Methods and compositions for inducing hematopoietic cell differentiation

Abstract

Methods and compositions are provided for differentiating stem cells into definitive hematopoiesis without relying on co-culture or serum-containing media and without requiring the formation of embryoid body aggregates as an intermediate. [Solution] The present invention provides methods and compositions for generating hematopoietic lineage cells via secondary hemogenic endothelium (HE) and secondary hematopoietic stem cells (HSC) derived from pluripotent stem cells, including hiPSCs, in a scalable monolayer culture platform under serum / feeder-free conditions without the need for EB formation. The present invention also provides methods and compositions for differentiating and expanding cells of the hematopoietic lineage from pluripotent stem cells in monolayer culture, comprising contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 62 / 107,517, filed January 26, 2015, and U.S. Provisional Application No. 62 / 251,016, filed November 4, 2015, the disclosures of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates generally to compositions and methods for producing cells of all hematopoietic lineages from pluripotent stem cells, and in particular to an improved culture platform for producing cells of all hematopoietic lineages from pluripotent stem cells, including human induced pluripotent stem cells. [Background technology]

[0003] (background) Human induced pluripotent stem cell (hiPSC) technology represents a highly promising and potentially limitless source of therapeutically viable hematopoietic cells for the treatment of numerous hematologic and non-hematologic malignancies, including cancer. To advance the promise of hiPSC and genetically engineered hiPSC technology as an allogeneic source for hematopoietic cell therapy, it is essential to be able to efficiently and reproducibly generate not only hematopoietic stem and progenitor cells (HSCs), but also immune effector populations, including diverse subsets of T cells, B cells, NKT cells, and NK lymphoid cells, as well as these progenitor cells.

[0004] The in vitro derivation of HSCs with lymphocyte-generating potential is complicated by the existence of at least two temporally and spatially distinct waves of blood formation during embryonic development: primitive and definitive hematopoiesis. Primitive hematopoiesis originates within the extraembryonic yolk sac and generates a transient, limited hematopoietic repertoire that primarily contains primitive erythroid and myeloid cells but does not contain HSCs. Nascent HSCs emerge only during the late secondary wave from specialized endothelial precursors within the arterial vasculature, termed definitive hemogenic endothelial (HE). Secondary HE then undergoes an endothelial-to-hematopoietic transition, giving rise to HSCs that ultimately migrate to the bone marrow and persist throughout adult lifespan through multilineage hematopoiesis, including T cells, B cells, NKT cells, and NK lymphoid cells. Thus, the generation of HSCs and lymphoid effector cells from pluripotent stem cells depends on the ability to accurately recapitulate the complex steps of early embryonic hematopoietic development into the secondary program through well-designed and validated methods and compositions. A limited number of studies have described the directed differentiation of hiPSCs into secondary embryonic stem cells in vitro. A major obstacle to utilizing hiPSCs for therapeutic purposes has been the requirement to first coculture these cells with mouse- or human-derived stromal cells in the presence of serum-containing medium of unknown composition to maintain pluripotency and induce differentiation. Additionally, existing protocols also employ strategies that involve culturing iPSCs to form embryoid bodies (EBs), heterogeneous cell aggregates containing a variety of differentiated cells, including ectodermal, mesodermal, and endodermal cells. These procedures require aggregation of pluripotent cells, for example, by spinning to form clumps, allowing cells to settle and aggregate within wells, or allowing passive aggregation and clump formation in suspension culture. The formed EBs are maintained under differentiation for a certain duration, typically induced in culture for 7-10 days to allow proper differentiation, and then the EBs are transferred to adherent culture for further maturation or dissociated into single cells for cell type selection to proceed to subsequent differentiation steps (Kennedy et al., Cell Reports, 2012, pp. 1722-1735; Knorr et al., Stem Cells Translational Medicine, 2013, vol. 2:274-283). For example, Kennedy et al. teach generating EBs for iPSC differentiation, treating pluripotent cells with collagenase and trypsin to allow scraping of the cells to form small aggregates, which are then cultured to form EBs. While EB formation has been shown to facilitate the differentiation of pluripotent stem cells, the requirement for forming aggregates and subsequent EBs is labor-intensive, expansion of cell numbers during this process is minimal, and exposure of the cellular contents in the three-dimensional EB aggregates to media factors is inconsistent and uneven, resulting in variable differentiation stages and heterogeneous cell products, severely compromising the scalability and reproducibility of the manufacturing process required for efficiency and rationalization. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kennedy et al., Cell Reports, 2012, pp. 1722-1735 [Non-patent document 2] Knorr et al., Stem Cells Translational Medicine, 2013, Vol. 2:274-283 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for methods and compositions for differentiating stem cells into definitive hematopoiesis without relying on co-culture or serum-containing media and without requiring the formation of embryoid body aggregates as an intermediate. [Means for solving the problem]

[0007] (Summary of the Invention) The present invention relates generally to cell culture conditions, media, culture platforms and methods for culturing and differentiating stem cells towards a hematopoietic cell fate.

[0008] Specifically, the present invention provides methods and compositions for generating hematopoietic lineage cells via secondary hemogenic endothelium (HE) and secondary hematopoietic stem cells (HSC) derived from pluripotent stem cells, including hiPSCs, in a scalable monolayer culture platform under serum / feeder-free conditions and without the need for EB formation. Cells that can be differentiated according to the methods of the present invention range from pluripotent stem cells to committed progenitor cells to specific terminally differentiated and transdifferentiated cells, to cells of diverse lineages that transition directly to a hematopoietic fate without passing through a pluripotent intermediate. Similarly, cells produced by stem cell differentiation range from multipotent stem cells or progenitor cells to terminally differentiated stem cells and all intervening hematopoietic lineage cells.

[0009] The present invention provides methods and compositions for differentiating and expanding hematopoietic cells from pluripotent stem cells in monolayer culture, comprising contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. Thus, mesodermal cells derived from the pluripotent stem cells are obtained and expanded from the pluripotent stem cells without forming embryoid bodies. The mesodermal cells are then placed in contact with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with secondary hemogenic endothelial (HE) potential from the pluripotent stem cells without forming embryoid bodies. Subsequent contact with bFGF and, optionally, a ROCK inhibitor and / or a WNT pathway activator differentiates the mesodermal cells with secondary HE potential into secondary HE cells, which are also expanded during differentiation.

[0010] The methods presented herein for obtaining cells of the hematopoietic system are superior to EB-mediated differentiation of pluripotent stem cells because there is little to minimal expansion of cells leading to the formation of EBs and they do not allow for cumbersome and inefficient monolayer culture, which is important for many applications and requires homogeneous expansion and homogeneous differentiation of cells within a population.

[0011] Provided herein is a monolayer differentiation platform that facilitates differentiation into secondary hemogenic endothelium, resulting in the derivation of hematopoietic stem cells and differentiated progeny cells, such as T cells, B cells, NKT cells, and NK cells. The proven monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion, enabling the delivery of therapeutically relevant numbers of pluripotent stem cell-derived hematopoietic cells for diverse therapeutic applications. Furthermore, the present invention also discloses that monolayer cultures using the methods presented herein yield functional hematopoietic cells capable of a full range of in vitro differentiation, ex vivo modulation, and long-term in vivo hematopoietic self-renewal, reconstitution, and engraftment.

[0012] One aspect of the present invention provides a culture platform for obtaining hematopoietic cells derived from pluripotent stem cells, the culture platform comprising Group I: (i) a culture medium suitable for differentiating and expanding secondary HSCs from secondary hemogenic endothelium, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, optionally lacking a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium from mesodermal cells, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for differentiating and expanding mesodermal cells from pluripotent stem cells, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor.

[0013] Alternatively, the culture platform for obtaining hematopoietic cells from pluripotent stem cells includes Group II: (i) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium from mesodermal cells with secondary hemogenic endothelial potential, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, optionally without a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for obtaining mesodermal cells with secondary hemogenic endothelial potential, comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for differentiating and expanding mesodermal cells from pluripotent stem cells, comprising a BMP activator and optionally bFGF. In some embodiments, the pluripotent stem cells of the above culture platform are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, group (II) of the above culture platform further comprises (iv) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and expanding pluripotent stem cells.

[0014] In some embodiments of the above culture platform, each of groups (I) and (II) further comprises additional culture medium.

[0015] Group (I) may further comprise (i) a culture medium suitable for differentiating pluripotent stem cell-derived T cell precursors into T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; and no BMP activators, or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary HSCs into T cell precursors, comprising a BMP activator; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; and one or more Notch pathway activators. These additional culture media are suitable for generating pluripotent stem cell-derived T lineage cells.

[0016] Group (II) can further include (i) a culture medium suitable for differentiating pluripotent stem cell-derived pre-T cell precursors into T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor; or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hematopoietic endothelium into pre-T cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7; these additional culture media are suitable for generating pluripotent stem cell-derived T lineage cells.

[0017] In some embodiments of the above culture platform, each of groups (I) and (II) still further comprises additional culture medium.

[0018] Group (I) can further comprise: (i) a culture medium suitable for differentiating pluripotent stem cell-derived NK cell precursors into NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15, and excluding a BMP activator; or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary HSCs into NK cell precursors, comprising a BMP activator; and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; these additional media are suitable for generating pluripotent stem cell-derived NK lineage cells.

[0019] With regard to group (II), in addition to the above-mentioned media, the media may further include (i) a medium suitable for differentiating pluripotent stem cell-derived pre-NK cell precursors into NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor; or (ii) a medium suitable for differentiating pluripotent stem cell-derived secondary hematopoietic endothelium into pre-NK cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15; these additional media are suitable for generating pluripotent stem cell-derived NK lineage cells.

[0020] In yet another embodiment, group (II) of the culture platform provided comprises: (i) a culture medium suitable for differentiating pre-HSCs derived from pluripotent stem cells into hematopoietic multipotent progenitors, comprising a BMP activator, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and not comprising a ROCK inhibitor; (ii) a culture medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells into pre-HSCs, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11; these culture media are provided for generating hematopoietic multipotent progenitors derived from pluripotent stem cells.

[0021] Another aspect of the present invention provides a composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, the composition comprising one or more of the following group (I) or (II):

[0022] Group (I): (i) a culture medium suitable for differentiating and expanding secondary HSCs from secondary hemogenic endothelium derived from pluripotent stem cells, comprising a BMP activator; one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and secondary hemogenic endothelium derived from pluripotent stem cells, optionally not comprising a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium from mesodermal cells derived from pluripotent stem cells, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; and mesodermal cells derived from pluripotent stem cells; and (iii) a culture medium suitable for differentiating and expanding mesodermal cells from pluripotent stem cells, comprising a GSK3 inhibitor, a BMP activator; and iPSCs.

[0023] Group (II): (i) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and mesodermal cells derived from pluripotent stem cells with the potential for secondary hematopoietic endothelium, optionally without a TGFβ receptor / ALK inhibitor, which is suitable for differentiating and expanding secondary hematopoietic endothelium from mesodermal cells derived from pluripotent stem cells with the potential for hematopoietic endothelium; (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not a TGFβ receptor / ALK inhibitor; and which is suitable for differentiating and expanding mesodermal cells with the potential for secondary hematopoietic endothelium from mesodermal cells derived from pluripotent stem cells; and (iii) a culture medium comprising a BMP activator, optionally bFGF, and iPSCs, which is suitable for differentiating and expanding mesodermal cells from pluripotent stem cells.

[0024] In some embodiments of the composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0025] In some embodiments of the composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, group (II) comprises (vi) a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and does not comprise a TGFβ receptor / ALK inhibitor; and further comprises a culture medium suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0026] In some embodiments of the above-described composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, group (I) additionally comprises (i) a culture medium suitable for differentiating the pluripotent stem cell-derived T cell precursors into T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; and a pluripotent stem cell-derived T cell precursor; and no BMP activator, or (ii) a culture medium suitable for differentiating the pluripotent stem cell-derived secondary HSCs into T cell precursors, comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; one or more Notch pathway activators; and a pluripotent stem cell-derived secondary HSC; these additional media are suitable for generating pluripotent stem cell-derived T lineage cells.

[0027] With respect to group (II) of compositions for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, some embodiments further include: (i) a culture medium suitable for differentiating pre-T cell precursors derived from pluripotent stem cells into T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor; and pre-T cell precursors derived from pluripotent stem cells; or (ii) a culture medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells into pre-T cell precursors. These additional media are suitable for generating T lineage cells derived from pluripotent stem cells.

[0028] In still other embodiments of the composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, group (I) further comprises: (i) a culture medium suitable for differentiating NK cell precursors into NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15; and NK cell precursors derived from pluripotent stem cells, the culture medium not comprising a BMP activator, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and secondary HSCs derived from pluripotent stem cells, the culture medium suitable for differentiating the secondary HSCs derived from pluripotent stem cells into NK cell precursors. These additional media are suitable for generating NK lineage cells derived from pluripotent stem cells. Alternatively, group (II) of the compositions for differentiating and expanding hematopoietic cells derived from pluripotent stem cells may further comprise: (i) a medium suitable for differentiating pre-NK cell precursors into NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor; and pluripotent stem cell-derived pre-NK cell precursors; or (ii) a medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells, comprising one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15, and pluripotent stem cell-derived secondary hemogenic endothelium, and pluripotent stem cell-derived secondary hemogenic endothelium. These culture media are suitable for generating NK lineage cells derived from pluripotent stem cells.

[0029] In still other embodiments, group (II) of the compositions for differentiating and expanding hematopoietic cells derived from pluripotent stem cells further comprises one or more media for generating hematopoietic multipotent progenitors derived from pluripotent stem cells, the media comprising (i) a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but not including a ROCK inhibitor and a pluripotent stem cell-derived hematopoietic progenitor. and / or (ii) a culture medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells into pre-HSCs, the culture medium being free of pluripotent stem cell-derived pre-HSCs and suitable for differentiating pre-HSCs into hematopoietic multipotent precursors; and / or (ii) a culture medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells into pre-HSCs, the culture medium being free of pluripotent stem cell-derived pre-HSCs and suitable for differentiating pre-HSCs into hematopoietic multipotent precursors;

[0030] One aspect of the present invention is a culture platform for generating T lineage cells derived from pluripotent stem cells, comprising Group I: (i) a culture medium comprising a GSK3 inhibitor, a BMP activator, and suitable for differentiating and expanding mesodermal cells derived from pluripotent stem cells from the pluripotent stem cells; (ii) a culture medium comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor, and suitable for differentiating and expanding secondary hematopoietic endothelium from mesodermal cells; (iii) a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and optionally not comprising a Wnt pathway activator and a TGFβ receptor / ALK inhibitor, and suitable for differentiating and expanding secondary hematopoietic endothelium from mesodermal cells. The present invention provides a culture platform comprising: a culture medium suitable for differentiating and expanding HSCs from secondary hemogenic endothelium; and a culture medium suitable for differentiating T cell precursors from secondary HSCs, the culture medium comprising: (iv) a BMP activator; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; and one or more Notch pathway activators; and, optionally, (v) a culture medium suitable for differentiating T cells from T cell precursors, the culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; and no BMP activators.

