 days, about 21 days may be 20, 20.5, 21, 21.5, or 22 days.

[0083] Numeric ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0084] The headings provided herein are not intended to limit the disclosure.

[0085] Throughout the specification, references to particular genes or proteins may be used interchangeably. A person skilled in the art will understand in the context whether the reference is intended to be a reference to the particular gene or the protein that is encoded by that gene.

[0086] The terms “pluripotent stem cell” and “PSC” refer to cells that display pluripotency. The terms “human pluripotent stem cell” and “hPSC” refer to cells derived, obtainable or originating from human tissue that display pluripotency. The hPSC may be a human embryonic stem cell or a human induced pluripotent stem cell (iPSC).

[0087] Human pluripotent stem cells may be derived from inner cell mass or reprogrammed using Yamanaka factors from many foetal or adult somatic cell types. The generation of hPSCs may be possible using somatic cell nuclear transfer.

[0088] The terms “human embryonic stem cell”, “hES cell” and “hESC” refer to cells derived, obtainable or originating from human embryos or blastocysts, which are self -renewing and pluri- or toti-potent, having the ability to yield all of the cell types present in a mature animal. Human embryonic stem cells (hESCs) can be isolated, for example, from human blastocysts obtained from human preimplantation embryos, in vitro fertilized embryos, or one-cell human embryos expanded to the blastocyst stage.

[0089] The terms “induced pluripotent stem cell” and “iPSC” and “hiPSC” (human iPSC) refer to cells derivable, obtainable or originating from adult somatic cells of any type reprogrammed to a pluripotent state through the expression of exogenous genes, such as transcription factors, including but not limited to a preferred combination of OCT4, SOX2, KLF4 and c-MYC. hiPSC show levels of pluripotency equivalent to hESC but can be derived from an individual for autologous therapy with or without concurrent gene correction prior to differentiation and cell delivery.

[0090] More generally, the method disclosed herein could be applied to any pluripotent stem cell derived from any individual or a hPSC subsequently modified to generate a mutant model using gene-editing or a mutant hPSC corrected using gene-editing. Gene-editing could be by way of CRISPR, TALEN or ZF nuclease technologies.

[0091] As used herein, the term “mesoderm” refers to one of the three germinal layers that appears during early embryogenesis and which gives rise to various specialized cell typesincluding blood cells of the circulatory system, muscles, the heart, the dermis, skeleton, and other supportive and connective tissues.

[0092] As used herein, the term “cell culture” refers to any in vitro culture of cells. The term “culturing” refers to the process of growing and / or maintaining and / or manipulating a cell. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos. As used herein, the terms “primary cell culture,” and “primary culture,” refer to cell cultures that have been directly obtained from cells in vivo, such as from a tissue specimen or biopsy from an animal or human. These cultures may be derived from adults as well as foetal tissue.

[0093] A "progenitor cell" is a cell which is capable of differentiating along one or a plurality of developmental pathways, with or without self -renewal. Typically, progenitor cells are unipotent or oligopotent and are capable of at least limited self- renewal.

[0094] The terms "differentiate", "differentiating" and "differentiated", relate to progression of a cell from an earlier or initial stage of a developmental pathway to a later or more mature stage of the developmental pathway. Thus, “undifferentiated”, in this context, relate to a cell from an earlier or initial stage of a developmental pathway or a cell that has not yet developed into a specialized cell type. It will be appreciated that in this context "differentiated' does not mean or imply that the cell is fully differentiated and has lost pluripotency or capacity to further progress along the developmental pathway or along other developmental pathways. Differentiation may be accompanied by cell division.

[0095] As will be well understood in the art, the stage or state of differentiation of a cell may be characterized by the expression and / or non-expression of one or more specific markers. In some embodiments, the expression of “signature” or “milestone” markers may be used in determining or defining the stage or state of differentiation instead of using the period of time defined in days and / or hours. In this context, by "markers" is meant nucleic acids or proteins that are encoded by the genome of a cell, cell population, lineage, compartment or subset, whose expression or pattern of expression changes throughout development. Nucleic acid marker expression may be detected or measured by any technique known in the art including nucleic acid sequence amplification (e.g. polymerase chain reaction) and nucleic acid hybridization (e.g. microarrays, Northernhybridization, in situ hybridization), although without limitation thereto. Protein marker expression may be detected or measured by any technique known in the art including flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein profiling (e.g. 2D gel electrophoresis), although without limitation thereto.

[0096] Such terms are commonplace and well-understood by the skilled person when characterizing cell phenotypes. By means of additional guidance, when a cell is said to be positive for or to express or comprise expression of a given marker, such as a given gene or gene product, a skilled person would conclude the presence or evidence of a distinct signal for the marker when carrying out a measurement capable of detecting or quantifying the marker in or on the cell. Suitably, the presence or evidence of the distinct signal for the marker would be concluded based on a comparison of the measurement result obtained for the cell to a result of the same measurement carried out for a negative control (for example, a cell known to not express the marker) and / or a positive control (for example, a cell known to express the marker). Where the measurement method allows for a quantitative assessment of the marker, a positive cell may generate a signal for the marker that is at least 1.5 -fold higher than a signal generated for the marker by a reference cell (e.g. negative control cell) or than an average signal generated for the marker by a population of reference or negative control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher, at least 100-fold higher, or even higher. Further, a positive cell may generate a signal for the marker that is 3.0 or more standard deviations, e.g., 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations, higher than an average signal generated for the marker by a population of reference or negative control cells.

[0097] As used herein, the terms “culture medium”, “cell culture medium”, “defined medium”, and the like refer to media that are suitable to support the growth of cells in vitro (i.e., cell cultures, cell lines, etc.). It is not intended that the term be limited to any particular culture medium. For example, it is intended that the definition encompass maintenance media as well as other media for the differentiation or specialization of cells. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures and cells of interest. In some embodiments, the cell culture medium used in various steps includes a basal medium which is supplemented. In some embodiments, the basal medium is SPELS medium. In one example, the SPELS medium is prepared by mixing 0.1% (or 0.05%) poly vinyl alcohol (PVA); linoleic and linolenic acid (125ng / mL each), soybean oil (Ipg / mL), alpha-tocopherol (50nM), L-ascorbic acid-2-phosphate (50pg / mL), L-ascorbic acid (50pg / mL, IxGlutaMAX, ITSE AF blood-free cell culture media supplement (50pgml-l), and lx Non Essential Amino Acids (MEM), in IMDM / F12 media.

[0098] As used herein the term “enriched” is used to refer to a population of cells which contains a significant proportion of a specific subset or subtype of cell, wherein the set of cells may contain 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95% or 100% of the specific subset / subtype of cell. “Enriched”, as in an enriched population of cells, can be defined phenotypically based upon the increased number of a specific subset or subtype of cells having a particular marker, or combination of markers, or having one or more markers and lacking one or more other markers, in a fractionated, or expanded, set of cells as compared with the number of cells having the marker, combination of markers, or having one or more markers and lacking one or more other markers, in the unfractionated or unexpanded set of cells.