[0031] Alternatively, the culture platform for generating T-lineage cells derived from pluripotent stem cells comprises Group II: (i) a culture medium suitable for differentiating and expanding mesodermal cells derived from pluripotent stem cells, comprising a BMP activator and, optionally, bFGF; (ii) a culture medium suitable for obtaining mesodermal cells having secondary HE potential from mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for obtaining mesodermal cells having secondary HE potential from mesodermal cells, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; optionally without a TGFβ receptor / ALK inhibitor, and for obtaining secondary hemogenic endothelial cells having secondary hemogenic endothelial potential. (iv) a culture medium suitable for differentiating secondary hemogenic endothelium into pre-T cell precursors, comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7, and secondary hemogenic endothelium derived from pluripotent stem cells; and (v) a culture medium suitable for differentiating pre-T cell precursors into T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor; and secondary hemogenic endothelium derived from pluripotent stem cells.

[0032] In some embodiments of the above-described culture platform for generating T-lineage cells derived from pluripotent stem cells, the Group II culture platform further comprises (vi) a culture medium suitable for seeding and expanding the pluripotent stem cells, comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and optionally, not comprising a TGFβ receptor / ALK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0033] Another aspect of the present invention is a culture platform for generating NK cells derived from pluripotent stem cells, comprising Group I: (i) a culture medium suitable for differentiating pluripotent stem cells into mesodermal cells, comprising a GSK3 inhibitor, a BMP activator; (ii) a culture medium suitable for differentiating mesodermal cells into secondary hematopoietic endothelium, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for differentiating mesodermal cells into secondary hematopoietic endothelium, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and optionally a Wnt pathway activator and a TGFβ receptor / ALK inhibitor. (iv) a culture medium suitable for differentiating secondary hemogenic endothelium into secondary HSCs, wherein the culture medium is free of a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and the culture medium is suitable for differentiating secondary HSCs into NK cell precursors; and, optionally, (v) a culture medium suitable for differentiating NK cell precursors into NK cells, wherein the culture medium is free of a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15.

[0034] Alternatively, the culture platform for generating NK cells derived from pluripotent stem cells comprises Group II: (i) a culture medium suitable for differentiating and expanding mesodermal cells from pluripotent stem cells, comprising a BMP activator, and optionally bFGF; (ii) a culture medium suitable for obtaining mesodermal cells having the potential of secondary hematopoietic endothelium from mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for obtaining mesodermal cells having the potential of secondary hematopoietic endothelium from mesodermal cells, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, optionally without a TGFβ receptor / ALK inhibitor. (iv) a culture medium suitable for differentiating secondary hematopoietic endothelium into pre-NK cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15; and (v) a culture medium suitable for differentiating pre-NK cell precursors into NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, but excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor.

[0035] In some embodiments of the above-described culture platform for generating NK cells derived from pluripotent stem cells, the Group II culture platform further comprises (vi) a culture medium suitable for seeding and expanding the pluripotent stem cells, comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0036] Yet another aspect of the present invention provides a culture platform for generating secondary hemogenic endothelium (iHE) derived from pluripotent stem cells, the culture platform comprising: (i) a culture medium suitable for differentiating and expanding mesodermal cells from pluripotent stem cells, the culture medium comprising a BMP activator, and optionally bFGF; (ii) a culture medium suitable for obtaining mesodermal cells having the potential for secondary hemogenic endothelium from mesodermal cells derived from pluripotent stem cells, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium from mesodermal cells having the potential for secondary hemogenic endothelium, the culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, the culture medium optionally without a TGFβ receptor / ALK inhibitor.

[0037] In some embodiments of the culture platform for generating secondary hemogenic endothelium (iHE) derived from pluripotent stem cells, the culture platform further comprises (iv) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not a TGFβ receptor / ALK inhibitor, suitable for seeding and expanding the pluripotent stem cells.

[0038] Yet another aspect of the present invention is a culture platform for generating hematopoietic multipotent progenitors derived from pluripotent stem cells, comprising: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from the pluripotent stem cells, the culture medium comprising a BMP activator, and optionally bFGF; (ii) a culture medium suitable for obtaining mesodermal cells having secondary hemogenic endothelial potential from pluripotent stem cell-derived mesodermal cells, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for obtaining mesodermal cells having secondary hemogenic endothelial potential from pluripotent stem cell-derived mesodermal cells, the culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and optionally without a TGFβ receptor / ALK inhibitor. (iv) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium from mesodermal cells having secondary hemogenic endothelial potential; (iv) a culture medium suitable for differentiating secondary hemogenic endothelium into pre-HSCs, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11; and (v) a culture medium suitable for differentiating pre-HSCs into hematopoietic multipotent progenitors, comprising a BMP activator, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but not a ROCK inhibitor. In some embodiments, the culture platform further comprises (vi) a culture medium containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not containing a TGFβ receptor / ALK inhibitor, suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0039] Another aspect of the present invention is a method for directing differentiation of pluripotent stem cells into secondary hematopoietic cells, comprising the steps of: (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor, a BMP activator to induce differentiation and expansion of pluripotent stem cell-derived mesodermal cells from the pluripotent stem cells; and (ii) contacting pluripotent stem cell-derived mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor to induce differentiation and expansion of pluripotent stem cell-derived secondary hematopoietic endothelial cells. (iii) contacting the pluripotent stem cell-derived secondary HSCs with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and optionally excluding a Wnt pathway activator and a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of the pluripotent stem cell-derived secondary HSCs from the hemogenic endothelial cells.

[0040] Alternatively, the method for directing the differentiation of pluripotent stem cells into secondary hematopoietic cells includes Group II: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to induce differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of mesodermal cells having secondary HE potential from the mesodermal cells; and (iii) contacting mesodermal cells having secondary HE potential with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of mesodermal cells having secondary HE potential from the mesodermal cells. contacting mesodermal cells with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and optionally, a composition not comprising a TGFβ receptor / ALK inhibitor to induce differentiation and expansion of secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having secondary hemogenic endothelial potential; and optionally, placing the pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells having hemogenic endothelium, and / or secondary hemogenic endothelium under a hypoxic tension of between about 2% and about 10%.

[0041] In some embodiments of the method for directing the differentiation of pluripotent stem cells into hematopoietic cells, the method of group (II) further comprises the step of contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0042] In some embodiments of methods for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of the hematopoietic lineage lacks the development of embryoid bodies and is in a monolayer culture format.

[0043] In some embodiments of the above methods, the obtained pluripotent stem cell-derived secondary hemogenic endothelial cells are CD34+. In some embodiments, the obtained secondary hemogenic endothelial cells are CD34+ CD43-. In some embodiments, the secondary hemogenic endothelial cells are CD34+ CD43- CXCR4- CD73-.

[0044] In some other embodiments of the above methods, Group I comprises (i) contacting secondary HSCs derived from pluripotent stem cells with a composition comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators to induce differentiation of the secondary HSCs derived from pluripotent stem cells into T cell precursors, and optionally contacting the T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; and one or more Notch pathway activators to induce differentiation of the T cell precursors into T cells. In some embodiments, group II of the methods further comprises: (i) contacting secondary hemogenic endothelium derived from pluripotent stem cells with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7 to induce differentiation of the secondary hemogenic endothelium into pre-T cell precursors; and optionally, (ii) contacting the pre-T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not including one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor to induce differentiation of the pre-T cell precursors into T cell precursors or T cells. In some embodiments of the methods, the T cell precursors derived from pluripotent stem cells are CD34+ CD7+.

[0045] In still other embodiments of the above-described methods for directing differentiation of pluripotent stem cells into cells of the hematopoietic lineage, group I of the methods further comprises: (i) contacting secondary HSCs derived from the pluripotent stem cells with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15 to induce differentiation of the secondary HSCs into NK cell precursors; and optionally, (ii) contacting the NK cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15, but not a BMP activator, to induce differentiation of the NK cell precursors into NK cells, or group II of the methods further comprises: (i) contacting secondary hemogenic endothelium derived from pluripotent stem cells with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15, to induce differentiation of the secondary hemogenic endothelium into pre-NK cell precursors; and optionally, (ii) contacting pre-NK cell precursors derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the medium does not include one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor, to induce differentiation of the pre-NK cell precursors into NK cell precursors or NK cells.In some embodiments, group II of the methods includes (i) contacting secondary hemogenic endothelium derived from pluripotent stem cells with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to induce differentiation of the secondary hemogenic endothelium into pre-HSCs; and (ii) further comprising a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but excluding a ROCK inhibitor, suitable for differentiating the pluripotent stem cell-derived pre-HSCs into hematopoietic multipotent precursors. In some embodiments, the pluripotent stem cell-derived secondary HSCs obtained using the above method are CD34+ CD45+ and suitable for long-term engraftment.

[0046] Another aspect of the present invention is a method for generating T lineage cells from pluripotent stem cells, comprising: Group I: (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor, a BMP activator to induce differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor to induce differentiation and expansion of secondary hemogenic endothelium from the mesodermal cells; and (iii) contacting the secondary hemogenic endothelium with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and not including a Wnt pathway activator and a TGFβ receptor / ALK inhibitor. (iv) contacting the secondary HSCs with a composition comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators to induce differentiation of the secondary HSCs into T cell precursors; and optionally, (v) contacting the T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; but no BMP activator to induce differentiation of the T cell precursors into T cells.

[0047] Alternatively, the method for generating T lineage cells from pluripotent stem cells includes Group II: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to induce differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not including a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of mesodermal cells having secondary HE potential from the mesodermal cells; and (iii) contacting mesodermal cells having secondary HE potential with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and not including a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of secondary hemogenic endothelium from mesodermal cells having secondary HE potential. (iv) contacting the secondary hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7 to induce differentiation of the secondary hemogenic endothelium into pre-T cell precursors; (v) contacting the pre-T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; and excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor to induce differentiation of the pre-T cell precursors into T cell precursors or T cells; optionally, the seeded pluripotent stem cells, mesodermal cells, mesodermal cells with secondary hemogenic potential, and / or secondary hemogenic endothelium can be placed under a hypoxic tension of between about 2% and about 10%. In some embodiments, group II of the above-mentioned method further comprises contacting iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not including a TGFβ receptor / ALK inhibitor, to seed and expand pluripotent stem cells.In some embodiments, the pluripotent stem cells are iPSCs.In some embodiments, the iPSCs are naive iPSCs.In some embodiments of the method, the differentiation of pluripotent stem cells into T lineage cells lacks embryoid body development and is in a monolayer culture format.

[0048] Yet another aspect of the present invention is a method for generating NK lineage cells from pluripotent stem cells, comprising: Group I: (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor, a BMP activator to induce differentiation of the pluripotent stem cells into mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor to induce differentiation of the mesodermal cells into secondary hemogenic endothelium; and (iii) contacting the secondary hemogenic endothelium with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and not including a Wnt pathway activator and a TGFβ receptor / ALK inhibitor. (iv) contacting the secondary HSCs with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15 to induce differentiation of the secondary HSCs into NK cell precursors; and optionally, (v) contacting the NK cell precursors derived from the pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15, but not a BMP activator, to induce differentiation of the NK cell precursors into NK cells.Alternatively, the method for generating NK lineage cells derived from pluripotent stem cells includes Group II: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to induce differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of mesodermal cells having secondary HE potential from the mesodermal cells; (iii) contacting mesodermal cells having secondary HE potential with a composition comprising one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and a ROCK inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of secondary hematopoietic endothelium derived from pluripotent stem cells with secondary HE potential from mesodermal cells derived from pluripotent stem cells; (iv) contacting the pluripotent stem cell-derived secondary hemogenic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15, a BMP activator, and a ROCK inhibitor to induce differentiation of the pluripotent stem cell-derived secondary hemogenic endothelium into pre-NK cell precursors; and (v) contacting the pluripotent stem cell-derived pre-NK cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15. and inducing differentiation of the pluripotent stem cell-derived pre-NK cell precursors into pluripotent stem cell-derived NK cell precursors or NK cells by contacting the pluripotent stem cell-derived pre-NK cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors; and optionally, placing the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, and / or secondary hemogenic endothelium under a hypoxic tension of between about 2% and about 10%.In some embodiments, the method for generating NK lineage cells from group II pluripotent stem cells further comprises contacting the iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, to seed and expand the iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method for generating NK lineage cells from pluripotent stem cells lacks embryoid body development and is in a monolayer culture format.

[0049] Another aspect of the present invention is a method for generating secondary hemogenic endothelium from pluripotent stem cells, comprising the steps of: (i) contacting iPSCs with a composition comprising a BMP activator and, optionally, bFGF, to induce differentiation and expansion of pluripotent stem cell-derived mesodermal cells from the pluripotent stem cells; (ii) contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of pluripotent stem cell-derived mesodermal cells having the potential for secondary HE from the pluripotent stem cell-derived mesodermal cells; and (iii) inducing differentiation and expansion of the pluripotent stem cell-derived mesodermal cells from the pluripotent stem cells, the mesodermal cells having the potential for secondary HE. and contacting pluripotent stem cell-derived mesodermal cells having secondary hemogenic potential with a composition comprising one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and a ROCK inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having secondary hemogenic potential; and optionally, placing the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, and / or secondary hemogenic endothelium under a hypoxic atmosphere of between about 2% and about 10%. In some embodiments, the method for generating pluripotent stem cell-derived secondary hemogenic endothelium further comprises contacting iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor / ALK inhibitor, to seed and expand the iPSCs, and / or the iPSCs are naive iPSCs. In some embodiments, the above-described methods of differentiating iPSCs into cells of secondary hemogenic endothelium lack embryoid body development and are in a monolayer culture format.

[0050] Another aspect of the present invention is a method for generating multipotent progenitors of the hematopoietic system from pluripotent stem cells, comprising the steps of: (i) contacting iPSCs with a composition comprising a BMP activator, and optionally bFGF, to induce differentiation and expansion of pluripotent stem cell-derived mesodermal cells from the iPSCs; (ii) contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of mesodermal cells with secondary HE potential from the mesodermal cells; and (iii) contacting the mesodermal cells with secondary HE potential with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, but not a TGFβ receptor / ALK inhibitor, to induce differentiation and expansion of mesodermal cells with secondary HE potential from secondary hemogenic endothelium. (iv) contacting the secondary hemogenic endothelium with a composition comprising a BMP activator, an IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to induce differentiation of the secondary hemogenic endothelium into pre-HSCs; and (v) contacting the pre-HSCs with a composition comprising a BMP activator, an IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to induce differentiation of the pre-HSCs into hematopoietic multipotent progenitors, but not a ROCK inhibitor. Optionally, the method comprises placing the seeded pluripotent stem cells, mesodermal cells, and / or secondary hemogenic endothelium under a hypoxic atmosphere of between about 2% and about 10%. In some embodiments, the method for generating hematopoietic multipotent progenitors from pluripotent stem cells further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs.In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the differentiation of pluripotent stem cells into hematopoietic multipotent progenitors using the above methods lacks embryoid body development and is in a monolayer culture format.

[0051] A further aspect of the present invention relates to one or more cell populations generated from the culture platform disclosed herein: (i) CD34+ secondary hemogenic endothelium (iCD34) derived from pluripotent stem cells, wherein the iCD34 cells have the ability to differentiate into multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, and NK cells and are CD34+ CD43−; (ii) secondary hemogenic endothelium (iHE), wherein the iHE cells are CD34+; (iii) secondary HSC derived from pluripotent stem cells, wherein the iHSCs are CD34+ CD45+; (iv) hematopoietic multipotent progenitor cells, wherein the iMPP cells are CD34+ CD45+; (v) T cell precursors that are CD34+ CD7+; (vi) T cells that are CD4+ or CD8+; (vii) CD56+ Compositions are provided that include (viii) NK cell precursors that are CD7+ CD161+; and (viii) NK cells that are CD56+ CD57+ CD16+ CD94-.

[0052] Yet further aspects of the present invention relate to one or more cell lines or clonal cells generated using the methods disclosed herein: (i) CD34+ secondary hemogenic endothelium (iCD34) derived from pluripotent stem cells, wherein the iCD34 cells have the ability to differentiate into multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, and NK cells and are CD34+ CD43-; (ii) secondary hemogenic endothelium (iHE), wherein the iHE cell line or clonal cells are CD34+; (iii) secondary HSC, wherein the iHSC are CD34+ CD45+; (iv) hematopoietic multipotent progenitor cells (iMPP), wherein the iMPP cells are CD34+ CD45+; (v) CD34+ (vi) T cell precursors that are CD7+; (vi) T cells that are CD4+ or CD8+; (vii) NK cell precursors that are CD56+ CD7+ CD161+; and (viii) NK cells that are CD56+ CD57+ CD16+ CD94-.

[0053] Another aspect of the invention relates to cell populations, cell lines, or clonal cells generated using the disclosed methods: (i) CD34+ secondary hemogenic endothelium (iCD34) derived from pluripotent stem cells, wherein the iCD34 cells have the ability to differentiate into multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, and NK cells and are CD34+ CD43-; (ii) secondary hemogenic endothelium (iHE), wherein the iHE cell line or clonal cells are CD34+; (iii) secondary HSC, wherein the iHSC are CD34+ CD45+; (iv) hematopoietic multipotent progenitor cells, wherein the iMPP cells are CD34+ CD45+; (v) T cell precursors that are CD34+ CD7+; (vi) T cells that are CD4+ or CD8+; (vii) CD56+ CD7+ and (viii) NK cells that are CD56+ CD57+ CD16+ CD94-.