[0099] As used herein, “tissue” means an aggregate of cells. In some embodiments, the cells in the tissue are cohered or fused.[000100] As used herein, “definitive haematopoietic stem / progenitor cell” refers to a cell or population of cells responsible for producing all mature blood cells throughout the lifespan of an organism. Clinically, they are important for transplantation in blood-related diseases, particularly in subjects undergoing myeloablative therapy. Definitive haematopoiesis is distinguished from primitive haematopoiesis that occurs transiently during early development and arises from the yolk sac.[000101] A definitive haematopoietic stem / progenitor cell described herein or produced by the methods described herein may be further characterized by gene and protein expression as detailed in the examples and figures.[000102] As used herein, “embryoid body” and “EB” refers to a three-dimensional aggregate of PSCs. Advantageously, EBs in may be cultured in suspension, thus making EB cultures scalable for clinical applications. Additionally, the three-dimensional structure of EBs, including the establishment of complex cell adhesions and paracrine signaling within the EB microenvironment, enables differentiation and morphogenesis which yields microtissues that are similar to native tissue structures, for example the AGM as disclosed herein. Methods for culturing EBs are knownin the art. A specific example of a spin or swirling EB culture method that may be used in the present invention is described in Ng et al. Nature Protocols 3, 768-776 (2008).[000103] The terms “decrease”, “reduced”, “reduction”, “to a lesser extent,” or “inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter, including by a statistically significant amount. In some embodiments, “reduced,” “reduction,” “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.[000104] The terms “increased”, “increase”, “increases”, or “enhance” or “activate” or “to a greater extent” are all used herein to mean an increase of a property, level, or other parameter, including by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase”, “to a greater extent,” “enhance" or “activate" can refer to an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100- fold increase, at least about a 1000-fold increase or more as compared to a reference level.[000105] As used herein, a “reference level” refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., cell, tissue, or a biological sample obtained from a patient prior to being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition asdisclosed herein). Alternatively, a reference level can also refer to the level of a given marker or parameter in a subject, organ, tissue, or cell, prior to administration of a treatment, e.g., with an agent or via administration of a composition.[000106] As used herein, a “control” or an “appropriate control” refers to an untreated, otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that was not contacted by an agent or composition described herein) relative to a cell, tissue, biological sample, or population contacted or treated with a given treatment. For example, an appropriate control can be a cell, tissue, organ or subject that has not been contacted with an agent or subjected to the same methods as described herein.[000107] In one or more embodiments described herein, assessing the expression of various genes includes comparing the fold change. In one embodiment, the fold change is used to measure the change in the expression level of genes. In one embodiment, the expression of a gene can be expressed as relative expression comparative to a housekeeping gene.[000108] The term “agonist” or “activator” may be used interchangeably and as used herein means an activator, for example, of a pathway or signalling molecule. An agonist of a molecule can retain substantially the same, or a subset, of the biological activities of the molecule (e.g. FGF). For example, an FGF agonist or FGF activator means a molecule that selectively activates FGF signalling.[000109] The term “antagonist” or “inhibitor” as used herein means a selective inhibitor, for example of a pathway or signalling molecule. An inhibitor or antagonist of a molecule (e.g. BMP4 inhibitor) can inhibit one or more of the activities of the naturally occurring form of the molecule. For example, a BMP4 inhibitor is a molecule that selectively inhibits BMP signalling mediated by BMP4.[000110] As used herein, a “condition, disease or disorder requiring HSC transplantation” may be a malignant condition, disease or disorder, for example acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), Non-Hodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, or other solid tumour, or may be a non- malignant condition, disease or disorder, for example thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.[000111] As used herein, “myeloablative therapy” refers to treatment, generally radiation or chemotherapy, that kills cells in the bone marrow, including cancer cells.[000112] As used herein, the term “therapeutic composition” refers to a composition comprising a haematopoietic stem / progenitor cell of or produced according to the invention that has been formulated for administration to a subject. Preferably, the therapeutic composition is sterile. In one embodiment, the therapeutic composition is pyrogen-free.[000113] The term “therapeutically effective amount” refers to an amount of the haematopoietic stem / progenitor cell of or produced according to the invention effective to treat a condition, disease or disorder in a subject.[000114] The terms “treat”, “treating” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent or ameliorate a condition, disease or disorder in a subject or slow down (lessen) progression of a condition, disease or disorder in a subject. Subjects in need of treatment include those already with the condition, disease or disorder as well as those in which the condition, disease or disorder is to be prevented.[000115] The terms “preventing”, “prevention”, “preventative” or “prophylactic” refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, a disease or disorder, including an abnormality or symptom. A subject in need of prevention may be prone to develop the condition, disease or disorder.[000116] The term “ameliorate” or “amelioration” refers to a decrease, reduction or elimination of a condition, a disease or disorder, including an abnormality or symptom. A subject in need of treatment may already have the condition, disease or disorder, or may be prone to have the condition, disease or disorder, or may be in whom the condition, disease or disorder is to be prevented.[000117] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. Any example or embodiment herein shall be taken to apply mutatis mutandis to any other example or embodiment unless specifically stated otherwise.[000118] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent methods and systems are clearly within the scope of the disclosure, as described herein.[000119] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.[000120] The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying drawings. Although the examples herein concern humans and the language is primarily directed to human concerns, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.[000121] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.Cell Culture components[000122] In some embodiments, the methods of the present invention comprise a WNT pathway activator or a WNT agonist. In some embodiments, the WNT pathway activator or WNT agonist is selected from the group consisting of CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)5-(5-methyl- lH-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), Wntl, Wnt-2, Wnt- 2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt48 8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-lOa, Wnt-lOb, Wnt-11, Wnt-16b, RSPO co-agonists, lithium chloride, TDZD8 (4-Benzyl-2-methyl-l, 2, 4-thiadiazolidine-3, 5-dione), BIO-Acetoxime ((2'Z,3'E)-6- Bromoindirubin-3 '-acetoxime) , A 1070722 ( 1 -(7 -Methoxy quinolin-4-yl)-3 -[6(trifluoromethyl)pyridin-2-yl]urea), HLY78 (4-Ethyl-5,6-Dihydro-5-methyl-[l,3]dioxolo[4,5j]phenanthridine), CID 11210285 hydrochloride (2-Amino-4- (3,4(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride), WAY - 316606, (hetero)arylpyrimidines, IQ1, QS11, SB-216763, and DCA. In a preferred embodiment the Wnt pathway activator is CHIR99021. In one embodiment, the WNT agonist in the cell culture media is CHIR99021 ((CHIR) CAS 252917-06-9). In some embodiments, the concentration of CHIR99021 in the medium is about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.1 pM, about 3.2 pM, about 3.3 pM, about 3.4 pM, about 3.5 pM, about 3.6 pM, about 3.7 pM, about 3.8 pM, about 3.9 pM, about 4 pM, about4.1 pM, about 4.2 pM, about 4.3 pM, about 4.4 pM, about 4.5 pM, about 4.6 pM, about 4.7 pM, about 4.8 pM, about 4.9 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM.[000123] In some embodiments, the methods of the present invention comprise a TGF-beta pathway activator, for example, Activin A. In another embodiment, the TGF-beta pathway activator is selected from the group consisting of Activin A, TGF-betal, TGF-beta2, TGF-beta3, IDE1 / 2 (IDE1 (l-[2-[(2Carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (Heptanedioic acid- 1 -(249 cyclopentylidenehydrazide)), and Nodal. In a preferred embodiment, the TGF-beta pathway activator is Activin A. In some embodiments, the concentration of Activin A in the medium is about 1 ng / mE, about 2 ng / mL, about 3 ng / mL about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL,about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, or about 50 ng / mL. In another preferred embodiment, the TGF-beta pathway activator is TGF-beta 1. In some embodiments, the concentration of TGF-beta in the medium used in the methods of the invention is about 0.1 ng / ml, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL.[000124] In some embodiments, the methods of the present invention comprise FGF. In some embodiments, the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20 and FGF23. In a preferred embodiment, the FGF is FGF2. In some embodiments, the concentration of FGF2, also known as basic fibroblast growth factor (bFGF), in the medium is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, about 50 ng / mL, about 51 ng / mL, about 52 ng / mL, about 53 ng / mL, about 54 ng / mL, about 55 ng / mL, about 56 ng / mL, about 57 ng / mL, about 58 ng / mL, about 59 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, or about 100 ng / mL.[000125] In some embodiments, the methods of the present invention comprise a TGF-beta pathway inhibitor or ACTIVIN antagonist. In some embodiments, the TGF-beta pathway inhibitor / ACTIVIN antagonist is selected from the group consisting of A-83-01 (3-(6-Methyl-2- pyridinyl)-N-phenyl-4-(4-quinolinyl)- 1 H-pyrazole- 1 carbothioamide) , D4476 (4- [4-(2,3 -Dihydro- l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lHimidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2- Pyridinyl)- lH-pyrazol-4-yl]-2-pyridinyl]-N(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridinyl)-lH-pyrazol-4-yl]quinoline), RepSox (2-(3-(6-Methylpyridine-2-yl)-lH-pyrazol-4-yl)-l,5-naphthyridine), SB431542 (4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH- imidazol-2-yl]benzamide), SB505124 (2-[4-(l,3-Benzodioxol-5-yl)-2-(l,l-dimethylethyl)-lH- imidazol-5-yl]-6-methylpyridine), SB 525334 (6-[2-(l,l-Dimethylethyl)-5-(6-methyl-2- pyridinyl)- lH-imidazol-4yl] quinoxaline), SD208 (2-(5-Chloro-2-fluorophenyl)-4-[(4- pyridyl)amino]pteridine), ITD1 (4[l,l'-Biphenyl]-4-yl-l,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5- oxo-3-quinolinecarboxylic acid ethyl ester), DAN / Fc, antibodies to TGF-beta and TGF-beta receptors, TGF-beta inhibitory nucleic acids. In a preferred embodiment the ACTIVIN antagonist is SB431542. In some embodiments, the concentration of SB431542 in the medium is about 0.1 pM, about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or about 10 pM.[000126] In some embodiments, the methods of the present invention comprise a Rho kinase inhibitor (ROCKi). In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, or pyrintegrin. In a preferred embodiment, the ROCK inhibitor is thiazovivin. In some embodiments, the concentration of Y-27263 in the medium is about 1 pM, about 2 pM, about 5 pM, about 8 pM, about 8.2 pM, about 8.4 pM, about 8.6 pM, about 8.8 pM, about 9 pM, about 9.2 pM, about 9.4 pM, about 9.6 pM, about 9.8 pM, about 10 pM, about 10.2 pM, about 10.4 pM, about 10.6 pM, about 10.8 pM, about 11 pM, about 11.2 pM, about 11.4 pM, about 11.6 pM, about 11.8 pM, about 12 pM, about 15 pM, about 20 pM, about 25 pM or about 50 pM. In some embodiments, the concentration of thiazovivin in the medium is about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.2 pM, about 2.4 pM, about 2.6 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.6 pM, about 4.8 pM or about 5 pM.[000127] In some embodiments, the methods of the present invention comprise vascular endothelial growth factor (VEGF). In one embodiment, the VEGF includes human VEGF family members such as VEGFA as well as non-human VEGF. In some embodiments, the concentration of VEGF in the medium is about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL,about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, or about 500 ng / mL.[000128] In some embodiments, the methods of the present invention comprise a retinoic acid signalling agent. In some embodiments, the retinoic acid signalling agent is a retinoid is selected from retinol or retinyl acetate (RETA). In a preferred embodiment, the retinoid is RETA. In some embodiments, the concentration of RETA in the medium is about 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM, 0.08 pM, 0.09 pM, 0.1 pM, 0.15 pM, 0.16 pM, 0.17 pM, 0.18 pM, 0.19 pM, 0.2 pM, 0.25 pM, 0.30 pM, 0.35 pM, 0.4 pM, 0.45 pM, 0.5 pM, 0.55 pM, 0.6 pM, 0.65 pM, 0.7 pM, 0.75 pM, 0.85 pM, 0.9 pM, 0.95 pM, 1 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, 1.5 pM, 1.6 pM, 1.7 pM, 1.8 pM, 1.9 pM, 2 pM, 2.1 pM, 2.2 pM, 2.3 pM, 2.4 pM,2.5 pM, 2.6 pM, 2.7 pM, 2.8 pM, 2.9 pM, 3 pM, 3.1 pM, 3.2 pM, 3.3 pM, 3.4 pM, 3.5 pM, 3.6 pM, 3.7 pM, 3.8 pM, 3.9 pM, 4 pM, 4.1 pM, 4.2 pM, 4.3 pM, 4.4 pM, 4.5 pM, 4.6 pM, 4.7 pM,4.8 pM, 4.9 pM, or 5pM.[000129] In some embodiments, the methods of the present invention comprise a BMP. In some embodiments, the BMP is selected from the group consisting of BMP4, BMP2 and BMP7. In a preferred embodiment, the BMP is BMP4. In some embodiments, the concentration of bone morphogenetic protein 4 (BMP4) in the medium used is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, or about 100 ng / mL.[000130] In some embodiments, the methods of the present invention comprise an IGF. In some embodiments, the IGF is selected from the group consisting of IGF1 and IGF2. In some embodiments, the medium comprises both IGF1 and IGF2. In some embodiments, the concentration of IGF and / or IGF2, in the medium used is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL,about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, about 50 ng / mL, about 51 ng / mL, about 52 ng / mL, about 53 ng / mL, about 54 ng / mL, about 55 ng / mL, about 56 ng / mL, about 57 ng / mL, about 58 ng / mL, about 59 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, or about 100 ng / mL.[000131] In some embodiments, the methods of the present invention comprise stem cell factor (SCF). The term “SCF” includes human SCF, non-human SCF and all naturally occurring variants thereof. In some embodiments, the concentration of SCF in the medium is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, about 50 ng / mL, about 51 ng / mL, about 52 ng / mL, about 53 ng / mL, about 54 ng / mL, about 55 ng / mL, about 56 ng / mL, about 57 ng / mL, about 58 ng / mL, about 59 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, or about 100 ng / mL.