[0054] In summary, the present invention provides methods and compositions that allow for the direct differentiation of pluripotent stem cells in monolayers without generating embryoid bodies from the pluripotent stem cells, thereby achieving differentiation and expansion of mesodermal cells, secondary HE, and secondary HSCs, which can be used to obtain other hematopoietic cells in a scalable and reliable manner with extremely high levels of efficiency. In certain aspects of the present invention, for example, the following items are provided: (Item 1) 1. A method for directing the differentiation of pluripotent stem cells into hematopoietic cells, comprising: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to obtain mesodermal cells; and (ii) contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator to obtain mesodermal cells with secondary hematopoietic endothelial (HE) potential, wherein the mesodermal cells with secondary hematopoietic endothelial (HE) potential can provide hematopoietic cells including hematopoietic stem and progenitor cells (HSCs), hematopoietic multipotent progenitor cells (MPPs), precursors of pre-T cells, precursors of pre-NK cells, precursors of T cells, precursors of NK cells, T cells, NK cells, NKT cells, or B cells; and wherein the mesodermal cells and mesodermal cells with secondary HE potential are obtained in steps (i) and (ii) without forming embryoid bodies. (Item 2) 2. The method of claim 1, further comprising contacting the mesodermal cells having the potential for secondary HE with a composition comprising bFGF and a ROCK inhibitor to obtain secondary HE cells. (Item 3) 3. The method of claim 2, further comprising contacting the secondary HE cells with a composition comprising a BMP activator, and optionally a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, to obtain hematopoietic multipotent progenitor cells (MPPs). (Item 4) 3. The method of claim 2, further comprising contacting the secondary HE cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; and optionally one or more of a BMP activator, a ROCK inhibitor, VEGF, and bFGF, to obtain pre-T cell precursors, T cell precursors, and / or T cells. (Item 5) 3. The method of claim 2, further comprising contacting the secondary HE cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15, and optionally one or more of a BMP activator, a ROCK inhibitor, VEGF, and bFGF, to obtain pre-NK cell precursors, NK cell precursors, and / or NK cells. (Item 6) 2. The method of claim 1, further comprising the step of contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the cells. (Item 7) Item 1. The method according to Item 1, further comprising the step of placing the pluripotent stem cells, the mesodermal cells, and / or the mesodermal cells having secondary hematopoietic endothelial potential under a low oxygen tension of between about 2% and about 10%. (Item 8) 2. The method according to item 1, wherein the differentiation of the pluripotent stem cells into hematopoietic cells does not contain or is essentially free of TGFβ receptor / ALK inhibitors. (Item 9) Item 2. The method according to item 1, wherein the differentiation of the pluripotent stem cells into hematopoietic cells is carried out under feeder-free conditions. (Item 10) Item 2. The method according to item 1, wherein the differentiation of the pluripotent stem cells into hematopoietic cells is carried out under stroma-free conditions. (Item 11) Item 10. The method of item 1, wherein the pluripotent stem cells comprise induced pluripotent stem cells (iPSCs). (Item 12) Item 12. The method of item 11, wherein the iPSCs are naive iPSCs. (Item 13) 2. The method of claim 1, wherein the WNT pathway activator is a GSK3 inhibitor. (Item 14) 14. The method of claim 13, wherein the GSK3 inhibitor is CHIR99021. (Item 15) 2. The method of item 1, wherein the BMP pathway activator is BMP4. (Item 16) 3. The method of claim 2, wherein the ROCK inhibitor is thiazovivin or Y-27632. (Item 17) 3. The method of claim 2, wherein the ROCK inhibitor is Y-27632. (Item 18) A method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, comprising: (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor and a BMP activator to obtain mesodermal cells; (ii) contacting the mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor to obtain a hemogenic endothelium; (iii) contacting the hemogenic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and a BMP activator to obtain secondary HSCs, wherein the composition optionally does not comprise a Wnt pathway activator or a TGFβ receptor / ALK inhibitor; A method comprising: (Item 19) (iv) contacting the secondary HSCs with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; a BMP activator; and one or more Notch pathway activators to obtain T cell precursors; and optionally, (v) contacting the T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; and one or more Notch pathway activators to obtain T cells; or (vi) contacting the secondary HSCs with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and a BMP activator to obtain NK cell precursors; and optionally, (vii) contacting the NK cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15 to obtain NK cells, wherein the composition does not comprise a BMP activator; Item 19. The method of item 18, further comprising: (Item 20) 19. The method of item 18, wherein the secondary HSCs are CD34+ CD45+. (Item 21) 21. The method of item 20, wherein the secondary HSCs are suitable for long-term engraftment. (Item 22) 20. The method of claim 19, wherein the T cell precursors are CD34+ CD7+. (Item 23) 20. The method of item 19, wherein the NK cell precursors are CD56+ CD7+ CD161+. (Item 24) 20. The method of claim 18, wherein directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage lacks the development of embryoid bodies. (Item 25) 20. The method according to item 18, wherein the differentiation of pluripotent stem cells into hematopoietic cells is directed in monolayer culture. (Item 26) Item 19. The method according to Item 18, wherein the differentiation of pluripotent stem cells into hematopoietic cells is directed under feeder-free conditions. (Item 27) Item 19. The method according to item 18, wherein the differentiation of pluripotent stem cells into hematopoietic cells is directed under stroma-free conditions. (Item 28) Item 19. The method of item 18, wherein the pluripotent stem cells are iPSCs. (Item 29) Item 29. The method of item 28, wherein the iPSCs are naive iPSCs. (Item 30) 1. A method for generating T lineage cells from pluripotent stem cells, said method comprising: (I) contacting T cell precursors derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; and one or more Notch pathway activators to obtain T cells; wherein the composition does not comprise a BMP activator. Includes; the pluripotent stem cell-derived T cell precursors are obtained by contacting pluripotent stem cell-derived secondary HSCs with a composition comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; and one or more Notch pathway activators to allow differentiation and expansion; the pluripotent stem cell-derived secondary HSCs are obtained by contacting pluripotent stem cell-derived hemogenic endothelium with a composition comprising a BMP activator; and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO to allow differentiation and expansion, wherein the composition optionally does not comprise a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; the pluripotent stem cell-derived secondary hemogenic endothelium is obtained by contacting pluripotent stem cell-derived mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor to allow differentiation and expansion; The mesodermal cells derived from the pluripotent stem cells are obtained by contacting the pluripotent stem cells with a composition comprising a GSK3 inhibitor, a BMP activator, and allowing differentiation and expansion; or the method comprises: (II) contacting pluripotent stem cell-derived pre-T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7 to obtain pluripotent stem cell-derived T cell precursors or T cells, wherein the composition does not comprise one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. Includes; the pluripotent stem cell-derived pre-T cell precursors are obtained by contacting pluripotent stem cell-derived secondary hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7 to allow differentiation and expansion of the cells; The pluripotent stem cell-derived secondary hemogenic endothelium is obtained by contacting pluripotent stem cell-derived mesodermal cells having secondary hemogenic potential with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11 to allow differentiation and expansion of the cells; and the composition does not comprise a TGFβ receptor / ALK inhibitor; The mesodermal cells derived from pluripotent stem cells having the potential for secondary HE are obtained by contacting the mesodermal cells derived from pluripotent stem cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to allow differentiation and expansion of the cells, wherein the composition does not comprise a TGFβ receptor / ALK inhibitor; The mesodermal cells derived from the pluripotent stem cells are obtained by contacting the pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to allow differentiation and expansion of the cells. method. (Item 31) contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the pluripotent stem cells. 31. The method of claim 30, further comprising: wherein the composition does not comprise a TGFβ receptor / ALK inhibitor. (Item 32) 31. The method of paragraph 30, wherein generating T lineage cells from pluripotent stem cells lacks embryoid body development. (Item 33) 31. The method of item 30, wherein generating T lineage cells derived from pluripotent stem cells is in monolayer culture. (Item 34) 31. The method of item 30, wherein generating T lineage cells derived from pluripotent stem cells is under feeder-free conditions. (Item 35) 31. The method of item 30, wherein generating T lineage cells derived from pluripotent stem cells is under stroma-free conditions. (Item 36) 31. The method of claim 30, wherein the pluripotent stem cells are iPSCs. (Item 37) Item 37. The method of item 36, wherein the iPSCs are naive iPSCs. (Item 38) 1. A method for generating NK lineage cells from pluripotent stem cells, said method comprising: (I) contacting pluripotent stem cell-derived NK cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15 to obtain pluripotent stem cell-derived NK cells, wherein the composition does not contain a BMP activator. Includes; the pluripotent stem cell-derived NK cell precursors are obtained by contacting pluripotent stem cell-derived secondary HSCs with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15 to allow differentiation and expansion of the cells; the pluripotent stem cell-derived secondary HSCs are obtained by contacting pluripotent stem cell-derived hemogenic endothelium with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO to allow differentiation and expansion of the cells, wherein the composition does not comprise a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; the pluripotent stem cell-derived hemogenic endothelium is obtained by contacting pluripotent stem cell-derived mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor to induce differentiation of the pluripotent stem cell-derived mesodermal cells and allow differentiation and expansion of the cells; The mesodermal cells derived from the pluripotent stem cells are obtained by contacting the pluripotent stem cells with a composition comprising a GSK3 inhibitor, a BMP activator, and allowing the cells to differentiate and expand; Or the method comprises: (II) contacting pluripotent stem cell-derived pre-NK cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 to obtain pluripotent stem cell-derived NK cell precursors or NK cells, wherein the composition does not comprise one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. Includes; the pluripotent stem cell-derived pre-NK cell precursors are obtained by contacting pluripotent stem cell-derived secondary hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15 to allow differentiation and expansion of the cells; Pluripotent stem cell-derived secondary hemogenic endothelium is obtained by contacting pluripotent stem cell-derived mesodermal cells, having secondary HE potential, with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, to allow differentiation and expansion of the cells, wherein the composition optionally does not comprise a TGFβ receptor / ALK inhibitor; the pluripotent stem cell-derived mesodermal cells having the potential for secondary HE are obtained by contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to allow differentiation and expansion of the cells, the composition optionally not comprising a TGFβ receptor / ALK inhibitor; The mesodermal cells derived from the pluripotent stem cells are obtained by contacting the pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to allow differentiation and expansion of the cells. method. (Item 39) Item 39. The method of Item 38, further comprising the step of placing the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells with hemogenic endothelial potential, and / or secondary hemogenic endothelium under a hypoxic tension of between about 2% and about 10%. (Item 40) 39. The method of claim 38, further comprising the step of contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the cells, wherein the composition does not comprise a TGFβ receptor / ALK inhibitor. (Item 41) 39. The method of paragraph 38, wherein generating NK lineage cells from pluripotent stem cells lacks embryoid body development. (Item 42) 39. The method of item 38, wherein generating NK lineage cells derived from pluripotent stem cells is in monolayer culture. (Item 43) 39. The method of item 38, wherein generating NK lineage cells derived from pluripotent stem cells is under feeder-free conditions. (Item 44) 39. The method of item 38, wherein generating NK lineage cells derived from pluripotent stem cells is under stroma-free conditions. (Item 45) Item 39. The method of item 38, wherein the pluripotent stem cells are iPSCs. (Item 46) Item 46. The method of item 45, wherein the iPSCs are naive iPSCs. (Item 47) 1. A method for generating secondary hemogenic endothelium from pluripotent stem cells, comprising: contacting pluripotent stem cell-derived mesodermal cells having the potential for secondary HE with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11 to obtain pluripotent stem cell-derived secondary hemogenic endothelium, wherein the composition optionally does not comprise a TGFβ receptor / ALK inhibitor; the pluripotent stem cell-derived mesodermal cells having the potential for secondary HE are obtained by contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to allow differentiation and expansion of the cells, the composition optionally not comprising a TGFβ receptor / ALK inhibitor; The mesodermal cells derived from the pluripotent stem cells are obtained by contacting the pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to allow differentiation and expansion of the cells. method. (Item 48) contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the pluripotent stem cells. 48. The method of claim 47, further comprising: wherein the composition does not comprise a TGFβ receptor / ALK inhibitor. (Item 49) 49. The method according to items 47 and 48, further comprising the step of placing the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells having secondary hemogenic endothelial potential, and / or secondary hemogenic endothelium under a hypoxic tension of between about 2% and about 10%. (Item 50) 48. The method of paragraph 47, wherein generating secondary hematopoietic endothelium from pluripotent stem cells lacks embryoid body development. (Item 51) 48. The method of item 47, wherein generating secondary hemogenic endothelium from pluripotent stem cells is in monolayer culture. (Item 52) 48. The method of item 47, wherein generating secondary hemogenic endothelium from pluripotent stem cells is under feeder-free conditions. (Item 53) 48. The method of item 47, wherein generating secondary hemogenic endothelium from pluripotent stem cells is under stroma-free conditions. (Item 54) Item 48. The method of item 47, wherein the pluripotent stem cells are iPSCs. (Item 55) Item 55. The method of item 54, wherein the iPSCs are naive iPSCs. (Item 56) 1. A method for generating multipotent progenitors of the hematopoietic system from pluripotent stem cells, comprising: contacting pre-HSCs derived from pluripotent stem cells with a composition comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to obtain multipotent progenitors derived from pluripotent stem cells, wherein the composition does not comprise a ROCK inhibitor; the pluripotent stem cell-derived pre-HSCs are obtained by contacting pluripotent stem cell-derived secondary hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to allow differentiation and expansion of the cells; the pluripotent stem cell-derived secondary hemogenic endothelium is obtained by contacting pluripotent stem cell-derived mesodermal cells having the potential for secondary HE with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11 to allow differentiation and expansion of the cells, wherein the composition does not comprise a TGFβ receptor / ALK inhibitor; the pluripotent stem cell-derived mesodermal cells having the potential for secondary HE are obtained by contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to allow differentiation and expansion of the cells, wherein the composition does not comprise a TGFβ receptor / ALK inhibitor; the pluripotent stem cell-derived mesodermal cells are obtained by contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to allow differentiation and expansion of the cells, and the composition does not comprise a TGFβ receptor / ALK inhibitor; The mesodermal cells derived from the pluripotent stem cells are obtained by contacting the pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to allow differentiation and expansion of the cells. method. (Item 57) contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the pluripotent stem cells. 57. The method of claim 56, further comprising: wherein the composition does not comprise a TGFβ receptor / ALK inhibitor. (Item 58) 58. The method of items 56 and 57, further comprising the step of placing the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells having secondary hemogenic endothelial potential, and / or secondary hemogenic endothelium under a hypoxic tension of between about 2% and about 10%. (Item 59) 59. The method of item 58, wherein the hematopoietic multipotent progenitors derived from pluripotent stem cells lack embryoid body development. (Item 60) 59. The method according to item 58, wherein the hematopoietic multipotent progenitors derived from pluripotent stem cells are in monolayer culture. (Item 61) 59. The method of item 58, wherein the hematopoietic multipotent progenitors derived from pluripotent stem cells are under feeder-free conditions. (Item 62) 59. The method of item 58, wherein the hematopoietic multipotent progenitors derived from pluripotent stem cells are under stroma-free conditions. (Item 63) Item 59. The method of item 58, wherein the pluripotent stem cells are iPSCs. (Item 64) Item 64. The method of item 63, wherein the iPSCs are naive iPSCs. (Item 65) 65. A method for promoting hematopoietic self-renewal, reconstitution, or engraftment using a composition comprising one or more of the cell populations, cell lines, or clonal cells generated using the method described in items 1 to 64, wherein the cell populations, cell lines, or clonal cells are: (i) CD34+ secondary hemogenic endothelium derived from pluripotent stem cells (iCD34 HE), wherein the iCD34 cells have the ability to differentiate into multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, and NK cells, and the iCD34 cells are CD34+ CD43-; (ii) secondary hemogenic endothelium (iHE) derived from pluripotent stem cells, wherein the iHE cell line or cloned cells are CD34+; (iii) secondary HSCs derived from pluripotent stem cells, wherein the iHSCs are CD34+ CD45+; (iv) multipotent progenitor cells derived from pluripotent stem cells, wherein the iMPP cells are CD34+ CD45+; (v) T cell precursors derived from pluripotent stem cells, which are CD34+ CD7+ T cell precursors; (vi) T cells derived from pluripotent stem cells, which are CD4+ or CD8+ T cells; (vii) NK cell precursors derived from pluripotent stem cells, which are CD56+ CD7+ CD161+ NK cell precursors; and (viii) NK cells derived from pluripotent stem cells, which are CD56+ CD57+ CD16+ CD94- NK cells Selected from: method. (Item 66) A culture platform for obtaining hematopoietic cells derived from pluripotent stem cells, comprising: I: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary HSCs from pluripotent stem cell-derived secondary hemogenic endothelium, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and optionally excluding a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, comprising a GSK3 inhibitor and a BMP activator; OR II: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having secondary hemogenic endothelium potential, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, and optionally not comprising a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for obtaining secondary hemogenic endothelial potential in pluripotent stem cell-derived mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from the pluripotent stem cells, the culture medium comprising a BMP activator and, optionally, bFGF. A culture platform comprising: (Item 67) Group II is (iv) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not comprising a TGFβ receptor / ALK inhibitor, and further comprising a culture medium suitable for seeding and expanding pluripotent stem cells; Item 67. The culture platform according to item 66. (Item 68) I: (i) a culture medium suitable for differentiating pluripotent stem cell-derived T cell precursors into pluripotent stem cell-derived T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; and no BMP activators; or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary HSCs into pluripotent stem cell-derived T cell precursors, comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; and one or more Notch pathway activators. further comprising whether the culture medium is suitable for generating T lineage cells derived from pluripotent stem cells; Or II: (i) a culture medium suitable for differentiating pluripotent stem cell-derived pre-T cell precursors into pluripotent stem cell-derived T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and excluding one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pluripotent stem cell-derived pre-T cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7. further comprising The culture medium is suitable for generating T-lineage cells derived from pluripotent stem cells. Item 67. The culture platform according to item 66. (Item 69) I: (i) a culture medium suitable for differentiating pluripotent stem cell-derived NK cell precursors into pluripotent stem cell-derived NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15, and not comprising BMP activators; or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary HSCs into pluripotent stem cell-derived NK cell precursors, the culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. further comprising whether the culture medium is suitable for generating NK lineage cells derived from pluripotent stem cells; Or II: (i) a medium suitable for differentiating pluripotent stem cell-derived pre-NK cell precursors into pluripotent stem cell-derived NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and excluding one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; or (ii) a medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells into pre-NK cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15. further comprising The culture medium is suitable for generating NK lineage cells derived from pluripotent stem cells. Item 67. The culture platform according to item 66. (Item 70) Group (II) is (i) a culture medium suitable for differentiating pluripotent stem cell-derived pre-HSCs into pluripotent stem cell-derived multipotent progenitors, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and not comprising a ROCK inhibitor; or (ii) a culture medium suitable for differentiating secondary hemogenic endothelium derived from pluripotent stem cells into pre-HSCs derived from pluripotent stem cells, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11. further including; 67. The culture platform of item 66, wherein the culture medium is suitable for generating hematopoietic multipotent progenitors derived from pluripotent stem cells. (Item 71) Item 67. The culture platform according to Item 66, wherein the pluripotent stem cells are iPSCs. (Item 72) Item 72. The culture platform of item 71, wherein the iPSCs are naive iPSCs. (Item 73) 1. A composition for obtaining and expanding hematopoietic cells derived from pluripotent stem cells, comprising: I: (i) a culture medium suitable for obtaining secondary HSCs derived from pluripotent stem cells from secondary hemogenic endothelium, comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and secondary hemogenic endothelium derived from pluripotent stem cells, optionally without a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for obtaining pluripotent stem cell-derived secondary hematopoietic endothelium from said pluripotent stem cell-derived mesodermal cells, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; and pluripotent stem cell-derived mesodermal cells; and (iii) a culture medium suitable for obtaining pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, the culture medium comprising a GSK3 inhibitor, a BMP activator, and iPSCs; OR II: (i) a culture medium suitable for obtaining pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells with secondary hemogenic endothelium potential, said culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; optionally, not comprising a TGFβ receptor / ALK inhibitor; and not comprising pluripotent stem cell-derived mesodermal cells with secondary hemogenic endothelium potential; (ii) a culture medium suitable for obtaining pluripotent stem cell-derived mesodermal cells having secondary hematopoietic endothelial potential from pluripotent stem cell-derived mesodermal cells, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not comprising a TGFβ receptor / ALK inhibitor; and pluripotent stem cell-derived mesodermal cells; and (iii) a culture medium suitable for obtaining pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, comprising a BMP activator and, optionally, bFGF; and pluripotent stem cells. A composition comprising one or more of: (Item 74) Group (II) is (vi) A culture medium suitable for seeding and expanding pluripotent stem cells, comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not comprising a TGFβ receptor / ALK inhibitor; and pluripotent stem cells. 74. The composition of claim 73, further comprising: (Item 75) I: (i) a culture medium suitable for differentiating pluripotent stem cell-derived T cell precursors into pluripotent stem cell-derived T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; and pluripotent stem cell-derived T cell precursors; and not comprising BMP activators; or (ii) a culture medium suitable for differentiating the pluripotent stem cell-derived secondary HSCs into pluripotent stem cell-derived T cell precursors and for generating pluripotent stem cell-derived T lineage cells, the culture medium comprising: a BMP activator; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6; one or more Notch pathway activators; and secondary HSCs derived from pluripotent stem cells. further comprising Or II: (i) a culture medium suitable for differentiating pluripotent stem cell-derived pre-T cell precursors into pluripotent stem cell-derived T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and excluding one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; and excluding pluripotent stem cell-derived pre-T cell precursors, or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pluripotent stem cell-derived pre-T cell precursors, the culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7; and pluripotent stem cell-derived secondary hemogenic endothelium. further comprising The culture medium is suitable for generating T-lineage cells derived from pluripotent stem cells. Item 74. The composition according to item 73. (Item 76) I: (i) a culture medium suitable for differentiating pluripotent stem cell-derived NK cell precursors into pluripotent stem cell-derived NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15; and pluripotent stem cell-derived NK cell precursors, wherein the culture medium does not contain BMP activators; or (ii) a culture medium suitable for differentiating the secondary HSCs derived from pluripotent stem cells into NK cell precursors derived from pluripotent stem cells, the culture medium comprising: a BMP activator; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and secondary HSCs derived from pluripotent stem cells. further comprising whether the culture medium is suitable for generating NK lineage cells derived from pluripotent stem cells; Or II: (i) a medium suitable for differentiating pluripotent stem cell-derived pre-NK cell precursors into pluripotent stem cell-derived NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and excluding one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; and pluripotent stem cell-derived pre-NK cell precursors; or (ii) a medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pre-NK cell precursors, the medium comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15, and secondary hemogenic endothelium derived from pluripotent stem cells. further comprising The culture medium is suitable for generating NK lineage cells derived from pluripotent stem cells. Item 74. The composition according to item 73. (Item 77) Group (II) further comprises one or more media for generating hematopoietic multipotent progenitors derived from pluripotent stem cells, the media comprising: (i) a culture medium suitable for differentiating pre-HSCs derived from pluripotent stem cells into multipotent progenitors derived from pluripotent stem cells, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and excluding a ROCK inhibitor; and pre-HSCs derived from pluripotent stem cells; and / or (ii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pluripotent stem cell-derived pre-HSCs, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11; and pluripotent stem cell-derived secondary hemogenic endothelium. 74. The composition according to item 73, comprising: (Item 78) Item 74. The culture platform of item 73, wherein the pluripotent stem cells are iPSCs. (Item 79) Item 79. The culture platform of item 78, wherein the iPSCs are naive iPSCs. (Item 80) 1. A culture platform for generating T lineage cells derived from pluripotent stem cells, comprising: I: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, the culture medium comprising a GSK3 inhibitor and a BMP activator; (ii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary HSCs from pluripotent stem cell-derived secondary hemogenic endothelium, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and optionally excluding a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; and (iv) a culture medium suitable for differentiating pluripotent stem cell-derived T cell precursors from pluripotent stem cell-derived secondary HSCs, comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators; and, optionally, (iii) a culture medium suitable for differentiating pluripotent stem cell-derived T cells from pluripotent stem cell-derived T cell precursors, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6; one or more Notch pathway activators; and no BMP activators; OR II: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, the culture medium comprising a BMP activator and, optionally, bFGF; (ii) a culture medium suitable for obtaining secondary HE potential in mesodermal cells derived from said pluripotent stem cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary hemogenic endothelium from said mesodermal cells with pluripotent stem cell-derived hemogenic endothelial potential, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, and optionally not comprising a TGFβ receptor / ALK inhibitor; (iv) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pluripotent stem cell-derived pre-T cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7; and (v) A platform comprising a culture medium suitable for differentiating pluripotent stem cell-derived pre-T cell precursors into pluripotent stem cell-derived T cell precursors or T cells, the culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and excluding one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. (Item 81) Group (II) is (vi) A culture medium suitable for seeding and expanding pluripotent stem cells, comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and optionally, not comprising a TGFβ receptor / ALK inhibitor. 81. The culture platform of item 80, further comprising: (Item 82) Item 81. The culture platform of item 80, wherein the pluripotent stem cells are iPSCs. (Item 83) Item 83. The culture platform of item 82, wherein the iPSCs are naive iPSCs. (Item 84) A culture platform for generating NK cells derived from pluripotent stem cells, comprising: I: (i) a culture medium suitable for differentiating pluripotent stem cells into pluripotent stem cell-derived mesodermal cells, comprising a GSK3 inhibitor and a BMP activator; (ii) a culture medium suitable for differentiating pluripotent stem cell-derived mesodermal cells into pluripotent stem cell-derived secondary hematopoietic endothelium, comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pluripotent stem cell-derived secondary HSCs, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and optionally, not comprising a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; and (iv) a culture medium suitable for differentiating pluripotent stem cell-derived secondary HSCs into pluripotent stem cell-derived NK cell precursors, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and, optionally, (iv) a culture medium suitable for differentiating pluripotent stem cell-derived NK cell precursors into pluripotent stem cell-derived NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15, and not comprising BMP activators; OR II: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, the culture medium comprising a BMP activator and, optionally, bFGF; (ii) a culture medium suitable for obtaining secondary hematopoietic endothelial potential in mesodermal cells derived from said pluripotent stem cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having secondary hemogenic endothelium potential, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, and optionally not comprising a TGFβ receptor / ALK inhibitor; (iv) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pre-NK cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15; and (v) a culture medium suitable for differentiating pluripotent stem cell-derived pre-NK cell precursors into pluripotent stem cell-derived NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and excluding one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. Platforms including. (Item 85) Group (II) is (vi) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not comprising a TGFβ receptor / ALK inhibitor, said culture medium further comprising a culture medium suitable for seeding and expanding pluripotent stem cells; 85. The culture platform of item 84. (Item 86) Item 85. The culture platform of item 84, wherein the pluripotent stem cells are iPSCs. (Item 87) Item 88. The culture platform according to Item 86, wherein the iPSCs are naive iPSCs. A culture platform for generating secondary hemogenic endothelium from pluripotent stem cells, comprising: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, the culture medium comprising a BMP activator and, optionally, bFGF; (ii) a culture medium suitable for obtaining secondary hemogenic endothelial potential in mesodermal cells derived from said pluripotent stem cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor; and (iii) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium derived from pluripotent stem cells from said mesodermal cells derived from pluripotent stem cells having the potential for secondary hemogenic endothelium, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and optionally, not comprising a TGFβ receptor / ALK inhibitor. A culture platform comprising: (Item 89) (iv) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not comprising a TGFβ receptor / ALK inhibitor, and further comprising a culture medium suitable for seeding and expanding pluripotent stem cells; Item 89. The culture platform according to item 88. (Item 90) Item 89. The culture platform of item 88, wherein the pluripotent stem cells are iPSCs. (Item 91) Item 91. The culture platform of item 90, wherein the iPSCs are naive iPSCs. (Item 92) 1. A culture platform for generating hematopoietic multipotent progenitors from multipotent stem cells, comprising: (i) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells, the culture medium comprising a BMP activator and, optionally, bFGF; (ii) a culture medium suitable for obtaining secondary hematopoietic endothelial potential in mesodermal cells derived from said pluripotent stem cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor; (iii) a culture medium suitable for differentiating and expanding pluripotent stem cell-derived secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having secondary hemogenic endothelium potential, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; and optionally, not comprising a TGFβ receptor / ALK inhibitor; (iv) a culture medium suitable for differentiating pluripotent stem cell-derived secondary hemogenic endothelium into pluripotent stem cell-derived pre-HSCs, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11; and (v) a culture medium suitable for differentiating pluripotent stem cell-derived pre-HSCs into pluripotent stem cell-derived multipotent progenitors, comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and not containing a ROCK inhibitor. A culture platform comprising: (Item 93) (vi) A culture medium suitable for seeding and expanding pluripotent stem cells, comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not containing a TGFβ receptor / ALK inhibitor. Item 93. The culture platform of item 92, further comprising: (Item 94) Item 93. The culture platform of item 92, wherein the pluripotent stem cells are iPSCs. (Item 95) Item 95. The culture platform of item 94, wherein the iPSCs are naive iPSCs. (Item 96) (a) optionally, a culture medium that does not contain a TGFβ receptor / ALK inhibitor; and (b) one or more cell populations generated from the culture platform described in items 4 to 7: (i) CD34+ secondary hemogenic endothelium derived from pluripotent stem cells (iCD34 HE), wherein the iCD34 cells have the ability to differentiate into multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, and NK cells, and are CD34+ CD43-; (ii) secondary hemogenic endothelium (iHE) derived from pluripotent stem cells, wherein the iHE cells are CD34+; (iii) secondary HSCs derived from pluripotent stem cells, wherein the iHSCs are CD34+CD45+; (iv) multipotent progenitor cells derived from pluripotent stem cells, wherein the iMPP cells are CD34+ CD45+; (v) T cell precursors derived from pluripotent stem cells, which are CD34+ CD7+ T cell precursors; (vi) T cells derived from pluripotent stem cells, which are CD4+ or CD8+ T cells; (vii) NK cell precursors derived from pluripotent stem cells, which are CD56+ CD7+ CD161+ NK cell precursors; and (viii) NK cells derived from pluripotent stem cells, which are CD56+ CD57+ CD16+ CD94- NK cells A composition comprising: (Item 97) (i) CD34+ HE cells (iCD34) and one or more culture media selected from iCD34-C, iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-A1, iNK-A2, iNK-B1, and iNK-B2; (ii) secondary hemogenic endothelium (iHE) and one or more culture media selected from iCD34-C, iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-A1, iNK-A2, iNK-B1, and iNK-B2; (iii) secondary HSCs and one or more culture media selected from iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-A1, iNK-A2, iNK-B1, and iNK-B2; (iv) multipotent progenitor cells (iMPPs) and iMPP-A; (v) T cell precursors (iproT) and one or more culture media selected from iTC-A1, iTC-A2, iTC-B1, and iTC-B2; (vi) T cells (iTC), and iTC-B1 or iTC-B2; (vii) a culture medium for NK cell precursors (iproNK), and one or more selected from iNK-A1, iNK-A2, iNK-B1, and iNK-B2; and (viii) NK cells (iNK) and iNK-B1 or iNK-B2 (ix) HSCs (iHSCs), as well as iHSC-A, iHSC-B, and iHSC-C A composition comprising hematopoietic cells derived from pluripotent stem cells and one or more culture media selected from the group consisting of: iHSC-A contains Wnt pathway activators and BMP activators; iHSC-B comprises a Wnt pathway activator, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor; the iHSC-Cs comprise a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; iTC-A1 comprises a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators selected from the group consisting of Jag1, Jag2, DLL-1, DLL-3, and DLL-4; iTC-B1 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators selected from the group consisting of Jag1, Jag2, DLL-1, DLL-3, and DLL-4; (iHSC platform); iNK-A1 comprises a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; iNK-B1 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, IL7, IL2, IL3, IL6, and IL15; iCD34-C comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11; iTC-A2 comprises a BMP activator, a ROCK inhibitor, VEGF, and bFGF; and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; iNK-A2 comprises one or more growth factors and cytokines selected from the group consisting of a BMP activator, a ROCK inhibitor, VEGF, and bFGF, and SCF, Flt3L, IL7, and IL15; iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15; composition. (Item 98) 98. The composition of item 97, wherein iHSC-C does not contain a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; iTC-A1 does not contain VEGF and / or IL15; iCD34-C does not contain a TGFβ receptor / ALK inhibitor; iTC-B1 does not contain a BMP activator; and / or iTC-B2 does not contain one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. [Brief explanation of the drawings]