[000132] In some embodiments, the methods of the present invention comprise thrombopoietin (TPO). In some embodiments, the concentration of TPO in the medium is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about48 ng / mL, about 49 ng / mL, about 50 ng / mL, about 51 ng / mL, about 52 ng / mL, about 53 ng / mL, about 54 ng / mL, about 55 ng / mL, about 56 ng / mL, about 57 ng / mL, about 58 ng / mL, about 59 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, or about 100 ng / mL.Methods for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs)[000133] In one aspect of the invention provides a method of using a multistage process to generate and expand definitive HSPCs. Generally, the method begins with a stage wherein a pluripotent stem cell is differentiated to a mesodermal cell, next the mesodermal cells are patterned to express HOXA genes, in a next stage, the patterned mesodermal cells are differentiated (and expanded) to hemogenic endothelium (HE). In the next stage, the HE cells are differentiated to undergo endothelial to haematopoietic transition into definitive HSPCs while being expanded at same time. The invention also provides a method of generating and expand definitive HSPCs that comprises differentiating pluripotent stem cell-derived mesodermal cells to give rise to HE, and differentiating the HE to give rise to HSPC.[000134] In one aspect, the present invention provides a method for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), the method comprising: a) culturing a population of mesoderm cells obtained from a population of PSCs in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; b) culturing the population of cells from step a) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step a); c) culturing the population of cells from step b) in a medium comprising a FGF, a BMP, an IGF, VEGF, and a retinoic acid signalling agent wherein the concentration of the retinoic acid signalling agent is 10 to 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b), and wherein the concentration of BMP is the same as that in themedium in step b), or alternatively up to about 20 fold lower, (preferably from 5 to 20 fold lower) than the concentration of BMP in the medium of step b); and d) culturing the population of cells from step c) in a medium comprising a stem cell factor (SCF) and thrombopoietin (TPO), FGF, an IGF, and a retinoic acid signalling agent to produce a population of definitive HSPCs.[000135] Time / duration[000136] In one embodiment, the culturing of the population of mesoderm cells in step a) is performed for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10. In a preferred embodiment, the culturing of the population of mesoderm cells in step a) is performed for a time sufficient for the generation of a population of cells expressing one or more of H0XA5, H0XA7, H0XA9, and HOXA10. In another embodiment, the culturing in step a) is performed for about 2 to about 72 hours, In one embodiment, the culturing in step a) is performed for about 2 days. Preferably, the culturing in step a) is performed for about 48 hours. In one embodiment, the WNT agonist is CHIR99021, optionally, wherein the medium comprises about 2 pM, to about 5 pM CHIR99021, preferably 4 pM CHIR99021. In another embodiment, the ACTIVIN antagonist is SB431542, optionally, wherein the medium comprises about 2 pM, to about 5 pM SB431542, preferably about 3 or 4 pM SB431542. In one embodiment, the medium comprises about 5 to about 50 ng / mL FGF, preferably about 20 ng / mL FGF. In one embodiment, the medium comprises about 5 to about 50 ng / mL VEGF, preferably about 25 ng / mL VEGF. In another embodiment, the retinoic acid signalling agent is RETA, optionally, wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA.[000137] In one embodiment, the culturing of the population of cells from step a) in step b) is performed for a time sufficient for generation of a population of cells expressing CD34 on the cell surface. In another embodiment, the culturing in step b) is performed for about 2 to about 72 hours, In one embodiment, the culturing in step b) is performed for about 2 days. Preferably, the culturing in step b) is performed for about 48 hours. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 20 ng / mL FGF. In one embodiment, the concentration of VEGF in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold,about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold greater than the concentration of VEGF in the medium of step a). In one embodiment, the concentration of VEGF in the medium is about 5 fold greater than the concentration of VEGF in the medium of step a). In one embodiment, the medium comprises about 50 to about 200 ng / mL VEGF, preferably about 150 ng / mL VEGF. In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a). In another embodiment, the retinoic acid signalling agent is RETA. In one embodiment, the medium comprises about 0.5 pM, to about 4 pM, RETA, preferably about 2 pM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of an IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL BMP4, preferably about 20 ng / mL BMP4.[000138] In one embodiment, the culturing of the population of cells from step b) in step c) is performed for a time sufficient for generation of a population of cells co-expressing CD34 and CXCR4 on the cell surface. In another embodiment, the culturing in step c) is performed for at least about 2 days and up to about 6 days. In one embodiment, the culturing in step c) is performed for about 6 days. In another embodiment, the culturing in step c) is performed for about 48 hours. In one embodiment, the medium comprises about 5 to about 50 ng / mL FGF, preferably about 20 ng / mL FGF. In one embodiment, the medium comprises about 50 ng / mL to about 200 ng / mL VEGF, preferably about 150 ng / mL VEGF. In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b). In another embodiment, the retinoic acid signalling agent is RETA. In one embodiment, the mediumcomprises about 50 nM to about 100 nM RETA, preferably about 100 nM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of an IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the concentration of the BMP4 in the medium is the same as that in the medium in step b). In one embodiment, the concentration of the BMP4 in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold lower than the concentration of BMP4 in the medium of step b). In one embodiment, the concentration of BMP4 in the medium is about 10 fold lower than the concentration of BMP4 in the medium of step b). In another embodiment, the medium comprises about 0.5 to about 5 ng / mL BMP4, preferably about 2 ng / mL BMP4.[000139] In one embodiment, the culturing of the population of cells from step c) in step d) is performed for a time sufficient for generation of a population of cells co-expressing CD34, CD90 and CD45 on the cell surface. In another embodiment, the culturing in step d) is performed for about 1 day up to about 5 days. In one embodiment, the culturing in step d) is performed for about 2 days. Preferably, the culturing in step d) is performed for about 48 hours. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 10 ng / mL FGF. In one embodiment, the medium does not comprise VEGF. In another embodiment, the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of an IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 5 to about 50 ng / mL TPO, preferably about 10 ng / mL TPO. In one embodiment, the medium comprises about 5 to about 50 ng / mL SCF, preferably about 10 ng / mL SCF. In one embodiment, the medium does not comprise BMP4. In another embodiment, the medium in step d) further comprises, a TGF-beta pathway activator, a BMP pathway activator, or a combination of one or more thereof.[000140] In one embodiment, step a) occurs from day 0 to day 2. In another embodiment, step b) occurs from day 2 to day 4. In another embodiment, step c) occurs from day 4 to day 10. In another embodiment, step d) occurs from day 10 to day 12, day 13 or day 14, or day 15. In one embodiment, step a) occurs from day 0 to day 2, step b) occurs from day 2 to day 4, step c)occurs from day 4 to day 10, and step d) occurs from day 10 to day 12, day 13 or day 14, or day 15.[000141] In one embodiment, the population of mesoderm cells are in embryoid bodies (EBs).[000142] According to another aspect, the present invention provides a method for differentiating a population of pluripotent stem cells (PSCs) into a population of definitive haematopoietic stem / progenitor cells (HSPCs), the method comprising: i) culturing the population of PSCs in a basal medium comprising a WNT agonist, a fibroblast growth factor (FGF), and Activin A; ii) culturing the population of cells comprising mesoderm cells from step i) in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; iii) culturing the population of cells from step ii) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step ii); iv) culturing the population of cells from step iii) in a medium comprising a FGF, a BMP, an IGF, VEGF, and a retinoic acid signalling agent wherein the concentration of the retinoic acid signalling agent is 10 to 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii), and wherein the concentration of BMP is 5 to 20 fold lower than the concentration of BMP in the medium of step iii), v) culturing the population of cells from step iv) in a medium as recited in step iv) excluding VEGF; and vi) culturing the population of cells from step v) in a medium comprising a stem cell factor (SCF) and thrombopoietin (TPO), FGF, an IGF, and a retinoic acid signalling agent to produce a population of definitive HSPCs.[000143] In one embodiment, the culturing of the population of cells in step i) is performed for a time sufficient to generate a population of mesoderm cells expressing CD13 and CD90 on the cell surface. In another embodiment, the culturing in step ii) is performed for about 2 to about 48 hours. In one embodiment, the culturing in step i) is performed for about 1 day. Preferably, the culturing in step i) is performed for about 24 hours. In one embodiment, the WNT agonist is CHIR99021, optionally wherein the medium comprises about 2 pM, to about 5 pM CHIR99021, preferably 4 pM. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 20 ng / mL FGF. In one embodiment, the concentration of ACTIVIN A in the medium of step i) is about 5 ng / ml to 50 ng / mL, preferably wherein the concentration is about 30 ng / mL. In another embodiment, the medium further comprises a BMP, preferably BMP4. In another embodiment, the concentration of ACTIVIN A in the medium of step i) is about 5 ng / ml and the medium further comprises a BMP, preferably BMP4, at a concentration of about 3 ng / mL. In yet a further embodiment, the medium also comprises a ROCK inhibitor. In a preferred embodiment, the ROCK inhibitor is thiazovivin. In another embodiment, the medium further comprises about 0.5 pM to about 5 pM, preferably about 1 pM ROCK inhibitor.[000144] In one embodiment, the culturing of the population of cells from step i) in step ii) is performed for a time sufficient for the generation of a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, and HOXA10. In a preferred embodiment, the culturing of the population of mesoderm cells in step ii) is performed for a time sufficient to generate a population of cells expressing one or more of H0XA5, H0XA7, H0XA9, and HOXA10. In another embodiment, the culturing in step ii) is performed for about 2 to about 72 hours. In one embodiment, the culturing in step ii) is performed for about 2 days. Preferably, the culturing in step ii) is performed for about 48 hours. In one embodiment, the WNT agonist is CHIR99021, optionally wherein the medium comprises about 2 pM, to about 5 pM CHIR99021, preferably 4 pM CHIR9902L In another embodiment, the ACTIVIN antagonist is SB431542, optionally wherein the medium comprises about 2 pM, to about 5 pM SB431542, preferably about 3 or 4 pM SB431542. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 20 ng / mL FGF. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL VEGF, preferably about 25 ng / mL VEGF. In another embodiment, the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA.[000145] In one embodiment, the culturing of the population of cells from step ii) in step iii) is performed for a time sufficient to generate a population of cells expressing CD34 on the cell surface. In another embodiment, the culturing in step iii) is performed for about 2 to about 72 hours. In one embodiment, the culturing in step iii) is performed for about 2 days. Preferably, the culturing in step iii) is performed for about 48 hours. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 20n g / mL FGF. In one embodiment, the concentration of VEGF in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold greater than the concentration of VEGF in the medium of step ii). In one embodiment, the concentration of VEGF in the medium is about 5 fold greater than the concentration of VEGF in the medium of step ii). In one embodiment, the medium comprises about 50 ng / mL to about 200 ng / mL VEGF, preferably about 150 ng / mL VEGF. In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii). In another embodiment, the retinoic acid signalling agent is RETA. In one embodiment, the medium comprises about 0.5 pM, to about 4 pM, RETA, preferably about 2 pM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of an IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL BMP4, preferably about 20n g / mL BMP4.[000146] In one embodiment, the culturing of the population of cells from step iii) in step iv) is performed for a time sufficient to generate a population of cells co-expressing CD34 and CXCR4 on the cell surface. In another embodiment, the culturing in step iv) is performed for at least about 2 days and up to about 6 days. In one embodiment, the culturing in step iv) is performed for about 2 days. In a preferred embodiment, the culturing in step iv) is performed for about 4 days. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 20 ng / mL FGF. In one embodiment, the medium comprises about 50 ng / mL to about 200 ng / mL VEGF, preferably about 150 ng / mL VEGF. In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 10 fold, about 15 fold, about 20 fold, about 25 fold,about 30 fold, about 35 fold, about 40 fold, about 45 fold, or about 50 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 20 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii). In one embodiment, the concentration of the retinoic acid signalling agent in the medium is about 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii). In another embodiment, the retinoic acid signalling agent is RETA. In one embodiment, the medium comprises about 50 nM to about 100 nM RETA, preferably about 100 nM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of an IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the concentration of the BMP4 in the medium is about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, or about 20 fold lower than the concentration of BMP4 in the medium of step iii). In one embodiment, the concentration of BMP4 in the medium is about 10 fold lower than the concentration of BMP4 in the medium of step iii). In another embodiment, the medium comprises about 0.5 ng / mL to about 5 ng / mL BMP4, preferably about 2 ng / mL BMP4.