[0055] [Figure 1] FIG. 1 shows an exemplary schematic diagram of the multi-step process for hematopoietic differentiation of human induced pluripotent stem cells (hiPSCs) into fully differentiated T cells.

[0056] [Figure 2] FIG. 2 shows an exemplary schematic diagram of the multi-step process for hematopoietic differentiation of hiPSCs into fully differentiated NK cells.

[0057] [Figure 3ABC] 3A-3E show morphological changes over the course of 9 days that support the transition from hiPSCs to hematopoietic cells. [Figure 3DE] 3A-3E show morphological changes over the course of 9 days that support the transition from hiPSCs to hematopoietic cells.

[0058] [Figure 4AB] 4A-4D show the expression profile of stem cells as they leave pluripotency and fully transition to a hematopoietic fate. [Figure 4CD] 4A-4D show the expression profile of stem cells as they leave pluripotency and fully transition to a hematopoietic fate.

[0059] [Figure 5AB] 5A-5C show the CD34 / CD45 expression profiles of hematopoietic cells differentiated from hiPSCs generated by various methods and starting inputs. [Figure 5C] 5A-5C show the CD34 / CD45 expression profiles of hematopoietic cells differentiated from hiPSCs generated by various methods and starting inputs.

[0060] [Figure 6A] 6A-6C show improved CD34 differentiation efficiency when intercalated as a monolayer in the differentiation culture medium described. [Figure 6B] 6A-6C show improved CD34 differentiation efficiency when intercalated as a monolayer in the differentiation culture medium described. [Figure 6C]6A-6C show improved CD34 differentiation efficiency when intercalated as a monolayer in the differentiation culture medium described.

[0061] [Figure 7] FIG. 7 shows the survival and proliferation of hematopoietic cells differentiated under ROCK inhibition after single cell dissociation.

[0062] [Figure 8] FIG. 8 shows maintenance of CD34 expression by hematopoietic cells differentiated from hiPSCs after 22 days of culture.

[0063] [Figure 9A] 9A-9B show in vivo reconstitution of engrafted CD34+ cells, as seen by the presence of cells exclusively expressing the human CD45 marker and the reconstitution of CD34+ cells with the presence of both T and B cells. The CD34+ cells were derived from naive hiPSCs cultured in a monolayer format with a GSK3 inhibitor, without EBs or aggregation intermediates. [Figure 9B] 9A-9B show in vivo reconstitution of engrafted CD34+ cells, as seen by the presence of cells exclusively expressing the human CD45 marker and the reconstitution of CD34+ cells with the presence of both T and B cells. The CD34+ cells were derived from naive hiPSCs cultured in a monolayer format with a GSK3 inhibitor, without EBs or aggregation intermediates.

[0064] [Figure 10] Figure 10 shows that CD56-positive cells and single CD4- and CD8-positive cells, derived by a CD56- / CD7+ / CD3+ / TCRαβ+ gating strategy, were identified when hiPSC-derived CD34-positive cells were further cultured in differentiation medium in the absence of stromal cells and in the presence of soluble DLL1 and DLL4 recombinant peptides (T cell-specific).

[0065] [Figure 11] FIG. 11 shows that hiPSC-derived CD34-positive cells can respond to pharmacological modulation, as seen by enhanced expression of PD-L1 surface expression.

[0066] [Figure 12] Figure 12 shows a schematic diagram of the multi-step process for hematopoietic differentiation of induced pluripotent stem cells (iPSCs) into de novo hemogenic endothelium (iHE) and multipotent progenitors (iMPPs). Note that cultures can be converted to fully defined cultures when Matrigel™ is substituted for Vitronectin.

[0067] [Figure 13] Figure 13 shows a schematic diagram of the multi-step process for hematopoietic differentiation of induced pluripotent stem cells into T cell precursors (iproT) and fully differentiated T (iT) cells. Note that the culture can be converted to fully defined by replacing Vitronecti with Matrigel™.

[0068] [Figure 14] Figure 14 shows a schematic diagram of the multi-step process for hematopoietic differentiation of induced pluripotent stem cells into NK cell precursors (iproNK) and fully differentiated NK (iNK) cells. Note that the culture can be completely converted to a fully defined culture medium when Vitronectin is replaced with Matrigel™.

[0069] [Figure 15A] 15A-C show flow cytometry profiles showing the emergence of iHE over the course of 10 days, and the output of iCD34 and iHE cells per iPSC differentiation. Calculations are based on snapshots of representative and non-optimized cultures. [Figure 15B]15A-C show flow cytometry profiles showing the emergence of iHE over the course of 10 days, and the output of iCD34 and iHE cells per iPSC differentiation. Calculations are based on snapshots of representative and non-optimized cultures. [Figure 15C] 15A-C show flow cytometry profiles showing the emergence of iHE over the course of 10 days, and the output of iCD34 and iHE cells per iPSC differentiation. Calculations are based on snapshots of representative and non-optimized cultures.

[0070] [Figure 16A] 16A-E show modifications to the protocol, including plating density and growth factor titration, that improve HE output at day 10. A) Plating density on day 0 affects the HE population at day 10. B) Concentration of BMP4 on days 2-6 affects the HE population at day 10. C) Concentration of CHIR on day 3.75 affects the HE population at day 10. D) Plating density on day 6 affects the HE population at day 10. E) Addition of IGF1 and EPO on day 8 reduces HE at day 10. [Figure 16B]16A-E show modifications to the protocol, including plating density and growth factor titration, that improve HE output at day 10. A) Plating density on day 0 affects the HE population at day 10. B) Concentration of BMP4 on days 2-6 affects the HE population at day 10. C) Concentration of CHIR on day 3.75 affects the HE population at day 10. D) Plating density on day 6 affects the HE population at day 10. E) Addition of IGF1 and EPO on day 8 reduces HE at day 10. [Figure 16C] 16A-E show modifications to the protocol, including plating density and growth factor titration, that improve HE output at day 10. A) Plating density on day 0 affects the HE population at day 10. B) Concentration of BMP4 on days 2-6 affects the HE population at day 10. C) Concentration of CHIR on day 3.75 affects the HE population at day 10. D) Plating density on day 6 affects the HE population at day 10. E) Addition of IGF1 and EPO on day 8 reduces HE at day 10. [Figure 16D]16A-E show modifications to the protocol, including plating density and growth factor titration, that improve HE output at day 10. A) Plating density on day 0 affects the HE population at day 10. B) Concentration of BMP4 on days 2-6 affects the HE population at day 10. C) Concentration of CHIR on day 3.75 affects the HE population at day 10. D) Plating density on day 6 affects the HE population at day 10. E) Addition of IGF1 and EPO on day 8 reduces HE at day 10. [Figure 16E] 16A-E show modifications to the protocol, including plating density and growth factor titration, that improve HE output at day 10. A) Plating density on day 0 affects the HE population at day 10. B) Concentration of BMP4 on days 2-6 affects the HE population at day 10. C) Concentration of CHIR on day 3.75 affects the HE population at day 10. D) Plating density on day 6 affects the HE population at day 10. E) Addition of IGF1 and EPO on day 8 reduces HE at day 10.

[0071] [Figure 17A] 17A-B show that day 10 HE represents definitive hematopoiesis that is multipotent and dependent on the Notch signaling pathway. A) Changes in morphology over a 7-day MPP assay are shown along with flow cytometry profiles of emerging CD45 hematopoietic cells. B) iPSC-derived CD34+ cells generate Notch-dependent secondary CD45+ cells in an iMPP assay. [Figure 17B]17A-B show that day 10 HE represents definitive hematopoiesis that is multipotent and dependent on the Notch signaling pathway. A) Changes in morphology over a 7-day MPP assay are shown along with flow cytometry profiles of emerging CD45 hematopoietic cells. B) iPSC-derived CD34+ cells generate Notch-dependent secondary CD45+ cells in an iMPP assay.

[0072] [Figure 18A] 18A-B show the effect of differentiation under hypoxic conditions on the generation of iHE and iMPP hematopoietic progenitors. A) Monolayer differentiation under hypoxic conditions increases the percentage of both iCD34-positive cells and iHE cells at day 10. B) Day 10 iCD34+ HE cells generated under hypoxic conditions can be further differentiated in an iMPP assay. [Figure 18B] 18A-B show the effect of differentiation under hypoxic conditions on the generation of iHE and iMPP hematopoietic progenitors. A) Monolayer differentiation under hypoxic conditions increases the percentage of both iCD34-positive cells and iHE cells at day 10. B) Day 10 iCD34+ HE cells generated under hypoxic conditions can be further differentiated in an iMPP assay.

[0073] [Figure 19A] Figures 19A-D show the ability of unsorted or sorted day 10 cultures to be cryopreserved and maintain hematopoietic potential. A) Cryopreserved day 10 unsorted differentiation cultures survive and can generate CD45+ hematopoietic cells in an iMPP assay. B), C), and D) Cryopreserved day 10 iCD34+ sorted cells survive and can generate CD45+ hematopoietic cells in an iMPP assay. [Figure 19B]Figures 19A-D show the ability of unsorted or sorted day 10 cultures to be cryopreserved and maintain hematopoietic potential. A) Cryopreserved day 10 unsorted differentiation cultures survive and can generate CD45+ hematopoietic cells in an iMPP assay. B), C), and D) Cryopreserved day 10 iCD34+ sorted cells survive and can generate CD45+ hematopoietic cells in an iMPP assay. [Figure 19C] Figures 19A-D show the ability of unsorted or sorted day 10 cultures to be cryopreserved and maintain hematopoietic potential. A) Cryopreserved day 10 unsorted differentiation cultures survive and can generate CD45+ hematopoietic cells in an iMPP assay. B), C), and D) Cryopreserved day 10 iCD34+ sorted cells survive and can generate CD45+ hematopoietic cells in an iMPP assay. [Figure 19D] Figures 19A-D show the ability of unsorted or sorted day 10 cultures to be cryopreserved and maintain hematopoietic potential. A) Cryopreserved day 10 unsorted differentiation cultures survive and can generate CD45+ hematopoietic cells in an iMPP assay. B), C), and D) Cryopreserved day 10 iCD34+ sorted cells survive and can generate CD45+ hematopoietic cells in an iMPP assay.

[0074] [Figure 20A]Figures 20A-B show that day 10 differentiation cultures can be shipped overnight at ambient temperature without losing HE potential. A) Cultures at day 7 were either maintained in the incubator (control) or processed for overnight shipping and then reintroduced into the incubator for an additional two days. Both cultures at day 10 were then analyzed for the presence of iCD34 and iHE cells. During overnight shipped cultures, T-flasks contained 30% culture medium with 70% basal medium or 100% culture medium. B) Calculation of cell number. [Figure 20B] Figures 20A-B show that day 10 differentiation cultures can be shipped overnight at ambient temperature without losing HE potential. A) Cultures at day 7 were either maintained in the incubator (control) or processed for overnight shipping and then reintroduced into the incubator for an additional two days. Both cultures at day 10 were then analyzed for the presence of iCD34 and iHE cells. During overnight shipped cultures, T-flasks contained 30% culture medium with 70% basal medium or 100% culture medium. B) Calculation of cell number.

[0075] [Figure 21AB] Figures 21A-C show early CD34+ CD7+ T cell precursors and mature CD4+ and CD8+ T cell subsets derived from hiPSCs using a CD45+ CD56-gating strategy. A) Early T lineage cell markers mark the presence of iproT cells defined by CD34+ / CD7+. B) Mature T cell markers mark the presence of mature T cells defined by CD4+ or CD8+ cells. C) Comparison of the iproT cell generating potential of cord blood-derived CD34+ cells and iCD34+ cells on day 5 of T cell differentiation. [Figure 21C]Figures 21A-C show early CD34+ CD7+ T cell precursors and mature CD4+ and CD8+ T cell subsets derived from hiPSCs using a CD45+ CD56-gating strategy. A) Early T lineage cell markers mark the presence of iproT cells defined by CD34+ / CD7+. B) Mature T cell markers mark the presence of mature T cells defined by CD4+ or CD8+ cells. C) Comparison of the iproT cell generating potential of cord blood-derived CD34+ cells and iCD34+ cells on day 5 of T cell differentiation.

[0076] [Figure 22A] Figures 22A-C show early CD56+ CD7+ CD161+ NK cell precursors and mature CD56+ CD16+ CD8+ NK cell subsets derived from hiPSCs using a CD45+ gating strategy. A) Early NK lineage cell markers mark the presence of iproNK cells, defined by CD7 and CD56. B) Mature NK lineage cell markers mark the presence of mature NK cells, defined by CD57, CD16, CD94, and CD56. C) Comparison of the iproNK cell generating potential of CD34+ cells derived from cord blood and iCD34+ cells on day 5 of NK cell differentiation. [Figure 22B]Figures 22A-C show early CD56+ CD7+ CD161+ NK cell precursors and mature CD56+ CD16+ CD8+ NK cell subsets derived from hiPSCs using a CD45+ gating strategy. A) Early NK lineage cell markers mark the presence of iproNK cells, defined by CD7 and CD56. B) Mature NK lineage cell markers mark the presence of mature NK cells, defined by CD57, CD16, CD94, and CD56. C) Comparison of the iproNK cell generating potential of CD34+ cells derived from cord blood and iCD34+ cells on day 5 of NK cell differentiation. [Figure 22C] Figures 22A-C show early CD56+ CD7+ CD161+ NK cell precursors and mature CD56+ CD16+ CD8+ NK cell subsets derived from hiPSCs using a CD45+ gating strategy. A) Early NK lineage cell markers mark the presence of iproNK cells, defined by CD7 and CD56. B) Mature NK lineage cell markers mark the presence of mature NK cells, defined by CD57, CD16, CD94, and CD56. C) Comparison of the iproNK cell generating potential of CD34+ cells derived from cord blood and iCD34+ cells on day 5 of NK cell differentiation.

[0077] [Figure 23A] Figures 23A-C show that the monolayer hiPSC hematopoietic differentiation platform enables a scalable expansion strategy not seen during EB formation. A. hiPSCs were aggregated to form embryoid bodies and differentiated for 14 days before being analyzed for CD34 and CD43 expression. B. hiPSCs were plated as a monolayer and differentiated for 8 days before being analyzed for CD34, CD43, CXCR4, and CD73. C. D34-positive cells were counted and plotted over time for both monolayer-mediated and EB-mediated hematopoietic differentiation. [Figure 23B] Figures 23A-C show that the monolayer hiPSC hematopoietic differentiation platform enables a scalable expansion strategy not seen during EB formation. A. hiPSCs were aggregated to form embryoid bodies and differentiated for 14 days before being analyzed for CD34 and CD43 expression. B. hiPSCs were plated as a monolayer and differentiated for 8 days before being analyzed for CD34, CD43, CXCR4, and CD73. C. D34-positive cells were counted and plotted over time for both monolayer-mediated and EB-mediated hematopoietic differentiation. [Figure 23C] Figures 23A-C show that the monolayer hiPSC hematopoietic differentiation platform enables a scalable expansion strategy not seen during EB formation. A. hiPSCs were aggregated to form embryoid bodies and differentiated for 14 days before being analyzed for CD34 and CD43 expression. B. hiPSCs were plated as a monolayer and differentiated for 8 days before being analyzed for CD34, CD43, CXCR4, and CD73. C. D34-positive cells were counted and plotted over time for both monolayer-mediated and EB-mediated hematopoietic differentiation.

[0078] [Figure 24] Figure 24 shows a schematic diagram of a scalable expansion strategy for off-the-shelf iNK and iT cell production using a monolayer hiPSC hematopoietic differentiation platform. Calculations are based on snapshots of representative and unoptimized cultures.

[0079] [Figure 25] FIG. 25 shows that hiPSC-derived CD34-positive cells have immunoregulatory properties by suppressing CD3+ T cell survival.

[0080] [Figure 26]FIG. 26 shows mature CD4+ and CD8+ T cell subsets derived from hiPSCs using a CD45+ CD56- gating strategy.

[0081] [Figure 27] FIG. 27 shows that feeder-based suspension culture supports the maturation of iCD34-derived NK cells.

[0082] [Figure 28] FIG. 28 shows that iCD34-derived iNKs can secrete pro-inflammatory cytokines in response to cytokine stimulation in a manner similar to peripheral blood NK cells.

[0083] [Figure 29] FIG. 29 shows that stroma-free differentiation of pro-NK cells derived from cord blood CD34-positive cells using a CD45+ gating strategy is more rapid than traditional stroma-based differentiation platforms.

[0084] [Figure 30] Figure 30 shows stroma-free differentiation of iPSC-derived iCD34+ cells into NK cells. Plate-bound DLL4 supports differentiation of CD56+ CD7+ CD161+ NK cell precursors, but not CD11b+ myeloid cells.

[0085] [Figure 31] FIG. 31 shows stroma-free differentiation of UCB CD34+ cells into T cells.

[0086] [Figure 32] FIG. 32 shows stroma-free differentiation of iPSC-derived iCD34+ cells into T cells.

[0087] [Figure 33] FIG. 33 shows engraftment of hiPSC-derived iCD34+ cells. DETAILED DESCRIPTION OF THE INVENTION

[0088] (Detailed Description of the Invention) The present invention generally relates to methods and compositions for differentiating stem cells toward a secondary hematopoietic cell fate. More specifically, the present invention provides a multi-stage differentiation platform that can induce iPSCs or iPSC-derived cells at various stages of development to fully differentiate hematopoietic cells, exhibiting a range of secondary hematopoietic phenotypes, from secondary hemogenic endothelium, including T cells, B cells, NKT cells, and NK cells. That is, the present invention provides methods and compositions for making cells more likely to exhibit a secondary hematopoietic fate, e.g., CD34+ secondary hematopoietic stem cells. Alternatively, the methods and compositions of the present invention generate secondary hemogenic endothelium (HE) from naive iPSCs in a scalable manner by avoiding the formation of EBs or aggregates.

[0089] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined below. As used herein, the articles "a," "an," and "the" are used to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0090] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0091] The term "and / or" should be understood to mean either or both of the alternatives.

[0092] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by at most 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0093] As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a range of quantities, levels, values, numbers, frequency, percentages, dimensions, sizes, amounts, weights, or lengths that is approximately the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0094] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of the specified substance or source thereof, such as a composition that is 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable as measured by conventional means. The term "free of" or "essentially free of" a particular component or substance in a composition also means that such component or substance (1) is not present in the composition at any concentration, or (2) is present in the composition at a low, but functionally inactive, concentration. A similar meaning applies to the term "absent," which refers to the absence of a particular substance or source thereof in a composition.

[0095] As used herein, the term "detectable" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of an event that is readily detectable by one or more standard methods. The terms "imperceptible" and "not detectable" and their equivalents refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of an event that is not readily detectable or undetectable by standard methods. In one embodiment, an event is not detectable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or 0.001% of the time.

[0096] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include," "having," "containing," and "comprise" are used interchangeably.

[0097] "Consisting of" means inclusive of, and limited to, the phrases that follow the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that no other elements may be present.

[0098] "Consisting essentially of" means including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0099] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "a further embodiment," or "an additional embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with that embodiment is incorporated in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0100] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of a living organism, preferably an environment that minimally alters natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues removed from a living organism and cultured in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours or more, depending on the circumstances. In certain embodiments, such tissues or cells can be harvested, frozen, and then thawed for ex vivo processing. Tissue culture experiments or procedures that take longer than several days and that use living cells or tissues are typically considered "in vitro," although in certain embodiments, the term can be used interchangeably with ex vivo.

[0101] The term "in vivo" generally refers to activities performed within a living organism.

[0102] As used herein, the term "mesoderm" refers to one of the three germ layers that emerge during early embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, skin, skeleton, and other supportive and connective tissues.

[0103] As used herein, the terms "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived secondary hemogenic endothelium" (iHE) refer to a subset of endothelium that gives rise to hematopoietic stem cells and progenitor cells in a process called the endothelial-to-hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially from lateral plate mesoderm, through hemangioblasts, to secondary hemogenic endothelium and hematopoietic precursors.

[0104] As used herein, the term "hematopoietic stem cell" or "secondary hematopoietic stem cell" refers to a CD34+ stem cell that can give rise to both mature myeloid and lymphoid cell types, including T cells, natural killer cells, and B cells.

[0105] As used herein, the terms "reprogramming," or "dedifferentiation," or "increasing cell potential," or "increasing developmental potential" refer to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell that has increased cell potential has greater developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an unreprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in an unreprogrammed state.

[0106] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as a blood cell or muscle cell. A differentiated or differentiation-induced cell is a cell that occupies a more specialized ("committed") position within the lineage of a cell. The term "committed" as applied to the differentiation process refers to a cell that has progressed to a point in the differentiation pathway that, under normal circumstances, will continue to differentiate into a specific cell type or subset of cell types and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.

[0107] As used herein, the term "differentiation marker gene" or "differentiation gene" refers to a gene whose expression indicates cellular differentiation occurring in a cell, such as a pluripotent cell. The differentiation marker genes were the following: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, and CD1D. These include, but are not limited to, FOXG1, LEFTY1, TUJ1, T gene (Brachyury), ZIC1, GATA1, GATA2, HDAC4, HDAC5, HDAC7, HDAC9, NOTCH1, NOTCH2, NOTCH4, PAX5, RBPJ, RUNX1, STAT1, and STAT3.

[0108] As used herein, the term "gene profile of differentiation markers," or "differentiation gene profile," "differentiation gene expression profile," "differentiation gene expression signature," "differentiation gene expression panel," "differentiation gene panel," or "differentiation gene signature" refers to the expression or expression levels of multiple differentiation marker genes.

[0109] As used herein, the term "potency" refers to the sum total of all developmental options accessible to a cell (i.e., developmental potential). The continuum of cell potential includes, but is not limited to, totipotent, pluripotent, multipotent, oligopotent, unipotent, and terminally differentiated cells.

[0110] As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that are incapable of giving rise to an entire organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to an entire organism.

[0111] As used herein, the term "induced pluripotent stem cells" or iPSCs means stem cells generated from differentiated adult, neonatal, or fetal cells that have been induced or changed, i.e., reprogrammed, into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. Generated iPSCs do not refer to cells found in nature.

[0112] The term "embryonic stem cell" as used herein refers to naturally occurring pluripotent stem cells from the inner cell mass of blastocyst.Embryonic stem cells are pluripotent and generate all derivatives of the three main germ layers: ectoderm, endoderm, and mesoderm during development.Embryonic stem cells do not contribute to extraembryonic membranes or placenta, i.e., they are not totipotent.

[0113] As used herein, the term "multipotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Thus, a multipotent cell can also be referred to as a "partially differentiated cell." Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Multipotency" indicates that a cell can form many types of cells within a given lineage but cannot form cells of other lineages. For example, a multipotent hematopoietic cell can form many different types of blood cells (e.g., red blood cells, white blood cells, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to a state of a cell with a degree of developmental potential that is less than totipotency and pluripotency.

[0114] The differentiation of pluripotent stem cells requires changes in the culture system, such as the addition of stimuli in the culture medium or changes in the physical state of the cells. Most conventional strategies utilize the formation of embryoid bodies (EBs) as a general and crucial intermediate for inducing lineage-specific differentiation. EBs are three-dimensional clusters that have been shown to mimic embryonic development because they give rise to multiple lineages within their three-dimensional space. Throughout the differentiation process, typically over hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point they are typically further processed over days to weeks to continue their differentiation. EB formation is induced by bringing pluripotent stem cells into close proximity with each other in three-dimensional, multilayered clusters of cells, which is typically achieved by one of several methods, including sedimenting pluripotent cells in droplets, sedimenting cells into "U"-bottom well-plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require additional differentiation cues to promote EB development. Therefore, pluripotent stem cell aggregates require transfer to a differentiation medium that provides induction cues for select lineages. EB-based culture of pluripotent stem cells typically results in the development of differentiated cell populations (ectoderm, mesoderm, and endoderm) with little proliferation within the EB cell clusters. Although proven to facilitate cell differentiation, EBs generate heterogeneous cells due to inconsistent exposure of cells in the three-dimensional structure to differentiation cues from the environment and variable differentiation states. Additionally, EBs are cumbersome to create and maintain. Furthermore, the limited cell expansion associated with differentiation through EBs also contributes to low differentiation efficiency.

[0115] In contrast, "aggregate formation," which is significantly different from "EB formation," can be used to induce differentiation of pluripotent stem cells and / or expand populations of cells derived from pluripotent stem cells. For example, in aggregate-based pluripotent stem cell expansion, culture media are selected to maintain proliferation and pluripotency. Cell proliferation generally increases aggregate size, forming large aggregates, which are routinely mechanically or enzymatically dissociated into smaller aggregates to maintain cell growth and expand cell numbers in culture. In significant contrast to EB cultures, cells cultured within aggregates in maintenance cultures maintain markers of pluripotency.

[0116] As used herein, "monolayer differentiation" refers to a differentiation method that is significantly different from differentiation via three-dimensional multilayered clusters of cells, i.e., "formation of EBs." Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to induce differentiation. Because monolayer culture does not mimic embryonic development, such as EB formation, differentiation into specific lineages is minimal compared to differentiation into all three germ layers in EBs.