[000147] In one embodiment, the culturing of the population of cells from step iv) in step v) is performed for a time sufficient to generate a population of cells expressing CD34 without CXCR4 on the cell surface. In another embodiment, the culturing in step v) is performed for about 1 day up to about 5 days. In one embodiment, the culturing in step v) is performed for about 2 days. Preferably, the culturing in step v) is performed for about 48 hours. In one embodiment, the medium comprises about 5 to about 50 ng / mL FGF, preferably about 10 ng / mL FGF. In another embodiment, the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of an IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 0.5 ng / mL to about 5 ng / mL BMP4, preferably about 2 ng / mL BMP4. In one embodiment, the medium in step v) is identical to that employed for step iv) except that VEGF is excluded. In another embodiment, the method further comprises including in the culture medium in step v) about 5 ng / mL to about 50 ng / mL SCF, preferably about 10 ng / mL SCF after about 24 hours, or after about 48h when the culturing in step iv) occurs for greater than 48 hours. In another embodiment, when the culturing in step iv) occurs for greater than 48 hours, the methodfurther comprises including in the culture medium in step v) after 48 hours a TGF-beta pathway activator, a BMP pathway activator, or a combination of one or more thereof.[000148] In one embodiment, the culturing of the population of cells from step v) in step vi) is performed for a time sufficient for to generate a population of definitive HSPCs. In another embodiment, the culturing in step vi) is performed from about 1 day up to about 10 days. In another embodiment, the culturing in step vi) is performed from about 1 day up to about 5 days. In one embodiment, the culturing in step vi) is performed for about 3 days. Preferably, the culturing in step vi) is performed for about 48 - 72 hours. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL FGF, preferably about 10 ng / mL FGF. In one embodiment, the medium does not comprise VEGF. In another embodiment, the retinoic acid signalling agent is RETA, optionally wherein the medium comprises about 50 nM to about 100 nM RETA, preferably about 50 nM RETA. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL of IGF, preferably about 10 ng / mL IGF1 and / or IGF2, even more preferably 10 ng / mL of each of IGF1 and IGF2. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL TPO, preferably about 10 ng / mL TPO. In one embodiment, the medium comprises about 5 ng / mL to about 50 ng / mL SCF, preferably about 10 ng / mL SCF. In one embodiment, the medium does not comprise BMP4. In yet a further embodiment, the medium further comprises StemRegenin 1 (SRI) (4-[2-[[2-benzo[b]thien-3-yl-9-(l-methylethyl)-9H-purin-6-yl]amino]ethyl]-phenol). In one embodiment, the medium comprises about 2 nM to about 500 nM, preferably about 100 nM StemRegenin. In another embodiment, the method further comprises including in the culture medium in step vi) a TGF-beta pathway activator, a BMP pathway activator, or a combination of one or more thereof.[000149] In one embodiment, step i) occurs from day 0 to day 1, step ii) occurs from day 1 to day 3, step iii) occurs from day 3 to day 5, step iv) occurs from day 5 to day 7, step v) occurs from day 7 to day 11, and step vi) occurs from day 11 to day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21 or for a time sufficient for generation of a population of definitive HSPCs.[000150] In one embodiment, the population of definitive HSPCs express one or more of HLF, SPINK2, HOXA. RUNX1, MECOM, MEET3, HLF, HOXA9. In a preferred embodiment, the population of definitive HSPCs are HLF+SP1NK2+. In another embodiment, the definitive the population of definitive HSPCs are CD34+. In another embodiment, the population of definitiveHSPCs express CD34, CD45, and one or more of CD90, CD44, KIT, CD201, ITGA3 and ITGA6 on the cell surface.[000151] In some embodiments, the above methods comprise culturing the population of cells using an embryoid body protocol. In another embodiment, the cells are cultured with swirling. In one embodiment, the cells are cultured according to a swirling Embryoid Body (SwB) protocol as described in Motazedian, A. et al. Nature cell biology 22, 60-73 (2020).[000152] In one embodiment of the above methods, about 2.0 x 106- about 2.0 x 107iPSCs are differentiated in 60 mm dishes. In one embodiment, dissociated iPSCs are transferred to a nontissue culture treated 60 mm dish and cultured in 5 ml of SPELS medium. In one embodiment, the cell culture dishes are placed on an orbital shaker. In one embodiment, the methods yield approximately 1.4 x 108CD34+hematopoietic cells by day 14, a yield of about 7 CD34+cells for each input iPSC.[000153] In one embodiment of the above methods, the method further comprises harvesting single cells, cell aggregates or clusters and / or EBs in suspension following step d) or step vi). In another embodiment, the method further comprises dissociating or disaggregating cell aggregates or EBs in suspension following step d) or step vi) and harvesting cells dissociated or disaggregated from the EBs or cell aggregates or clusters. In another embodiment of the above methods, the method further comprising enriching, or sorting the harvested cells, such as for CD34+cells. In one embodiment, the cells are enriched or sorted using magnetic bead separation. In another embodiment, the cells are enriched using cell sorting. In one embodiment, the cells are enriched for CD34+cells.[000154] In some embodiments, the above methods further comprise sorting the obtained cells, such as following step d) or step vi) or following any harvesting or dissociation or disaggregation, using CD34, CD44, CD45, CD73, CD90, KIT, CD201, ITGA3, ITGA6 and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positive. In some embodiments, the sorting uses CD34 positive and CD44 positive. In some embodiments, the sorting uses CD34 positive and CD45 positive. In some embodiments, the sorting uses CD34 positive, CD44 positive and CD45 positive. In some embodiments, the sorting uses CD34 positive, CD44 positive, CD45 positive and CD90 positive. In some embodiments, the sorting uses CD34 positive, CD44 positive, CD45 positive, CD90 positive and KIT positive. In some embodiments,the sorting uses CD34 positive and CD73 negative. In some embodiments, the sorting uses CD34 positive, CD73 negative, and CXCR4 lo / negative. In some embodiments, the sorting uses CD34 positive and / or CD201 positive, ITGA3 positive, or ITGA6 positive.[000155] In one embodiment of the above methods, the population of cells is cultured under hypoxic conditions, or low oxygen tension, preferably between about 2% and about 10% oxygen.[000156] In another embodiment, the above methods comprise a step of cry opreserving the harvested, enriched and / or sorted cells. That is, the population of definitive HSPCs may be optionally cryopreserved and subsequently thawed and further cultured or administered to a subject. In a preferred embodiment, the method further comprises cryopreserving the population of HSPCs following any of the aforementioned methods. In a further embodiment, there is provided a cryopreserved population of HSPCs obtained according to the methods described herein. In another embodiment, there is provided a composition comprising the cryopreserved population of HSPCs.[000157] As disclosed herein, the present invention provides culture methods that yield definitive HSPCs capable of in vitro differentiation, ex vivo modulation, and the capacity to engraft multiple lineages long term.[000158] Medium[000159] Any pluripotent stem cell population, including a human embryonic stem cell population (hESC) or a human induced pluripotent stem cell population (iPSCs), can be used as the starting material to derive HSPCs using the methods described herein. In one embodiment, the population of pluripotent progenitor cells is a human iPSC population. In one embodiment, the iPSC cells grown in feeder-free conditions. In another embodiment, cells obtained from a subject can be subjected to methods to generate patient specific iPSCs which can then be differentiated using the methods described herein.[000160] In another embodiment, in the methods of the invention, the cells undergoing haematopoietic differentiation are cultured in SPELS medium, comprising IMDM / F12 media supplemented with 0.1% (or 0.05%) poly vinyl alcohol (PVA); linoleic and linolenic acid (125 ng / mL each), soybean oil (1 pg / mL), alpha-tocopherol (50 nM) , L-ascorbic acid-2-phosphate (50pg / mL), L-ascorbic acid (50|jg / mL, IxGlutaMAX, ITSE AF blood-free cell culture media supplement (50 pgml-1), and lx Non Essential Amino Acids (MEM).Methods of treatment[000161] A haematopoietic stem / progenitor cell produced by or obtained according to the methods described herein or a therapeutic composition comprising such a cell may be used for treating a condition, disease or disorder requiring HSC transplantation following myeloablative therapy in a subject. Alternatively, a haematopoietic stem / progenitor cell produced according to the methods described herein or a therapeutic composition comprising such a cell may be used for treating a condition, disease or disorder requiring HSC transplantation that does not require myeloablative therapy in a subject.[000162] Accordingly, in one aspect, the present invention provides a method for treating a condition, disease or disorder requiring HSC transplantation, the method comprising administering to a subject the cell or population of cells or a therapeutic composition comprising the cell or population of cells, wherein the cell or population of cells is produced according to the methods described herein.[000163] In another aspect, the present invention provides a use of a cell or population of cells, or a therapeutic composition comprising the cell or population of cells, in the manufacture of a medicament for treating a condition, disease or disorder requiring HSC transplantation, wherein the cell or population of cells is produced according to the methods described herein.[000164] In another aspect, the present invention provides a cell or population of cells for use in treating a condition, disease or disorder requiring HSC transplantation, wherein the cell or population of cells is produced according to the methods described herein.[000165] In one embodiment of the foregoing aspects pertaining to methods of treatment and therapeutic uses of cells produced according to the methods described herein, the condition, disease or disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), Non-Hodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, other solid tumor, thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.[000166] It will be appreciated by the person skilled in the art that the exact manner of administering to a subject a therapeutically effective amount of a haematopoietic stem / progenitor cell produced according to the invention for treating a condition, disease or disorder will be at the discretion of the medical practitioner. The mode of administration, including dosage, combination with other agents, timing and frequency of administration, and the like, may be affected by the diagnosis of a subject’s likely responsiveness to treatment with the haematopoietic stem / progenitor cell produced according to the invention, as well as the subject’s condition and history.[000167] The haematopoietic stem / progenitor cell produced according to the invention will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular type of condition, disease or disorder being treated, the particular subject being treated, the clinical condition of the subject, the site of administration, the method of administration, the scheduling of administration, possible sideeffects and other factors known to medical practitioners. The therapeutically effective amount of the haematopoietic stem / progenitor cell produced according to the invention to be administered will be governed by such considerations.[000168] The haematopoietic stem / progenitor cell may be administered to a subject by any suitable method including intravenous (IV), intra-arterial, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous (SC), intra- articular, intrasynovial, intrathecal, intracoronary, transendocardial, surgical implantation, topical and inhalation (e.g. intrapulmonary) routes. Most preferably, the haematopoietic stem / progenitor cell produced according to the invention is administered IV.Kits / Culture Systems[000169] Also provided herein are kits comprising one or more of a cell or population of cells produced according to a method described herein, a product or composition comprising a cell or population of cells produced, optionally comprising a further therapeutic agent, or optionally wherein the cell comprises a reporter system or other modification, according to a method described herein. The kits may further comprise one or more of components selected from an agonist, inhibitor, medium, apparatus or other component that can be used in a method described herein, instructions for use, for example instructions on how to generate the cells, perform an assay, harvest, isolate, or administer the cell or population of cells, composition, or product, and a vial orother container for housing one of these aforementioned cells, compositions, products, agonists, inhibitors, media etc.[000170] In one embodiment, there is provided a kit for use in generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), wherein said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF), thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor. In another embodiment, the kit components are provided in amounts as described in the paragraphs above referring to “cell culture components”. In another embodiment, the kit further comprises one or more basal media.[000171] In another embodiment, there is provided a kit when used for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), wherein said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF), thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor. In another embodiment, the kit components are provided in amounts as described in the paragraphs above referring to “cell culture components”. In another embodiment, the kit comprises one or more basal media.[000172] In another embodiment, the kit of the foregoing embodiments, comprises SPELS medium or components to provide SPELS medium, wherein said medium comprises IMDM / F12 media supplemented with 0.1% (or 0.05%) poly vinyl alcohol (PVA); linoleic and linolenic acid (125 ng / mL each), soybean oil (1 pg / mL), alpha-tocopherol (50 nM) , L-ascorbic acid-2-phosphate (50 pg / mL), L-ascorbic acid (50 pg / mL, IxGlutaMAX, ITSE AF blood-free cell culture media supplement (50 pg / mL), and lx Non Essential Amino Acids (MEM).ExamplesMaterials and Methods[000173] Induced pluripotent stem cell (iPSC) culture and maintenance. RM TOM iPSCs, constitutively expressing a tdTOMATO transgene from the GAPDH locus, were derived from human foreskin fibroblasts purchased from ATCC and reprogrammed using the hSTEMCCAloxP four-factor lentiviral vector, and integrated vector sequences were removed using Cre recombinase. PB1.1 (male), PB 10.5 (male) and PB5.1 (female) iPSCs were reprogrammed from the peripheral blood of a healthy volunteers with Sendai virus carrying the reprogramming factors POU5F1, SOX2, KLF4 and MYC. PB1.1 was engineered to express mTagBFP2 from the GAPDH locus. Following vector integration, Cre recombinase was used to excise the antibiotic selectable marker from this version of the targeting vector. Human iPSC lines were maintained by coculture with mouse embryo fibroblasts in KOSR medium (Thermofisher), or adapted to culture on Matrigel (Coming) in Essential 8 medium (Thermofisher). Molecular karyotyping by single nucleotide polymorphism array was performed at regular intervals using the Illumina Infinium GSA-24 v3.0 chip with a resolution of 0.50Mb, with no clinically significant genomic imbalance detected. Mycoplasma contamination was excluded by regular testing.