[0117] Pluripotency can be determined, in part, by assessing cells for pluripotent characteristics, including, but not limited to, (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50; (iv) the ability to differentiate into all three somatic lineage cells (ectoderm, mesoderm, and endoderm); (v) teratoma formation composed of cells from the three somatic lineages; and (vi) formation of embryoid bodies composed of cells derived from the three somatic lineages.

[0118] Two types of pluripotency have been described: a "primed" or "metastable" pluripotent state, analogous to the epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" pluripotent state, analogous to the inner cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells; (ii) improved clonability and survival during single-cell culture; (iii) global reduction in DNA methylation; (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmentally regulated gene promoters; and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard cell reprogramming methods in which an exogenous pluripotency gene is introduced into a somatic cell, expressed, and then silenced or removed from the resulting pluripotent cell are generally seen to have characteristics of a primed state of pluripotency. Under standard pluripotent cell culture conditions, characteristics of the ground state are observed; unless expression of the exogenous transgene is maintained, such cells remain in a primed state.

[0119] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological features of embryonic stem cells, which are characterized by a spherical shape, small size, a large nucleus-to-cytoplasm ratio, the notable presence of nucleoli, and typical intercellular spacing.

[0120] As used herein, "feeder cells" or "feeders" are terms used to describe cells of one type that are co-cultured with cells of a second type, providing an environment in which the second cell type can grow, since the feeder cells provide growth factors and nutrients to support the second cell type. Feeder cells are optionally derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. Feeder cells are typically inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemic cells. Without limiting the foregoing, one specific feeder cell type can be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, a variety of feeder cells can be used to partially maintain pluripotency, direct differentiation down certain lineages, and promote maturation into specialized cell types, such as effector cells.

[0121] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is essentially free of feeder cells or stromal cells and / or has not been previously conditioned by culturing feeder cells. A "pre-conditioned" medium refers to a medium that has been collected after culturing feeder cells in the medium for a period of time, such as at least one day. A pre-conditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells cultured in the medium.

[0122] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domestic animal.

[0123] "Pluripotency factors" or "reprogramming factors" refer to agents that, alone or in combination with other agents, can increase the developmental potential of a cell. Pluripotency factors include, without limitation, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0124] "Adherent to" refers to cells that adhere to a container, e.g., cells that adhere to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. Certain classes of cells will not survive or grow in culture unless they adhere to a cell culture container. Certain classes of cells ("non-adherent cells") will maintain and / or proliferate in culture without adhering.

[0125] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case, a single "medium"), "supplement," and "medium supplement" refer to nutritional compositions in which cell cultures are cultivated.

[0126] "Culturing" or "maintaining" refers to keeping cells alive, propagating (growing), and / or differentiating outside a tissue or body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" may utilize culture medium as a source of nutrients, hormones, and / or other factors that help the cells grow and / or survive.

[0127] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or surfaces (e.g., culture plate surfaces). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, in Cells that aggregate in vitro can be enzymatically or mechanically dissociated from each other, such as by dissociating into suspensions of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from culture plates or other surfaces. Thus, dissociation can involve disrupting the interaction of cells with the extracellular matrix (ECM) and substrate (e.g., culture surface), or disrupting the ECM between cells.

[0128] B. Overview The present invention generally relates to a multistep process for differentiating naive pluripotent cells into non-pluripotent or partially differentiated cells, including mesodermal cells, secondary hemogenic endothelium, secondary hematopoietic stem or progenitor cells, CD34+ cells, multipotent progenitors (MPPs) (capable of differentiating into myeloid cells, including neutrophil precursors), T cell precursors, NK cell precursors; or fully differentiated definitive hematopoietic cells, such as T cells, B cells, NKT cells, or NK cells. The present invention also relates to compositions used in the disclosed methods; and to cell populations, cell lines, or clonal cells generated using the disclosed methods.

[0129] In contrast to methods used in the art, the present invention avoids the formation of EBs during iPSC differentiation. As demonstrated, hematopoietic cells derived from iPSCs were obtained by seeding clonal iPSCs in TGFβ-free culture medium to maintain their pluripotent ground state or naive state, differentiating the clonal iPSCs in a monolayer format without EB formation, and utilizing a stepwise strategy to apply appropriate combinations of small molecule chemicals, growth factors, and cytokines at early and intermediate stages of differentiation. Thus, the present invention allows for the direct transfer of expanded clonal iPSCs into adherent culture in a monolayer format for immediate differentiation without requiring EB formation from iPSCs.

[0130] Thus, the present invention provides a culture platform that enables highly efficient differentiation of stem cells into definitive hematopoietic cells and functional hematopoietic cells without the use of TGFβ receptor / ALK inhibitors, including SB431532. Furthermore, unlike previous studies, the present invention also provides a culture platform that uses feeder-free, serum-free conditions to support the direct differentiation of iPSCs in monolayer culture without the need for iPSC-derived EBs or aggregation intermediates.

[0131] C. Culture Platform Existing methods for culturing pluripotent cells rely heavily on feeder cells or media preconditioned with feeder cells and containing fetal bovine serum, but such environments may not be suitable for producing cells for clinical and therapeutic use. For example, exposure to animal components may pose a significant risk of immune rejection and the transmission of unidentified pathogens to treated patients, potentially reactivating animal retroviruses, so cells cultured in such heterogeneous environments are generally considered unsuitable for human cell transplantation. A culture system using an animal-free culture medium, such as the feeder-free environment envisioned herein, facilitates the production of clinical-grade cell lines, particularly hESCs, hiPSCs, and pluripotent stem cell-derived HSCs, T cell lines, B cell lines, NKT cell lines, or NK cell lines.

[0132] In certain embodiments, the feeder-free environment is essentially free of human feeder cells and has not been previously conditioned with feeder cells, including, but not limited to, mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells. The feeder-free cell culture medium is suitable for use in culturing pluripotent cells, reprogramming cells, culturing single cells, dissociating, and subculturing pluripotent cells, sorting pluripotent cells, generating ground-state pluripotent cells, maintaining ground-state pluripotency, and inducing differentiation of pluripotent cells. In certain embodiments, the feeder-free environment is used to induce pluripotency, improve reprogramming efficiency, increase or maintain cell potency, and / or induce differentiation. In certain embodiments, the feeder-free environment is also substantially free of growth factors, including cytokines and bFGF.

[0133] In some aspects of the present invention, one or more of the steps of iPSC differentiation described above can be performed under feeder-free conditions. Such feeder-free conditions can be in the form of, but are not limited to, monolayer culture and suspension culture. In one embodiment of the present invention, the differentiation of pluripotent cells into mesodermal cells is performed under monolayer feeder-free conditions. In another embodiment of the present invention, the differentiation of mesodermal cells into secondary hemogenic endothelium is performed under monolayer feeder-free conditions. In yet another embodiment of the present invention, the differentiation of secondary hemogenic endothelium into hematopoietic stem cells is performed under monolayer feeder-free conditions. In one embodiment of the present invention, the differentiation of secondary hematopoietic stem cells into multipotent progenitors, T cell progenitors, or NK cell progenitors is performed under suspension feeder-free conditions or monolayer feeder-free conditions followed by suspension feeder-free conditions. In another embodiment of the present invention, the differentiation of T cell progenitors into fully differentiated T cells or NK cell progenitors into fully differentiated NK cells is performed under suspension feeder-free conditions or monolayer feeder-free conditions followed by suspension feeder-free conditions.

[0134] Any suitable vessel or cell culture container can be used as a support for cell culture in basal medium and / or cell culture supplements. However, in some embodiments, coating the surface of the culture vessel with an adhesion-promoting matrix / substrate (e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.) can promote cell attachment and, in certain embodiments, enhance the effectiveness of the cell culture medium and supplements disclosed herein. Suitable substrates for culturing and subculturing cells are known in the art and include, but are not limited to, mixtures of matrices produced by naturally occurring cell lines, such as vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombospondin, and Matrigel™, with synthetic or artificial surfaces, such as polyamine monolayers and carboxy-terminated monolayers. In some embodiments, providing feeder-free conditions involves culturing cells on a matrix-coated surface. In one embodiment, the culture platform contemplated herein comprises a matrix / substrate comprising Matrigel™ or vitronectin. Some embodiments of the cultures use Matrigel™, and therefore the cultures are completely compositionally defined.

[0135] In some embodiments of the present invention, one or more of the differentiation steps described above can be performed under serum-free conditions. Examples of commercially available serum-free media suitable for cell attachment and / or induction include mTeSR™1 or TeSR™2 from Stem Cell Technologies (Vancouver, Canada), Primate ES / iPS cell medium from ReproCELL (Boston, Massachusetts), StemPro™-34 from Invitrogen (Carlsbad, Calif.), StemPro™ hESC SFM from Invitrogen, and X-VIVO™ from Lonza (Basel, Switzerland).

[0136] In further embodiments, one or more of the culture platform media are feeder-free environments, optionally substantially free of cytokines and / or growth factors. In other embodiments, the cell culture media contain supplements such as serum, extracts, growth factors, hormones, and cytokines. Generally, the culture platform includes one or more stage-specific, feeder-free, serum-free media, each of which further includes one or more of the following: nutrients / extracts, growth factors, hormones, cytokines, and media additives. Suitable nutrients / extracts may include, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), a basal medium widely used to support the growth of many different mammalian cells; KOSR (knockout serum replacement); L-glutamine; and NEAA (non-essential amino acids). Other media additives may include, but are not limited to, MTG, ITS, βME, and antioxidants (e.g., ascorbic acid). In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, various interleukins (such as IL-1 through IL-18), various colony-stimulating factors (such as granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (such as IFN-γ), and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines are commercially available, for example, from R&D Systems (Minneapolis, Minn.), and may be natural or recombinant.In some other embodiments, the culture media of the present invention comprise one or more of one or more cytokines derived from bone morphogenetic protein 4 (BMP4), insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factors (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (Flt3L); and leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors / mitogens and cytokines are stage- and / or cell-type-specific, at concentrations determined empirically or as guided by established cytokine art.

[0137] Generally, techniques for differentiating induced pluripotent cells involve the direct or indirect modulation of specific cellular pathways using polynucleotide-, polypeptide-, and / or small molecule-based approaches. The developmental potential of a cell can be modulated, for example, by contacting the cell with one or more modulators. As used herein, "contacting" can involve culturing the cell in the presence of one or more factors (e.g., small molecules, proteins, peptides, etc.). In some embodiments, the cell is contacted with one or more agents to induce differentiation of the cell. Such contacting can be achieved, for example, by introducing one or more agents into the cell during in vitro culture. Thus, contacting can be achieved by introducing one or more agents into the cell in a nutrient cell culture medium. The cell can be maintained in the culture medium containing the one or more agents for a sufficient time for the cell to achieve the desired differentiation phenotype. In some other embodiments, "contacting" occurs when one or more factors are introduced into the cell via a vector. In some embodiments, the one or more vectors are introduced by retrovirus, Sendai virus, and adenovirus, episomes, minicircles, vector systems with expression cassettes, or mRNA.

[0138] In other embodiments, one or more of the stage-specific, feeder-free, serum-free media of the culture platforms disclosed herein further comprise one or more small molecules. In some embodiments, the culture platform comprises a cell culture medium comprising a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and does not consist of or include a small molecule inhibitor of the TGFβ / activin signaling pathway, including but not limited to an inhibitor of the TGFβ receptor or ALK5.

[0139] The culture platform contemplated herein also offers numerous advantages by utilizing a homogenous population of commercial-grade or clinical-grade pluripotent cells that have reduced spontaneous differentiation and / or achieved ground-state pluripotency. In one embodiment, homogenous iPSCs are maintained in a composition comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, but the composition does not include a TGFβ receptor / ALK inhibitor. As used herein, the term "homogeneous" refers to a population of cells in which each cell is identical or substantially identical to the other cells in the population. In one embodiment, a cell is identical to the other cells in the population if each cell expresses one or more of the same pluripotency markers contemplated herein, e.g., SSEA4 and TRA1-81. In one embodiment, a population is homogenous if at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the cells are identical or substantially identical to the other cells in the population.

[0140] In various embodiments, the cell culture medium of the culture platform for generating hematopoietic lineage cells via secondary hemogenic endothelium herein does not contain or is essentially free of inhibitors of the TGFβ / activin signaling pathway, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In one embodiment, the culture platform includes seeding with a medium for maintaining naive hiPSCs, the medium containing a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor. Without wishing to be bound by any particular theory, the inventors have discovered that TGFβR / ALK5 inhibitors increase the efficiency of reprogramming but are counter to the long-term maintenance, quality, and homogeneity of the pluripotent cell population. That is, while inhibition of TGFβ pathway signaling improved the efficiency of cell reprogramming, removal of this inhibition contributes to the subsequent maintenance of pluripotent cell populations within in vitro culture systems, particularly those using feeder-cell-free, single-cell, enzymatic passaging, which reduce spontaneous differentiation and favor a homogenous pluripotent population that remains in a "basal" or "naive" pluripotent state. As used herein, the term "long-term," as measured by the number of passages, is not limited, but often refers to at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more passages. As defined, "passaging" refers to the act of dividing and seeding cells onto multiple cell culture surfaces or cell culture vessels once the cells have proliferated to a desired extent. Additionally, the metastable pluripotent cells are cultured in a medium comprising a GSK3 inhibitor, a MEK inhibitor, and optionally a ROCK inhibitor, but not a TGFβR / ALK5 inhibitor, as disclosed herein, to achieve reduced spontaneous differentiation and / or transition the pluripotent cells to achieve ground-state pluripotency.

[0141] Achieving ground-state or naive pluripotency of iPSCs is also important for obtaining hematopoietic cells by differentiating iPSCs without forming EB intermediates. Additionally, the efficiency of differentiation of naive iPSCs into secondary HE is also significantly affected by the use of monolayer cultures that do not form EBs and their aggregates. In some embodiments, the culture platform comprises a medium containing a ROCK inhibitor but not, or essentially not, a TGFβR / ALK5 inhibitor. In other embodiments, the culture platform comprises a medium containing a GSK3 inhibitor but not a TGFβR / ALK5 inhibitor, which promotes the generation of secondary HE and / or secondary HSC cells using the culture platform presented herein.