[000174] Harvest of iPSCs for initiation of differentiation. Hematopoietic differentiation was performed using the swirling embryoid body (SwB) method as described (Calvanese, V. et al. Nature 604, 534-540 (2022)). Cells were dissociated using Accutase cell dissociation reagent (Merck) and resuspended in SPELS differentiation medium, an evolution of APEL and STAPEL media. SPELS medium (defined hereinabove) includes non-essential amino acids, but not albumin or Protein Free Hybridoma Medium. SPELS medium was supplemented during differentiation with various cell culture components as described in detail herein.[000175] Approximately 2 x 106dissociated cells were transferred to each non-tissue culture treated 60 mm dish in 5 ml of SPELS medium. The dishes were then placed on a digital orbital shaker (Heathrow Scientific) rotating at 60 rpm in a 5% CO2 incubator at 37°C.[000176] Identification of differentiation conditions that produce CD34+hematopoietic cells multilineage engraftment ability: screening protocol#! and mouse cohort#!. Versions of screening protocol#! (encompassing 12 differentiation conditions) were analysed in mouse cohort#l transplantations, to identify conditions that generated iHSCs. As shown diagrammatically (Figures 7 and 9a, and Figure 2a), mesoderm was induced on day 0 of differentiation by a combination of 1, 2 or 4 pM CHIR99021 (Tocris Biosciences), 0 or 3 ng / ml recombinant human (rh) bone morphogenetic protein 4 (BMP4, R&D Systems), 5 or 30 ng / ml rhACTIVIN A (R&D Systems). All conditions included 20 ng / ml rh fibroblast growth factor FGF2 (PeproTech) and 1 pM Thiazovivin (Selleck Chem). Starting at day 1, medium changes occurred every 2 days during the differentiation. From day 1 - day 3, mesoderm was patterned to HOXA expression with 3 pM CHIR99021 (Tocris Biosciences) and 4 pM SB431542 (Cayman Chemicals or Selleck Chemical), 25 ng / ml rh vascular endothelial growth factor (VEGF, PeproTech), 25 ng / ml rh stem cell factor (SCF, PeproTech) and 20 ng / ml rh FGF2. On day 3, the medium was supplemented with 20 ng / ml rh BMP4, 50 ng / ml rh VEGF, 20 ng / ml rh FGF2, 50 ng / ml rh SCF and 10 ng / ml rh insulin-like growth factor 2 (IGF2, PeproTech). In selected transplantation experiments, cultures were supplemented at day 3 of differentiation with 2 pM retinol (ROL) or 2 pM retinyl acetate (RETA), which was removed at the day 5 medium change. At day 5, rh BMP4 was reduced to 2 ng / ml, while the other growth factors were unchanged. In early experiments, 10 ng / ml APELIN peptide (Merck) was included from day 5 to day 9. From day 11 of differentiation, growth factors were modified to include 50 ng / ml rh VEGF, 50 ng / ml rh SCF, 50 ng / ml rh thrombopoietin (TPO, PeproTech), 10 ng / ml rh FGF2 and 20 nM Stemregeninl (SRI, Selleck Chemical). Early experiments also included 10 ng / ml rh FLT3 receptor ligand (FLT3L, PeproTech) and 10 ng / ml rh IL3 (PeproTech). Blood cells were shed into the medium after 10-12 days of differentiation. After day 14 - day 16, cultures were harvested. Cells shed into the medium (suspension hematopoietic cells) were analyzed separately from cells dissociated from the swirling embryoid bodies (SwBs). Embryoid bodies were disaggregated by 45 min incubation with 2 mg / ml Collagenase Type I (Worthington) at 37°C. Suspension hematopoietic cells and disaggregated embryoid bodies were analyzed by flow cytometry, and RNA was extracted or cells were cryopreserved in 10%DMSO / CJ2 medium (Stachecki, J. J., et al. Cryobiology 37, 346-354 (1998)) prior to transplantation. For some transplantation experiments, CD34 antibody-conjugated magnetic beads (Miltenyi Biotec) were used according to the manufacturer's instructions, to enrich CD34+cells from disaggregated embryoid bodies and deplete cultures of stromal cells prior to cryopreservation.[000177] Determination of retinoid treatments for generating iHSCs: screening protocol#! and mouse cohorts#! and#3. On day 0, mesoderm was patterned using 4 pM CHIR99021, 3 ng / ml rhBMP4, 5 ng / ml rh Activin A, 20 ng / ml rhFGF2 and 1 pM thiazovivin or 4 pM CHIR99021 with 30 ng / ml rh Activin A, 20 ng / ml rhFGF2 and 1 pM thiazovivin. HOXA expression was induced as above (3 pM CHIR99021, 4 pM SB431542, 25 ng / ml rh VEGF, 25 ng / ml rh SCF, and 20 ng / ml rh FGF2). On day 3, the medium was supplemented with 20 ng / ml rh BMP4, 50 ng / ml rh VEGF, 20 ng / ml rh FGF2, 50 ng / ml rh SCF 10 ng / ml rh IGF2, and 2 pM RETA. The retinoidwas removed at the day 5 medium change (control), or RETA supplementation was repeated at 2 day intervals during the differentiation as shown in Figure 4a at concentrations between 100 nM and 2 pM. On day 5, the medium was supplemented with 2 ng / ml rh BMP4, 50 ng / ml rh VEGF, 20 ng / ml rh FGF2, 50 ng / ml rh SCF and 10 ng / ml rh IGF2 with or without RETA at 2 pM or 100 nM. From day 11 of differentiation, growth factors included were 50 ng / ml rh VEGF, 50 ng / ml rh SCF, 50 ng / ml rh thrombopoietin (TPO, PeproTech), 10 ng / ml rh FGF2 and 20 nM SRI, with and without RETA at 2 pM or 100 nM. From day 14 to 16, suspension haematopoietic cells were pooled in some experiments with MACS-enriched CD34+cells from disaggregated SwBs, analysed by FACS, and cryopreserved for transplantation.[000178] Development of a protocol for the generation of hematopoietic cells containing iHSCs from multiple iPSC lines: protocol#3 and mouse cohorts#4-#7. Similar to previous versions, mesoderm was induced with 4 pM CHIR99021, 30 ng / ml Activin A, 20 ng / ml rh FGF2 and 1 pM thiazovivin and patterned to HOXA expression (day 1-3) with 3 pM CHIR99021, 4 pM SB431542, 25 ng / ml rh VEGF, 20 ng / ml rh FGF2 and 50 nM RETA. On day 3, the medium was supplemented with 20 ng / ml rh BMP4, 2 pM RETA, 150 ng / ml rh VEGF, 20 ng / ml rh FGF2, 10 ng / ml rh IGF2 and 10 ng / ml rh insulin-like growth factor 1 (IGF1, PeproTech). At day 5, rh BMP4 and RETA were reduced to 2 ng / ml and 100 nM respectively and all other cytokines were as for day 3. At day 7, rh VEGF was removed, rh BMP4 and RETA were retained at 2 ng / ml 100 nM respectively, while rh FGF2, rh IGF1 and rh IGF2 were supplemented at 10 ng / ml. At day 9, rh SCF was included at 10 ng / ml, and all other cytokines were as for day 7. From day 11 onwards, rh BMP was removed. SwBs were cultured in 10 ng / ml rh SCF, rh thrombopoietin (TPO PeproTech), rh FGF2, rh IGF1, rh IGF2, and 100 nM RETA, and 20 nM Stemregenin 1 (SRI, Selleck Chem). From day 14 to 16, suspension haematopoietic cells were analysed by FACS before being cryopreserved for transplantation.[000179] Flow cytometry. Suspension hematopoietic cells and disaggregated embryoid bodies were analyzed by flow cytometry. Cells shed into the medium (suspension hematopoietic cells) were analyzed separately from cells dissociated from the swirling embryoid bodies. Embryoid bodies were disaggregated by 45 min incubation with 2 mg / ml Collagenase Type I (Worthington) at 37°C. For analysis of mouse tissues, hematopoietic cells were flushed from bone marrow, spleen and thymus using a 25 G needle and 2 mF syringe with phosphate buffered saline to generate single cell suspensions. Red cell lysis of peripheral blood samples was performed by incubating 100 pF of blood with 10 mF of Ammonium chloride lysis buffer (155 mM NH4CI / 12 mMNaHCCh / 0.1 mM EDTA) at 37°C for 15 min. Cells were pelleted and washed with phosphate buffered saline. For analysis, all samples were resuspended in phosphate buffered saline supplemented with 2% fetal calf serum (PBS / 2%FCS). Directly conjugated antibodies directed against cell surface antigens, were used to identify dissociated cells by flow cytometric analysis during differentiation and in single cell suspensions from hematopoietic tissues and peripheral blood samples from transplanted mice. Samples were incubated with the indicated dilution of antibodies in a volume of 25 pF of PBS / 2%FCS for 15 min at 4°C, washed twice with 2 mF PBS / 2%FCS and resuspended in 300 pF PBS / 2%FCS with 1 pg / mF propidium iodide to detect dead cells. Flow cytometric analysis used a four laser BD FSR Fortessa™ analyser (Becton Dickinson). A panel of negative controls for flow cytometry are shown in Figure 8. FlowFogic 8 (Inivai Technologies) was used to analyze data and prepare figures.[000180] Cord blood cells. Samples of human umbilical CB from healthy subjects were obtained from the BDMI National Cord Blood Bank, Royal Children's Hospital, Parkville 3052, under auspices of the Royal Children's Hospital Human Research Ethics Committee (reference 34170A / ID 42470). Mononuclear cells were isolated and cryopreserved for use in transplantation assays.[000181] Mice. NOD.B6. PrkdcscldIl2r "nl''vil / S'1KilW4l / W41(NBSGW) mice were sourced from JAX Mice and Services (stock number 0266220) at The Jackson Eaboratory (Maine, USA) and a colony was established at the Murdoch Children's Research Institute. The Murdoch Children's Research Institute animal ethics committee approved all animal protocols (reference A885 and A954), and experiments were carried out under its guidelines for the care and use of laboratory animals.[000182] Transplantation experiments. Differentiated CD34+suspension hematopoietic cells, CD34-enriched swirling embryoid bodies, or a combination of both, were harvested and cryopreserved prior to transplantation. Cells in most experiments (>85%) were differentiated for 14 - 16 days before harvesting. Cells were thawed and male and female mice aged between 8 and 13 weeks were transplanted by intravenous injection into the tail vein with 5 x 105- 2 x 106cells. In some experiments cryopreserved CB mononuclear cells from four independent cords (0.7% - 2.7% CD34+) were thawed and mononuclear cells estimated to contain 3.5 x 102- 2.7 x 104CD34+cells were transplanted in a similar manner. Tissues were harvested for analysis from most recipients at least 16 weeks, and up to 24 weeks, post engraftment. Single cell suspensions were generated from peripheral blood, bone marrow (femurs and tibiae), spleen and, where visible,thymic tissue. Cells were analyzed by flow cytometry for surface antigens indicative of erythroid, myeloid, B cell, T cell and stem cell compartments. Residual bone marrow and spleen samples from repopulated mice were cryopreserved for further analyses including secondary transplantation.[000183] Cryopreserved bone marrow samples from selected multilineage engrafted mice were transplanted (3 x 105- 2 x 106total bone marrow cells per mouse) into NBSGW recipients by tail vein injection, and bone marrow and spleen analyzed after 13 - 20 weeks.[000184] Transcriptional profiling using scRNA sequencing. Single cell transcriptomic sequencing was performed after 14 days differentiation on a total of 28 samples from RM TOM and PB 1.1 BFP samples as outlined in Figure 3a. Data from suspension hematopoietic cells and swirling embryoid body cells were collected separately. Embryoid bodies were disaggregated by a 45 min incubation with Collagenase Type I (Worthington) at 37°C. Single cell suspensions were prepared at 1 x 106cells / ml with at least 90% cell viability and processed by The Victorian Clinical Genetics Service who prepared the libraries following the lOx Genomics Cell Preparation Guide (www.10xgenomics.com). Sequencing of single cell RNA was performed using an Illumina Novaseq-6000, aiming for a target of -300 million reads per sample comprising 6000 - 10000 cells with -50,000 reads per cell. Selected data from samples from the RM TOM line that were not treated with RETA was shown previously in Calvanese, V. et al. Nature 604, 534-540 (2022).[000185] The fastq files generated from the Illumina sequencing were mapped against the human reference genome GRCh38-1.20 using the 10X Cellranger software version 6.0.2 with the 'Cellranger count' function. Data from the two iPSC lines were aggregated with the 'Cellranger aggr' function allowing for convenient visualization of genes expressed via the Loupe browser (10X genomics). Other output files generated that were used for bioinformatic analysis consisted of the matrices, barcode and features file found in the 'filtered_gene_bc_matrices' folder. Both the Loupe browser and mapped unprocessed files are accessible through this Github link: https: / / github.com / jackyyishengli / Ng-2023 / .[000186] Visualizations from Figure 3 and Figures 10 - 14 were generated on the R platform. Seurat version 4.1.2 was used for pre-processing quality control and downstream analysis. Analysis was completed through the basic Seurat pipeline with quality control metrics applied to the raw data. Cells that expressed more than 8 x 103or fewer than 2 x 102genes, more than 5 x104or fewer than 1 x 103counts, along with cells that expressed more than 20% mitochondrial, 40% ribosomal and 1.5% mitoribosomal genes were excluded. Following quality control, the standard Seurat downstream processes were carried out with normalization first then followed by identification of the most variable genes of each sample. Integration through the 'FindAnchors' and 'IntegrateData' functions was performed across all samples to minimize the batch effects seen throughout the 28 PSC samples. 'SelectlntegrationFeatures' was used to determine a list of 2 x 103genes used in the integration matrix. Following integration, the number of dimensions was reduced with principal component analysis (PCA) and clustering was completed with the 'FindClusters' function using the Louvain clustering algorithm. In total, 252,607 cells, comprising 12 RM TOM and 16 PB 1.1 BFP samples, passed quality controls.[000187] To identify each cluster within the integrated 28 samples, 'FindAllMarkers' generated a list of cluster specific genes for each cluster. These genes were then compared with known markers of a cell type to assign cluster identities. Differential gene analysis between clusters was completed with the 'FindMarkers' function, whilst differential genes expressed between samples utilized a pseudobulk method based on average counts. Here, each single cell within a preselected cell cluster acted as a replicate thus allowing for the RNA expression level across the cluster to be treated as a bulk RNA sample. Differing conditions of the same cluster could therefore utilize the same analysis strategies as used in bulk RNA sequencing. To identify the effects of retinoid supplementation, the Voom / limma method on the Degust web portal was used to identify differentially expressed genes in the 'Artl', 'HE' and 'HSPC1' clusters identified in Figure 11. Cells from these three clusters were also pooled and reclustered with a higher resolution to investigate the endothelial to hematopoietic transition.[000188] ACTINN version 2 was used as an unsupervised neural network based method to identify subsets of the haematopoietic ally differentiated iPSC population based on a comparison to a reference dataset of human embryonic and CB derived endothelial and hematopoietic cell populations (Calvanese, V. et al. Nature 604, 534-540 (2022)). The ACTINN data sets used in Figure 3h comprised 27 samples of hematovascular cells from gestational day 22-24 (CS 10-11) embryo and yolk sac, day 29-36 (CS 14-15) AGM, yolk sac, embryonic liver and placenta, week 6, 8, 11 and 15 embryonic and fetal liver HSPCs, and CB HSCs and progenitor cells. The expression matrix for the reference training data and the cell type annotation of the cells are accessible in GitHub (https: / / github.com / mikkolalab / Human-HSC-Ontogeny). Cells expressingHLF and SPINK2 from the differentiated iPSCs were matched to 19 of the 27 reference samples. Results from these 19 data sets that are shown in Figure 3h.[000189] Images. Confocal Images were captured using a Zeiss LSM 900 laser scanning confocal microscope with Zeiss Blue software (Zeiss, version 2.1). Images for figures were assembled in Adobe Illustrator 2020 (version 24.1). Adjustments to brightness and contrast were the only image manipulations performed. Diagrams in Figures la, 2a, 3a, and 4a and Figures 7, and 17 were created in part using BioRender.com.[000190] Statistical analysis. Experiments were analyzed using GraphPad Prism versions 7 - 10 (GraphPad Software Inc.) and Microsoft Excel (Microsoft corporation). Means and standard errors of the mean are shown with the number of independent replicates in each case in the figure legend or on the figure or in the text. Tests for statistical significance are listed with the figure legend of each experiment. One-way ANOVA based statistics (Kruskal Wallis for non parametric distributions) were used for experiments with multiple comparisons of one or more grouped variables, accompanied by the post hoc tests indicated as appropriate by the software (Dunn's). Two-tailed Fisher's exact test was used to compare groups in contingency tables. Mann-Whitney two-tailed tests were used to compare unpaired non parametric groups and two-tailed Wilcoxon signed rank tests were used for paired non parametric groups. The reproducibility of the data is captured by the number of experimental replicates, as listed in figure legends. No statistical method was used to predetermine sample size.Example 1 - Differentiation of iPSCs to CD34 expressing hematopoietic cells[000191] Induced pluripotent stem cells were differentiated to hematopoietic cells using a swirling embryoid body (EB) protocol, in which iPSCs were seeded into dishes that were incubated on a rotating platform (Calvanese, V. et al. Nature 604, 534-540 (2022) and Motazedian, A. et al. Nature cell biology 22, 60-73 (2020)) (see Figure 7 for protocol details, Figure la, b). Mesoderm was induced in albumin-free SPELS medium (made in house, see Methods) using a combination of the WNT-agonist CHIR99021 (CHIR), FGF2, BMP4 and / or ACTIVIN A for 24 hours (Figure la). Subsequent patterning to induce the expression of HOXA genes was achieved by culture for 48 hours with CHIR and the ALK-kinase inhibitor SB431542 (SB). From day 3, mesoderm was further differentiated to hemogenic endothelium, with or without a 2 -day pulse of a retinoic acid precursor, retinyl acetate (RETA) or retinol (ROL). From day 7, cells undergoing an endothelialto hematopoietic transition were visible as protrusions on the surface of the embryoid bodies, reminiscent of intra-arterial hematopoietic clusters of blood cells that are seen emerging from the aorta in the embryonic AGM (Figure 1c). From day 9, these cellular accumulations broke away from the embryoid bodies, shedding blood cells were into the medium from day 11 (Figure 1c). Cultures at day 14 comprised a dominant blood cell suspension with most cells expressing CD34, CD90, CD44 and KIT (Figure Id and Figure 8). The embryoid body-derived fraction consisted of stroma, endothelium, and hematopoietic cells that resembled those shed into the medium (Figure le and Figure 8). A small proportion of the hematopoietic cells expressed CXCR4 or CD73, reflecting their recent emergence from an endothelial precursor (Figure le). The endothelial populations present at this time included CXCR4+CD73+arterial cells, CXCR4 CD73hlvenous cells and CXCR4 CD73’ hemogenic cells. From day 14 - 16, the suspension hematopoietic cells were cryopreserved for further analyses (Figure Id). In some experiments, CD34+cells enriched from embryoid bodies by magnetic bead separation (Figure le) were also cryopreserved.[000192] Typically, 2.0 x 107iPSCs were differentiated in 60 mm dishes to give approximately 1.4 x 108CD34+hematopoietic cells by day 14, a yield of 7 CD34+cells (7.1 ± 0.8, n=3) for each input iPSC.Example 2 - Multilineage engrafting cells require retinoids during iPSC differentiation[000193] The inventors assessed combinations of CHIR, ACTIVIN A, BMP4 and a retinoid during the mesoderm induction and patterning stages (screening protocol #1, see Figure 7 and Figure 9a), to determine whether any supported the generation of engraftable human hematopoietic cells. The CD34+hematopoietic cells generated from an iPSC line constitutively expressing a tandem TOMATO fluorescent protein (RM TOM) (Figure 1c), were cryopreserved prior to thawing and injection into the tail vein of NOD,B6.Prk<7cscldtw4i / W4i (NBSGW) mice, mimicking the workflow in clinical HSC transplantation.[000194] In this series of experiments, groups of mice (totaling 134, denoted cohort #1) were injected with cells differentiated under one of 12 mesoderm induction and patterning protocols in screening protocol #1. Groups of mice (totaling 134, denoted cohort #1) were injected with RM TOM cells differentiated under one of 12 mesoderm induction and patterning protocols (Figure 9a). Human cell contributions in the bone marrow and spleen were determined post transplantation based on coincident expression of the TOMATO reporter with human lympho-myeloid (CD45and / or CD43) and erythroid (GYPA) cell surface markers by flow cytometry. For cohort #1, most animals were analysed > 12 w after transplantation (85%) and half were > 16 w post transplantation (49%). Across the 12 conditions, 103 / 134 (76.9%) of animals had detectable human cells in the bone marrow with 45 / 103 (43.7%) also having human cells in the spleen (Figure 9b). Flow cytometry revealed different patterns of lineages in individual mice, suggesting that there was a hierarchy of engrafting cells. Most frequent were myeloid restricted stem cells that gave low-level bone marrow engraftment (human cells, 0.7+0.1%) in 58 / 134 transplant recipients, or myelo- lymphoid stem cells that led to mixture of bone marrow myeloid cells (human cells, 0.5+0.1%) accompanied by B lymphocytes in the spleen (human cells, 0.02+0.003%) in 30 / 134 mice (Figure 9c). A small number of mice (3 / 134) were engrafted predominantly by a higher proportion of B lymphoid cells (8.7+4.4% human cells in bone marrow, and 0.4+0.3% in spleen) (Figure 3c).[000195] Analysis showed that some mice (12 / 134) were engrafted by stem cells displaying multilineage differentiation resulting in erythroid, myeloid and lymphoid reconstitution (denoted multilineage engraftment, MLE). The mouse groups in which MLE engraftment occurred were predominantly those receiving cells in which mesoderm was induced with 4 pM CHIR on day 0, and a pulse of a retinoic acid precursor (ROL or RETA) was included from day 3 - day 5 of differentiation (Figure 2a-d). Indeed, 17.6% (9 / 51) mice transplanted with cells treated with the combination of 4 pM CHIR and retinoid showed erythroid, myeloid and lymphoid cell engraftment (Figure 2e). There were over 80% human cells occupying the bone marrow in some of these MLE cohort #1 recipients (average 46.5+10.0% human cells in bone marrow, and 11.9+5.1% in spleen) (Figure 2e). Hereafter, these functionally-defined, iPSC-derived multipotent hematopoietic cells with the capacity to engraft multiple lineages long-term are referred to as 'iHSCs'.Example 3 - Transcriptional similarity of in vitro differentiated iPSCs and human AGM[000196] Single cell RNA sequencing (scRNA seq) of differentiated iPSCs was performed to search for transcriptional signatures accounting for functional differences between cells differentiated with and without retinoid and to allow comparisons with published data sets of human AGM (e.g. Calvanese, V. et al. Nature 604, 534-540 (2022)). Two iPSC lines were profiled, the RM TOM line described above and an independent line in which the mTagBFP2 fluorescent protein was expressed from the GAPDH locus of PB 1.1 iPSCs (denoted PB1.1 BFP). Mesoderm was induced with 4 pM CHIR and either 3 ng / ml BMP4 and 5 ng / ml ACTIVIN A (4CH 3B5A), or 30 ng / ml ACTIVIN A (4CH 30A), because it had been found that both these combinations offactors, plus a retinoid, supported the generation of MLE mice (Figure 2b). The iPSC lines were differentiated for 14 days, with or without RETA. Some cultures were treated with RETA from day 3 - day 5, others for a more prolonged period (RETA added every 2 days from day 3 - day 11, day 13 or day 14) to reflect embryo data indicating that AGM-derived HSCs develop in a retinoid conditioned milieu (Figure 3a). After 14 days, blood cells in suspension and disaggregated embryoid bodies were subjected to scRNA seq using the 10X Genomics platform. In total, 252,607 cells, comprising 12 RM TOM and 16 PB 1.1 BFP samples were analyzed. Uniform manifold approximation projection (UMAP) plots of integrated samples from both cell lines, followed by cluster analysis, allowed allocation of cells to stromal, endothelial, hemogenic, and hematopoietic lineages (Figure 3b - e).[000197] Reclustering cells within arterial (Artl), hemogenic (HE) and HLF+SPINK2+cells within the hematopoietic stem cell (HSPC) clusters confirmed that the hematopoietic stem cell signature genes (RUNX1 , MECOM, MEET3, HEF, HOXA9, SP1NK2) recently identified in human AGM (Figure 10a, b) were also expressed in iPSC-derived cells (Figure 3f). The percentages of expressing cells and levels of expression were very similar in the RM TOM and PB 1.1 BFP cell lines under both mesoderm induction conditions (4CH 3B5A and 4CH 30A) (Figure 10c). The expected pattern of HOXA gene expression in response to the SB / CHIR patterning in both cell lines was also confirmed (Figure lOd).[000198] The addition of retinoids minimally impacted stem cell gene expression (Figure 12a), but most notably influenced genes associated with retinoic acid metabolism such as CYP26B1, DHRS3, CRABP2, RARB and RARG, modulators of WNT and FGF signalling such as SHISA3, DKK1, RSPO1 and WNT4, as well as genes associated with vascular and hematopoietic development such as FOXC2 and CD38 (Figures 11 and 12). Retinoid metabolism genes were similarly up-regulated in the HE cluster, as were genes related to Tube Development (gene ontology term G0:0035295, FDR 3.8 x 10-6) (Figure Ilf). Genes enriched in the HSPC1 cluster included those associated with Positive Regulation of Multicellular Organismal Process (gene ontology term G0:0051240, FDR 1.04 x 10-16), and Genes Upregulated in HL-60 Myeloid Cells in Response to Retinoic Acid (FDR 1.03 x 10-23) (Figure Ilf). Interestingly, expression of many retinoid responsive genes was only induced if the retinoids were included until at least day 11 of differentiation (Figure 12b).[000199] The inventors compared the transcriptional profiles of the iPSC-derived HLF*SPINK2+HSPC cells to similar HLF+SP1NK2+stem cell-like populations from human embryos at CS 14 and 15, examining the expression of a selected range of relevant genes (Figure 3g). For a more extensive comparison between in vitro and human embryo derived samples, a suite of scorecards developed in profiling studies of hematopoietic development in human embryos (Calvanese, V. et al. Nature 604, 534-540 (2022)) (Example 4 and Figure 13 and 14). These studies benchmarked the HLP+SP1NK2+HSPCs generated using the methods of the present invention against CS14 and CS 15 human embryo cells, demonstrating a high level of concordance between the transcriptional profiles across the 9 scorecards of genes examined.[000200] A machine learning algorithm, ACTINN (Ma, F. & Pellegrini, M. Bioinformatics 36, 533-538 (2020)), was employed to compare the expression profiles of day 14 differentiated iPSCs to a human reference data set comprising hematovascular cells from gestational day 22-24 (CS 10- 11) embryo and yolk sac, day 29-36 (CS 14-15) AGM, yolk sac, embryonic liver and placenta, week 6, 8, 11 and 15 embryonic and fetal liver HSPCs, and cord blood (CB) stem and progenitor cells (Calvanese, V. et al. Nature 604, 534-540 (2022)). This analysis confirmed that HLB+SP1NK2+HSPCs were most closely related to cells categorized as hematopoietic stem and progenitor cells (HSPCs) in CS 14-15 AGM, placenta and yolk sac (Figure 3h). Dissecting the allocation of cells to these categories from the two cell lines and the different durations of retinoid treatment, revealed that the RM TOM line mapped predominantly to the CS 14 / 15 AGM HSPCs whilst the PB 1.1 BFP cells were more similar to CS 14 / 15 YS and placental HSPCs. For both lines, the longer duration of retinoid increased the proportion of CS 14 / 15 AGM HSPCs and decreased the CS 14 / 15 YS and placental HSPCs (Figure 3h).Example 4 - Transcriptional profiles of iPSC-derived hematopoietic cells resemble those of hematopoietic cells from the AGM.[000201] To determine the similarity between the iPSC-derived hematopoietic stem cell like populations and similar populations found in the human embryo, the transcriptomes of iPSC- derived cells from the HSPC 1-3 clusters that co-expressed HLF and SPINK2, were compared with those of HLF+SPINK2+cells from CS14 and CS15 embryos, using the scorecards developed by the Mikkola laboratory as templates (Calvanese, V. et al. Nature 604, 534-540 (2022)) (Figures 13 and 14). Examining the 'Nascent HSC scorecard, the inventors confirmed expression of the six HSC signature genes in the iPSC-derived cells, although the proportion of cells expressingH0XA9, MEETS and MECOM were a little lower than in the CS14 and CS15 embryo reference samples. Similarly, there were some genes enriched in HSCs or shared with endothelium that were expressed in a higher percentage of cells in embryo samples, such as STAT5A, GATA2, SEEP. AEDH1A1, PROCR and EMCN (Figure 13).[000202] Both the 'HSC transcription factor' and 'HSC maturation' scorecards showed high degrees of concordance in gene expression between embryo and iPSC-derived samples Figure 13). The percentage of GATA3, HOXA7, PBX1, PVEAP and CSF1R expressing cells was a little less in the iPSC-derived samples.[000203] The expression pattern of genes in the 'HSPC waves' scorecard reflected similarity between the embryo and iPSC-derived samples, but it was observed that the percentage of cells expressing definitive transcriptional regulation and definitive HSC genes was lower in the iPSC- derived samples (Figure 13). The 'Hematopoietic cell identity' scorecard confirmed the predominant expression of HSC rather than lineage marking genes in the iPSC-derived cells (Figure 13). Expression of the 'Liver SPINK2+ genes' were very low in all samples with the exception of PKIB (Figure 14). The 'Proliferation and metabolic activity' scorecard showed similar gene expression between cell sources. The 'Signaling' scorecard showed broad concordance between embryo and iPSC-derived samples with the notable exceptions of the retinoid metabolising enzyme ALDH1A1, the BMP-responsive transcription factor ID3, and some differences in the balance of JAK-STAT signaling genes (Figure 14). The transcriptional regulators and the hematopoietic lineage genes in the 'Endothelial to hematopoietic transition' scorecards (Figure 14d-f) were concordantly expressed in iPSC-derived and embryo samples. However, iPSC-derived arterial cells (Figure 14f) expressed very low levels of the aortic (pre- hemogenic endothelium) genes.[000204] Some differences were observed between the RM TOM and PB 1.1 BFP cell lines, with a higher percentage of cells in the RM TOM differentiated cells expressing genes from the 'HSC enriched' and 'HSC shared with endothelium' sections of the 'Nascent HSC scorecard (Figure 13). Similarly, increased proportions of RM TOM cells expressed 'HSC maturation' scorecard genes and one of the genes associated with early stage HSCs, DDIT4, a regulator of cell growth and survival, on the 'HSPC waves' scorecard (Figure 13). From the 'Hematopoietic cell identity' scorecard, CDH5 and LYZ were expressed in a higher percentage of RM TOM cells (Figure 13). In the 'Liver SP1NK2+genes' scorecard, LTB was expressed more highly in PB 1.1 BFP derivedcells. Examination of the 'Signaling' scorecard sowed that downstream NOTCH signaling targets HES1 and HES4, RARA, RXRA were more prominent in RM TOM cells. Higher expression of the endothelial gene TJP1 was also seen in RM TOM cells in the 'Endothelial to hematopoietic transition' scorecard. Few genes from the scorecard analyses appeared to be retinoid signaling responsive (the HE gene KCNK17, the transcription factor GATA2. and the BMP4 target ID]). all of which were expressed in a higher percentage of RM TOM cells (Figure 14).Example 5 - Multilineage engrafting cells are generated from cultures treated with retinoids throughout differentiation[000205] The functional ramifications of the ACTINN results were explored further by varying the duration of retinoid exposure in a second series of transplantation experiments. In 10 experiments using cells sourced from 6 independent differentiations, 103 animals (cohort #2) were injected with differentiated RM TOM hematopoietic cells exposed to increasing durations of retinoid treatment (screening protocol#2 in Figure 7, Figures 4a, b). Data from mice receiving cells in which mesoderm was induced with 4CH 3B5A or 4CH 30A were pooled, given that both variations displayed similar expression of HSC signature genes (Figure 10c). Multi-lineage engraftment was seen in 6 / 25 (24%) mice transplanted with cells treated with RETA from day 3 - day 5, the duration of RETA that was successful for engraftment in cohort#l, and in 7 / 19 (36.8%) mice receiving cells exposed to RETA treatment from day 3 - day 13, although the difference did not achieve statistical significance. All mice with MLE were analysed >16 weeks post transplantation, with one exception (15.7 w). Importantly, these experiments confirmed that prolonged treatment with retinoid was compatible with the in vitro generation of multilineage engrafting iHSCs from iPSCs and consistent with our transcriptomic data showing that prolonged exposure to retinoid was required for the expression of retinoid responsive genes (Figure 12b), and embryo data indicating that AGM-derived HSCs develop in a retinoid conditioned milieu (Figure 12c).[000206] A similar of series experiments were performed using the second transcriptionally profiled human iPSC line, PB1.1 BFP, transplanting 79 mice in 8 experiments derived from 6 independent differentiation experiments (cohort #3). Bone marrow engraftment was observed in 44.3% of recipients with predominantly myeloid-restricted engraftment, although one mouse demonstrated MLE after 19 weeks (Figure 15a - c). This data demonstrated that the differentiation protocol enabled generation of multilineage engraftable cells from a second independent iPSC line.Example 6 - Flow cytometry characterization of multilineage engrafted recipients of iHSCs[000207] The contribution and lineage distribution of human cells in the bone marrow, spleen, thymus and peripheral blood of MLE animals identified in cohorts #1-3 was analysed (Figure 4 c-g and Figure 20). Confocal analysis showed readily observable TOMATO+human cells in the bone marrow (Figure 4c) whilst flow cytometry analysis revealed the presence of erythroid, myeloid and B lymphoid cells in the bone marrow, as well as splenic B and T cells and developing thymic T cells in some animals (Figure 4d - g, Figures 15b, and Figure 16a, b). Human cells were present in the peripheral blood at 12 week post transplantation in MLE recipients (Figure 16a). Immature CD3“ thymocytes passed from the CD4 CD8’ stage via an intermediate single positive CD4+stage to CD4 and CD8 double positive thymocytes, and CD3+double positive thymocytes gave rise to single positive CD4 and CD8 expressing T cells (Figure 4g, Figure 16b). Erythroid cells in the bone marrow stained for cell surface GYPA and CD43, and predominantly expressed low levels of the TOMATO or BFP reporter genes (Figure 4d, Figure 15b). This was consistent with prior observations that maturing erythroid cells preferentially transcribed globin genes and reduced expression from the GAPDH locus. Another defining characteristic of the MLE animals was the presence of a bone marrow CD45+CD34+CD38lo / _HSC-like population (Figure 4d, Figure 15b, Figure 16a).Example 7 - Modulating VEGF signaling enhances multilineage engraftment in recipients from multiple independent iPSC lines[000208] Evidence from the human embryo suggests that HSCs arise from arterially patterned hemogenic endothelium. VEGF acts in a dose-dependent manner to drive endothelium generation and arterialization in differentiating pluripotent stem cells. However, published data showed that VEGF suppressed hematopoietic progenitor development from endothelium by blocking the upregulation of RUNX1 expression, a critical marker of hemogenic endothelium and HSCs in the human embryo. To explore these opposing effects, a range of VEGF concentrations were trialed from day 3 - day 7, during endothelial generation, followed by continuing or removing VEGF to determine which best enhanced the endothelial to hematopoietic transition. The inventors demonstrated a VEGF dose-dependent increase in CD34+CXCR4+arterial endothelial cell generation, followed by a rapid loss of the arterial marker CXCR4 after the removal of VEGF at day 7 of differentiation (Figure 17). Gene expression analysis revealed that the combination of high VEGF from day 3 followed by its removal at day 7 of differentiation increased the expressionof aortic endothelial genes AGTR2, IL33, EDNl), reduced ALDH1A2 and increased ALDH1A 1, accelerated the endothelial to hematopoietic transition evidenced by reduction in CXCR4 and DLL4, and increased RUNX1 and HLF expression (Figure 18). Importantly, the inventors had identified many of these genes as being more lowly expressed in the iPSC-derived cells compared to the human embryo in an endothelial to hematopoietic transition scorecard (Figure 14f).[000209] Incorporating these modifications into the differentiation protocol (protocol#3, Figure 7), the functional consequences were explored in further transplantation experiments. In mice transplanted with RM TOM cells (cohort#4), improved engraftment was observed compared with the earlier experiments (cohort#land #2), recording 30 / 62 (48.4%) mice with multilineage engraftment, with 61 / 62 recipients analysed >16 w post-transplantation (Figure 5a). Similar engraftment results were seen in three additional human iPSC lines, including the PB1.1 BFP line that had transplanted poorly in the earlier experiments (cohort#3). Multilineage engraftment was observed in 11 / 23 (47.8%) of PB 1.1 BFP (cohort#5), 4 / 15 (26.7%) of PB5.1 (cohort#6) and 3 / 8 (37.5%) of PB 10.5 (cohort#7), analysed >16 w post-transplantation in 41 / 46 cases (Figure 5b-d). A direct comparison of the effects of modulating the VEGF concentration on bone marrow engraftment is shown in Figure 23. Bone marrow engraftment by cells from RM TOM and PB 1.1 BFP iPSC lines differentiated with 50 ng / ml VEGF throughout the differentiation (+VEGF) or with 150 ng / ml VEGF limited to days 3- 7 (-VEGF d7) revealed that both the proportion of mice exhibiting multilineage engraftment, and the average level of human engrafted cells, was higher in recipients of cells receiving 150 ng / ml VEGF limited to days 3- 7.[000210] These results indicated that protocol#3 generated more robustly engrafting cells. Limit dilution transplantation experiments with this protocol were not performed, but analysis of the overall engraftment results given above suggests that the frequency of multilineage engrafting cells is still low, at 1 / 3.0 x 106for the PB TOM, 1 / 3.1 x 106for the PB 1.1 BFP, 1 / 6.2 x 106for the PB5.1 and 1 / 4.3 x 106for the PB 10.5. There was some variability in outcomes between experiments, with estimated engraftment frequencies as high as 1 / 1.3 x 106for a PB TOM differentiation experiment E#427 in which 7 / 9 recipients displayed MLE.[000211] The contribution and lineage distribution of human cells in the bone marrow, spleen, thymus and peripheral blood of the 48 MLE animals receiving cells differentiated under protocol#3 was analyzed (Figure 5e-h and Figure 19). In most cases, human cells were present in the peripheral blood at 12 week post transplantation (38 / 46 mice analysed) (Figure 5e-h), andevaluation of paired samples at 16 week revealed an increase in human cells in 28 / 35 mice analysed across the four cell lines (Figure 19a). There was an evident sex bias in BM engraftment, most prominent in the RM TOM line recipients (Figure 19b), with significantly higher levels of human cells in female than male recipients, consistent with published literature. Over all experiments (cohorts #1-7), multilineage engrafted mice were seen in 19.6% of recipients transplanted with CD34+suspension blood cells, 23.8% of those receiving CD34 enriched cells from the embryoid bodies, and 24.5% of mice that received both suspension blood cells and CD34- enriched cells from the embryoid bodies. These results demonstrated that similar proportions of stem cells were present in CD34+cells from both sources.[000212] Flow cytometry analysis revealed the presence of bone marrow erythroid, myeloid, B and T lymphoid cells, and CD45+CD34+CD38lo / “ HSC-like cells, splenic B and T cells, and thymic T cells, similar to MLE animals in cohorts #1 - 3 (Figure 5i, j and Figure 19c-e). Comparison between mice engrafted with RM TOM cells under the different protocols revealed higher percentages of human cells in BM, SPL and PB in recipients of protocol#3 differentiated cells, with a persistent bias towards engraftment in female mice (Figure 20a, b). The proportions of erythroid, myeloid, B cell and stem cells were similar in male and female recipients but the proportion of T cells in the bone marrow and spleen were greater in engrafted female mice (Figure 20c, d).[000213] Where T cell development was observed in the spleen and bone marrow, there was rarely a macroscopically identifiable bi-lobed thymus, but small amounts of putative lymphoid tissue were frequently present in the mediastinum. This usually contained single positive CD4 and CD8 expressing cells, with few CD4+CD8+double positive thymic cells, and often a population of CD19+B cells (Figure 5j and Figure 19e). The low percentage of CD4+CD8+double positive cells was particularly marked in the more highly engrafted cohort#4 female (1.8+0.7% CD4+CD8+cells) compared to male (23.7+6.9% CD4+CD8+cells) mice receiving cells from protocol#3, and contrasted with the high proportion of CD4+CD8+cells seen in female (52.7+12.1%) and male (mean 75.7+4.1%) mice receiving protocol#! and#2 differentiated cells (Figure 20c, d). This might reflect inability to sustain thymic tissue in aging immune deficient mice, with also likely sampling of mediastinal lymph nodes to account for the presence of B cells (Figure 5j). This dimorphic pattern of T cell engraftment has been observed in immune deficient recipients of CB CD34+cells, in which a major CD4+CD8+thymic population was only seen in 10 / 19 mice with T cell engraftment with low CD4+CD8+cell numbers in the remainder.Example 8 - Lineage composition in multilineage engrafted recipients of iHSCs[000214] Lineage contributions varied between different MLE recipients, and between recipients of independent iPSC lines (Figures 16 and 19). The dominant population in the bone marrow of most mice receiving RM TOM cells in cohort#l-3 were B cells (Figure 21a), although there were some with predominantly erythroid engraftment and the cohort#4 mice receiving cells differentiated under protocol #3 also frequently displayed T cell engraftment (Figure 6a). Recipients of PB1.1 BFP differentiated cells (cohort#5) displayed dominant erythroid engraftment whilst the smaller number of recipients of PB5.1 (cohort#6) and PB 10.5 (cohort#?) lines showed more balanced engraftment patterns (Figures 6a, 16 and 19). It was observed that engraftment was maintained in bone marrow and spleen in animals evaluated for >16 weeks, consistent with stable engraftment by long-term repopulating cells (Figure 22).Example 9 - Umbilical cord blood mononuclear cells display dose dependent engraftment[000215] The engraftment phenotypes of iPSC-derived iHSCs were compared with that of CB cells, a clinically validated source of transplantable hematopoietic stem cells. A total of 39 mice were transplanted with 5 x 104- 2.5 x 106CB mononuclear cells isolated from four separate cords that comprised 0.7 - 2.7% CD34+cells, resulting in transplantation of 3.5 x 102- 2.7 x 104CD34+cells. Multilineage engraftment was observed in most recipients (14 / 15) of mononuclear cells estimated to contain > 6.0 x 103CD34+cells (Figure 6b), and the estimated frequency of repopulating CB stem cells was 1 / 6.3 x 103CD34+cells by limit dilution assay (Figure 21b), consistent with reports in the literature. Similar to the findings observed with iPSC derived iHSC transplants, higher levels of engraftment were observed in female mice engrafted with CB cells (Figure 21b, c). Mice receiving fewer than 6.0 x 103CB CD34+cells showed lower total proportions of human cells in the bone marrow and frequently displayed restricted lineage engraftment with myeloid or myeloid and lymphoid lineages (Figure 6b). This positive correlation between engraftment level and lineage complexity in recipients of CB stem cells mirrored the similar correlation observed in the iPSC-derived blood cell transplants. The observation that restricted lineage engraftment in CB recipients was seen with lower numbers of transplanted CD34+cells, is suggestive of a hierarchy of stem cells read out by the transplantation assay. Multilineage engrafting cells with high proliferative capacity are less abundant than myeloid or myeloid / lymphoid restricted stem cells with low proliferative capacity.[000216] Comparing the proportions of human cells in bone marrow and spleen in multilineage engrafted CB with iHSC engrafted mice, the profile of engrafted lineages was similar between CB and RM TOM, PB5.1 and PB 10.5 recipients, although T cell engraftment was greater in the RM TOM mice and the stem cell compartment was smaller (compare Figure 6c-g with Figure 19a-d). PB 1.1 BFP recipients displayed prominent erythroid engraftment in the BM and SPL, with commensurately lower levels of lymphoid and myeloid engraftment, although their stem cell compartment was maintained (Figure 19c-d). Heterogeneity in the distribution of lineages in individual multilineage engrafted CB mice can be appreciated in the bar graphs in Figure 6h, where the most abundant lineages were B and myeloid cells, with few mice displaying large erythroid populations and few cases of T cell engraftment.[000217] Secondary engraftment was observed following transplantation of bone marrow cells from primary recipients engrafted with either CB or iPSC derived HSCs; 6 / 12 primary mice engrafted with iHSCs and 2 / 5 primary mice engrafted with CB HSCs. Engraftment was at a low level and restricted to myeloid lineages, although one iHSC secondary transplant recipient displayed B, T and myeloid lineages in the BM, spleen and thymus. It is likely that the levels of secondary engraftment reflected the low dose of primary bone marrow used (0.3 - 2.0 x 106BM cells), combined with the suboptimal niche provided by the mouse bone marrow environment.Example 10 - Treating acute myeloid leukaemia[000218] Haematopoietic stem / progenitor cells produced according to the present disclosure will be administered to a subject diagnosed with acute myeloid leukaemia (AML) at a dose of 3 x 106cells / kg who has undergone myeloablative therapy according to standard protocols prior to allogeneic HSC transplantation. Administration of the haematopoietic stem / progenitor cells will result in an equivalent or improved response in the subject.Example 11 - Treating acute lymphocytic leukaemia[000219] Haematopoietic stem / progenitor cells produced according to the present disclosure will be administered to a subject diagnosed with acute lymphocytic leukaemia (ALL) at a dose of 3 x 106cells / kg who has undergone myeloablative therapy according to standard protocols prior to allogeneic HSC transplantation. Administration of the haematopoietic stem / progenitor cells will result in an equivalent or improved response in the subject.

Claims

CLAIMS1. A method for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), the method comprising: a) culturing a population of mesoderm cells obtained from a population of PSCs in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent; b) culturing the population of cells from step a) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step a), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step a); c) culturing the population of cells from step b) in a medium comprising a FGF, a BMP, an IGF, VEGF, and a retinoic acid signalling agent wherein the concentration of the retinoic acid signalling agent is 10 to 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step b); and d) culturing the population of cells from step c) in a medium comprising a stem cell factor (SCF) and thrombopoietin (TPO), FGF, an IGF, and a retinoic acid signalling agent to produce a population of definitive HSPCs.

2. The method of claim 1, wherein step a) comprises culturing the population of cells for a time sufficient to generate a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10, preferably one or more of H0XA5, H0XA7, H0XA9, and HOXA10, even more preferably H0XA5, H0XA7, H0XA9, and HOXA10.

3. The method of claim 1 or 2, wherein step a) comprises culturing the population of cells for a period of about 2 days.

4. The method of any one of claims 1 to 3, wherein step b) comprises culturing the population of cells for a time sufficient to generate of a population of cells expressing CD34 on the cell surface.

5. The method of any one of claims 1 to 4, wherein step b) comprises culturing the population of cells for a period of about 2 days.

6. The method of any one of claims 1 to 5, wherein step c) comprises culturing the population of cells for a time sufficient to generate a population of cells co-expressing CD34 and CXCR4 on the cell surface.

7. The method of any one of claims 1 to 6, wherein step c) comprises culturing the population of cells for a period of at least about 2 days.

8. The method of any one of claims 1 to 7, wherein the concentration of BMP in the medium in step c) is the same as in the medium of step b), or alternatively up to 20 fold lower than the concentration of BMP in the medium of step b).

9. The method of any one of claims 1 to 8, wherein step d) comprises culturing the population of cells for a time sufficient to generate a population of cells co-expressing CD34, CD90 and CD45 on the cell surface.

10. The method of any one of claims 1 to 8, wherein step d) comprises culturing the population of cells for a period of about 3 days.

11. A method for differentiating a population of pluripotent stem cells (PSCs) into a population of definitive haematopoietic stem / progenitor cells (HSPCs), the method comprising: i) culturing the population of PSCs in a basal medium comprising a WNT agonist, a fibroblast growth factor (FGF), and Activin A; ii) culturing the population of cells comprising mesoderm cells from step i) in a medium comprising a WNT agonist, an ACTIVIN antagonist, a FGF, vascular endothelial growth factor (VEGF), and a retinoic acid signalling agent;iii) culturing the population of cells from step ii) in a medium comprising a FGF, VEGF, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), and a retinoic acid signalling agent, wherein the concentration of the retinoic acid signalling agent is 10 to 40 fold greater than the concentration of the retinoic acid signalling agent in the medium of step ii), and wherein the concentration of VEGF is 2 to 10 fold greater than the concentration of VEGF in the medium of step ii); iv) culturing the population of cells from step iii) in a medium comprising a FGF, a BMP, an IGF, VEGF, and a retinoic acid signalling agent wherein the concentration of the retinoic acid signalling agent is 10 to 40 fold lower than the concentration of the retinoic acid signalling agent in the medium of step iii), and wherein the concentration of BMP is 5 to 20 fold lower than the concentration of BMP in the medium of step iii); v) culturing the population of cells from step iv) in a medium as recited in step iv) excluding VEGF; and vi) culturing the population of cells from step v) in a medium comprising a stem cell factor (SCF) and thrombopoietin (TPO), FGF, an IGF, and a retinoic acid signalling agent to produce a population of definitive HSPCs.

12. The method of claim 11, wherein step i) comprises culturing the population of cells for a time sufficient to generate a population of mesoderm cells expressing CD13 and CD90 on the cell surface.

13. The method of claim 11 or 12, wherein step i) comprises culturing the population of cells for a period of about 1 day.

14. The method of any one of claims 11 to 13, wherein step ii) comprises culturing the population of cells for a time sufficient to generate a population of cells expressing a pattern of HOXA genes including one or more of H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, H0XA6, H0XA7, H0XA9, HOXA10, preferably one or more of H0XA5, H0XA7, H0XA9, and HOXA10, even more preferably H0XA5, H0XA7, H0XA9, and HOXA10.

15. The method of any one of claims 11 to 14, wherein step ii) comprises culturing the population of cells for a period of about 2 days.

16. The method of any one of claims 11 to 15, wherein step iii) comprises culturing the population of cells for a time sufficient to generate a population of cells expressing CD34 on the cell surface.

17. The method of any one of claims 11 to 16, wherein step iii) comprises culturing the population of cells for a period of about 2 days.

18. The method of any one of claims 11 to 17, wherein step iv) comprises culturing the population of cells for a time sufficient to generate a population of cells co-expressing CD34 and CXCR4 on the cell surface.

19. The method of any one of claims 11 to 18, wherein step iv) comprises culturing the population of cells for a period of about 2 days.

20. The method of any one of claims 11 to 19, wherein step v) comprises culturing the population of cells for a time sufficient to generate a population of cells expressing CD34 without CXCR4 on the cell surface.

21. The method of any one of claims 11 to 20, wherein step v) comprises culturing the population of cells for a period of about 4 days.

22. The method of any one of claims 11 to 21, wherein step vi) comprises culturing the population of cells for a time sufficient to generate a population of definitive HSPCs.

23. The method of any one of claims 11 to 21, wherein step vi) comprises culturing the population of cells for a period of about 3 days.

24. The method of any one of claims 1 to 23, wherein the WNT agonist is CHIR99021.

25. The method of claim 24, wherein CHIR99021 is present in said medium at a concentration of about 4 pM.

26. The method of any one of claims 11 to 25, wherein the concentration of ACTIVIN A in the medium of step i) is about 5 ng / mL to 50 ng / mL, preferably wherein the concentration is about 30 ng / mL.

27. The method of claim 26, wherein the concentration of ACTIVIN A in the medium of step i) is about 5ng / ml and the medium further comprises BMP at a concentration of about 3 ng / mL.

28. The method of any one of claims 1 to 27, wherein the ACTIVIN antagonist is SB431542.

29. The method of claim 28, wherein SB431542 is present in said medium at a concentration of about 3-4 pM.

30. The method of any one of claims 1 to 29, wherein the retinoic acid signalling agent is a retinoid or retinoic acid analogue, preferably retinol or retinyl acetate (RETA).

31. The method of claim 30, wherein the concentration of the retinoid or retinoic acid analogue in the medium of step a) or step ii) is about 50 nM to lOOnM, preferably about 50nM of RETA.

32. The method of any one of claims 1 to 31, wherein the concentration of VEGF in the medium of step a) or step ii) is about 50 ng / mL.

33. The method of any one of claims 1 to 32, wherein the concentration of VEGF in the medium of step b) or step iii) is about 100 ng / mL to about 200 ng / mL.

34. The method of any one of claims 1 to 33, wherein the bone morphogenic protein is BMP4.

35. The method of any one of claims 1 to 34, wherein the concentration of BMP in the medium of step b) or step iii) is about 20 ng / mL.

36. The method of any one of claims 1 to 35, wherein the medium of steps a) to c) or steps i) to v) do not comprise SCF.

37. The method of any one of claims 1 to 36, wherein the medium of step d) or step vi) comprises SCF at a concentration of about 10 ng / mL and TPO at a concentration of about 10 ng / mL.

38. The method of any one of claims 1 to 37, wherein the medium of step d) or step vi) comprises StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), and / or erythropoietin (EPO).

39. The method of any one of claims 1 to 38, wherein the FGF is FGF2, and / or wherein IGF comprises IGF1 and / or IGF2, optionally in equal concentrations when IGF1 and IGF2 are both present.

40. The method of any one of claims 11 to 39, wherein the medium of step i) comprises a ROCK inhibitor, optionally wherein the ROCK inhibitor is thiazovivin or Y-27632.

41. The method of any one of claims 1 to 40, the population of cells is cultured as embryoid bodies (EBs).

42. The method of claim 41, wherein the embryoid bodies are cultured with swirling.

43. The method of any one of claims 1 to 42, wherein the basal medium is SPELS medium.

44. The method of any one of claims 1 to 43, wherein the PSCs are embryonic stem cell (ESC) or induced PSCs (iPSCs).

45. The method of any one of claims 1 to 44, wherein the PSC is human, preferably a human induced pluripotent stem cell iPSC.

46. The method of any one of claims 1 to 45, wherein in any one of the recited steps, the population of cells is cultured under hypoxic conditions, preferably between about 2% and about 10% oxygen.

47. The method of any one of claims 1 to 46, wherein the population of definitive HSPCs are HLF+and SPINK+.

48. The method of any one of claims 1 to 47, wherein the population of definitive HSPCs express CD34 and CD45, and one or more of CD90, CD44, KIT, CD201, ITGA3 and ITGA6 on the cell surface.

49. The method of any one of claims 1 to 48, further comprising harvesting cells in suspension following step d) or step vi).

50. The method of any one of claims 1 to 49, further comprising dissociating EBs in suspension following step d) or step vi) and harvesting cells dissociated from the EBs.

51. The method of claim 49 or 50, further comprising enriching, or sorting the harvested cells for CD34+cells.

52. A definitive haematopoietic stem / progenitor cell, or population of such cells obtained by the method of any one of claims 1 to 51.

53. A therapeutic composition comprising the cell or population of cells of claim 52.

54. An infusion bag comprising the cell or population of claim 52 or the therapeutic composition of claim 53.

55. A method for treating a condition, disease or disorder requiring HSC transplantation, the method comprising administering to a subject the cell or population of claim 52 or the therapeutic composition of claim 53.

56. Use of the cell, or population of cells, of claim 52 in the manufacture of a medicament for treating a condition, disease or disorder requiring HSC transplantation.

57. A definitive haematopoietic stem / progenitor cell, or population of cells, of claim 52 for use in treating a condition, disease or disorder requiring HSC transplantation.

58. The method of claim 55, the use of claim 56, or the cell or population of cells for use in treating a condition, disease or disorder requiring HSC transplantation of claim 57, wherein the condition, disease or disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), NonHodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, other solid tumour, thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.

59. A kit for use in generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF) and thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor.

60. A kit when used for generating a population of definitive haematopoietic stem / progenitor cells (HSPCs), said kit comprising one or more components selected from the group consisting of: a WNT agonist, a fibroblast growth factor (FGF), Activin A, an ACTIVIN antagonist, vascular endothelial growth factor (VEGF), a retinoic acid signalling agent, a bone morphogenic protein (BMP), an insulin-like growth factor (IGF), a stem cell factor (SCF) and thrombopoietin (TPO), StemRegenninl (SRI), FLT3 receptor ligand (FLT3L), interleukin 3 (IL-3), erythropoietin (EPO), and a ROCK inhibitor.