[0142] 1. TGFβ receptor / ALK inhibitor TGFβ receptor (e.g., ALK5) inhibitors may include antibodies against TGFβ receptors (e.g., ALK5), dominant-negative mutants thereof, and antisense nucleic acids that suppress their expression. Exemplary TGFβ receptor / ALK inhibitors include SB431542 (see, e.g., Inman et al., Molecular Pharmacology, 62(1):65-74 (2002)); A-83-01, also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (see, e.g., Tojo et al., Cancer Science, 96(11):791-800); (2005); commercially available, e.g., from Toicris Bioscience; 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine; Wnt3a / BIO (see, e.g., Dalton et al., WO 2008 / 094597, incorporated herein by reference); GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide) (see, e.g., Gellibert et al., Journal of Medicinal Chemistry, 49(7):2210-2221 (2006)); SM16 (see, e.g., Suzuki et al., Cancer Research, 67(5):2351-2359 (2007); IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, e.g., Kim et al., Xenobiotica, 38(3):325-339 (2008); GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., de Gouville et al., Drug News Perspective, 19(2):8 5-90 (2006); SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, e.g., DaCosta et al., Molecular Pharmacology, 65(3):744-752 (2004)); and pyrimidine derivatives (see, e.g., the pyrimidine derivatives listed in Stiefl et al., WO 2008 / 006583, incorporated herein by reference). Furthermore, "ALK5 inhibitor" is not intended to encompass non-specific kinase inhibitors, although "ALK5 inhibitor" may include, for example, SB-431542 (see, e.g., Inman et al., J. It should be understood that the present invention encompasses inhibitors that inhibit not only ALK5 but also ALK4 and / or ALK7, such as those described in Mol. Pharmacol., vol. 62(1):65-74 (2002). Without intending to limit the scope of the present invention, it is believed that ALK5 inhibitors affect the mesenchymal-to-epithelial transition / transition (MET) process. The TGFβ / activin pathway is a driving factor for epithelial-to-mesenchymal transition (EMT). Therefore, inhibiting the TGFβ / activin pathway can promote the MET (i.e., reprogramming) process.

[0143] Considering the data showing the effect of inhibiting ALK5, it is believed that inhibiting the TGFβ / activin pathway will have a similar effect of inhibiting ALK5.Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or instead of an ALK5 inhibitor, as described in each paragraph of this specification.Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, inhibitors of SMAD2 / 3 phosphorylation, inhibitors of the interaction between SMAD2 / 3 and SMAD4, and activators / agonists of SMAD6 and SMAD7.Furthermore, the classifications described below are for structural purposes, and those skilled in the art will know that a compound may affect one or more points in the pathway, and therefore, a compound may function in more than one of the defined categories.

[0144] TGFβ receptor (TGFβR) inhibitors may include antibodies against TGFβ receptors, dominant-negative mutants thereof, and siRNA or antisense nucleic acids targeting them. Specific examples of TGFβ receptor inhibitors include SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumab (CAT-152); methelimumab (CAT-192); GC-1008; ID11 ; AP-12009; AP-11014; LY550410; LY580276; LY364947; LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (Morin); activin-M108A; P144; soluble TBR2-Fc; and antisense-transfected tumor cells targeting the TGFβ receptor (e.g., Wrzesinski et al., Clinical Cancer Research, 13(18):5262-5270 (2007); Kaminska et al., Acta Biochimica Polonica 52(2):329-337 (2005); and Chang et al., Frontiers in Bioscience 12:4393-4401 (2007)).

[0145] Inhibitors of SMAD2 / 3 phosphorylation may include antibodies against SMAD2 or SMAD3, dominant-negative mutants thereof, and antisense nucleic acids targeting these. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (Morin) (see, e.g., Wrzesinski, supra; Kaminska, supra; Shimanuki et al., Oncogene, 26:3311-3320, which are incorporated herein by reference). (2007); and Kataoka et al., see EP1992360).

[0146] Inhibitors of the interaction between SMAD2 / 3 and SMAD4 may include antibodies against SMAD2, SMAD3, and / or smad4, dominant-negative mutants thereof, and antisense nucleic acids targeting these. Specific examples of inhibitors of the interaction between SMAD2 / 3 and SMAD4 include Trx-SARA, Trx-xFoxH1b, and Trx-Lef1 (see, e.g., Cui et al., Oncogene, 24:3864-3874 (2005)). and Zhao et al., Molecular Biology of the Cell, 17:3819-3831 (See, e.g., p. (2006)).

[0147] Activators / agonists of SMAD6 and SMAD7 include, but are not limited to, antibodies against SMAD6 or SMAD7, dominant-negative mutants thereof, and antisense nucleic acids targeting them. Specific examples of inhibitors include, but are not limited to, Smad7 (as PTO oligonucleotides) (see, e.g., Miyazono et al., US6534476, and Steinbrecher et al., US2005119203, both of which are incorporated herein by reference).

[0148] 2. Wnt pathway agonists As used herein, the terms "Wnt signal promoter," "Wnt pathway activator," "Wnt pathway activator," or "Wnt pathway agonist" refer to agonists of the Wnt signaling pathway, including, but not limited to, agonists of one or more of Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, and Wnt16. Wnt pathway agonists further include, but are not limited to, one or more of the following polypeptides or fragments thereof: Dkk polypeptide, Crescent polypeptide, Cerberus polypeptide, Axin polypeptide, Frzb polypeptide, T-cell factor polypeptide, or dominant-negative Disheveled polypeptide.

[0149] Further non-limiting examples of Wnt pathway agonists include one or more of the following: a nucleic acid comprising a nucleotide sequence encoding a Wnt polypeptide, a polypeptide comprising the amino acid sequence of a Wnt polypeptide, a nucleic acid comprising a nucleotide sequence encoding an activated Wnt receptor, a polypeptide comprising the amino acid sequence of an activated Wnt receptor, a small organic molecule that promotes Wnt / β-catenin signaling, a small organic molecule that inhibits the expression or activity of a Wnt antagonist, an antisense oligonucleotide that inhibits the expression of a Wnt antagonist, a ribozyme that inhibits the expression of a Wnt antagonist, an RNAi construct, siRNA, or shRNA that inhibits the expression of a Wnt antagonist, an antibody that binds to and inhibits the activity of a Wnt antagonist, a nucleic acid comprising a nucleotide sequence encoding a β-catenin polypeptide, a polypeptide comprising the amino acid sequence of a β-catenin polypeptide, a nucleic acid comprising a nucleotide sequence encoding a Lef-1 polypeptide, and a polypeptide comprising the amino acid sequence of a Lef-1 polypeptide.

[0150] Wnt pathway agonists further include GSK3 inhibitors, such as, for example, a dominant negative GSK-3 polypeptide, a GSK3α polypeptide, or a nucleic acid comprising a nucleotide sequence encoding a GSK3 polypeptide, a dominant negative GSK-3 polypeptide, a GSK3α polypeptide, or a polypeptide comprising the amino acid sequence of a GSK3 polypeptide, a small organic molecule that binds to GSK-3, GSK3α, or GSK3 and inhibits their expression or activity, an RNAi construct, siRNA, or shRNA that binds to GSK-3, GSK3α, or GSK3 and inhibits their expression and / or activity, an antisense oligonucleotide that binds to GSK-3, GSK3α, or GSK3 and inhibits their expression, an antibody that binds to GSK-3, GSK3α, or GSK3 and inhibits their expression and / or activity, a ribozyme that binds to GSK-3, GSK3α, or GSK3 and inhibits their expression, and any GSK-3-independent reagent that activates β-catenin target genes and is similar in effect to inhibiting GSK-3.

[0151] 3.GSK3 inhibitors GSK3 inhibitors are specific exemplary Wnt pathway agonists suitable for use in the compositions contemplated herein and may include, but are not limited to, polynucleotides, polypeptides, and small molecules. GSK3 inhibitors contemplated herein may reduce the expression and / or activity of GSK3α / β. Illustrative examples of GSK3 inhibitors contemplated herein include, but are not limited to, anti-GSK3 antibodies targeting GSK3, dominant-negative GSK3 mutants, siRNA, shRNA, miRNA, and antisense nucleic acids.

[0152] Other exemplary GSK3 inhibitors include kenpaullone, 1-azakempaullone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, lithium, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopenadienyl ruthenium complex, TDZD-8 4-benzyl-2-methyl-1,2,4-thiazolinone These include, but are not limited to, diazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, alpha-4-dibromocetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, TWS1 19 pyrrolopyrimidine compound, L803H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, RO318220, TDZD-8, TIBPO, and OTDZT.

[0153] In certain exemplary embodiments, the GSK3 inhibitor is CHIR99021, BIO, or Kenpaullone.

[0154] In a preferred embodiment, the GSK3 inhibitor is CHIR99021.

[0155] In another embodiment, the GSK3 inhibitor is BRD0705.

[0156] 4. ERK / MEK inhibitors ERK / MEK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ERK / MEK inhibitors contemplated herein may attenuate MEK or ERK expression and / or MEK or ERK activity. Illustrative examples of MEK / ERK inhibitors contemplated herein include, but are not limited to, anti-MEK or anti-ERK antibodies that target MEK or ERK, dominant-negative MEK or ERK mutants, siRNA, shRNA, miRNA, and antisense nucleic acids.

[0157] Other exemplary ERK / MEK inhibitors include, but are not limited to, PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSK1 120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEA1 19, AZD6244, FR180204, and PTK787.

[0158] Further exemplary MEK / ERK inhibitors include compounds disclosed in International Patent Publication Nos. WO99 / 01426, WO02 / 06213, WO03 / 077914, WO05 / 051301, and WO2007 / 044084.

[0159] Further illustrative examples of MEK / ERK inhibitors include the following compounds: 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo-2-chlorophenyl) 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide si)-amide, 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(2,4-dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, hereinafter referred to as MEK inhibitor 1; 5-carboxylic acid (2-hydroxy-ethoxy)-amide, 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (hereinafter referred to as MEK inhibitor 2), 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide, and pharmaceutically acceptable salts thereof.

[0160] In a preferred embodiment, the MEK / ERK inhibitor is PD98059.

[0161] 5.ROCK inhibitors Rho-associated kinase (ROCK) is a serine / threonine kinase that acts as a downstream effector of Rho kinase, of which three isoforms exist: RhoA, RhoB, and RhoC. ROCK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ROCK inhibitors contemplated herein may attenuate ROCK expression and / or ROCK activity. Illustrative examples of ROCK inhibitors contemplated herein include, but are not limited to, anti-ROCK antibodies targeting ROCK, dominant-negative ROCK mutants, siRNA, shRNA, miRNA, and antisense nucleic acids.

[0162] Exemplary ROCK inhibitors contemplated herein include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, Y27632, H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, as well as the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated by reference in its entirety.

[0163] In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, or pyrintegrin.

[0164] In a preferred embodiment, the ROCK inhibitor is thiazovivin.

[0165] The amount of small molecule in the compositions and cell culture media contemplated herein can be varied and optimized according to the specific culture conditions, including the specific molecules and combinations used, the type of cells cultured in the media, and the specific application. In one embodiment, the small molecule is present in the composition at a concentration sufficient to induce pluripotency, improve the efficiency of reprogramming, increase or maintain the potency of cells, or induce or maintain ground-state pluripotency.

[0166] Another aspect of the present invention relates to Notch activators for use in the present invention. Notch encompasses all members of the Notch receptor family, including, but not limited to, Notch1. Notch activators include, but are not limited to, agonists of Notch receptors. Notch agonists bind to Notch receptors and induce or mediate signaling events associated with the Notch receptor, such as cleaving the intracellular domain of Notch and translocating it to the nucleus. Notch activators include, but are not limited to, Jag1, Jag2, DLL-1, DLL-3, and DLL-4. Notch activators include, but are not limited to, those disclosed in EP2606884, US6689744, and US5780300, the disclosures of which are incorporated herein by reference. In some embodiments, one or more of the Notch ligands can be introduced as soluble peptides or immobilized on a solid material. The solid material can include, but is not limited to, polystyrene plates or beads. Beads for immobilizing Notch ligands can be agarose beads, magnetic beads, and latex beads. In one embodiment, the Notch ligand peptide is conjugated / immobilized to beads. In another embodiment, the Notch ligand peptide is conjugated / immobilized to the surface of a polystyrene plate. In some embodiments, the Notch ligand is immobilized non-covalently. In some embodiments, the Notch ligand peptide is presented by cells.

[0167] Yet another aspect of the present invention relates to BMP pathway activators, including agents disclosed in the following publications: WO2014011540, WO2014062138, and WO2005117994, the disclosures of which are incorporated herein by reference. BMP pathway activators for use in the present invention include, but are not limited to, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In a non-limiting embodiment of the present invention, the BMP pathway activator is BMP-4. BMP is a multifunctional cytokine that is a member of the transforming growth factor beta superfamily. Bone morphogenetic protein (BMP) receptors mediate BMP signaling through the activation of Smads. BBMP ligands bind to the BMP receptors BMPRI and BMPRII. BMPRII is phosphorylated, followed by activation of BMPRI. Phosphorylation of BMPRI subsequently phosphorylates receptor-activated Smad (R-Smad) proteins, which associate with common mediator-Smads (co-Smads) and enter the nucleus, where they regulate gene expression. In one embodiment, the BMP pathway activator is BMP4.

[0168] Although the present invention provides compositions for obtaining hematopoietic cells from iPSCs via secondary HSCs differentiated from iPSCs or via secondary hemogenic endothelium differentiated from iPSCs, each of these approaches lacks the formation of EBs from iPSCs for differentiation of the desired cells.

[0169] 6. hiPSC Differentiation Platform I. iHSC Platform One aspect of the present invention provides a culture medium for obtaining mesodermal cells from pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, the culture medium comprises a Wnt pathway activator and a BMP activator. In one embodiment, the culture medium comprises an iHSC basal medium containing a Wnt pathway activator and a BMP activator. In one embodiment, the Wnt pathway activator in the culture medium is a GSK3 inhibitor. In one embodiment, the GSK3 inhibitor is CHIR99021. In one embodiment, the BMP activator is BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 1. In some embodiments, the culture medium is fully defined when using Matrigel™ instead of Vitronectin. [Table 1]

[0170] One aspect of the present invention provides a culture medium for obtaining secondary hemogenic endothelium from mesodermal cells. In one embodiment, the culture medium comprises a Wnt pathway activator, a BMP activator, and optionally a TGFβ receptor / ALK inhibitor. In one embodiment, the culture medium comprises a Wnt pathway activator and a BMP activator, but does not contain or is essentially free of a TGFβ receptor / ALK inhibitor. In one embodiment, the culture medium comprises an iHSC basal medium containing a Wnt pathway activator, optionally a TGFβ receptor / ALK inhibitor, and a BMP activator. In one embodiment, the Wnt pathway activator in the culture medium is a GSK3 inhibitor. In one embodiment, the GSK3 inhibitor is CHIR99021. In one embodiment, the BMP activator is BMP4. In one embodiment, the optional TGFβ receptor / ALK inhibitor is SB431542. In some embodiments, the culture medium herein comprises an extracellular matrix protein. In other embodiments, the culture medium contains small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 2. In some embodiments, the culture medium is fully defined when Matrigel™ is substituted for Vitronectin. [Table 2]

[0171] One aspect of the present invention provides a culture medium for obtaining secondary HSCs from hemogenic endothelium. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. In one embodiment, the culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO does not comprise a Wnt pathway activator and a TGFβ receptor / ALK inhibitor. In one embodiment, the culture medium comprises an iHSC basal medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. In one embodiment, the BMP activator is BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture media herein contain small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 3. In some embodiments, the culture media is completely defined when Matrigel™ is substituted for Vitronectin. [Table 3]

[0172] One aspect of the present invention provides a culture medium for obtaining T cell precursors from secondary HSCs. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. In one embodiment, the culture medium comprises an iTC basal medium comprising SCF, Flt3L, IL7, a BMP activator, one or more growth factors and cytokines selected from the group consisting of IL2, IL3, and IL6, and one or more Notch pathway activators. In one embodiment, the iTC basal medium comprises a combination of IL2, IL3, IL6, and one or more Notch pathway activators. In one embodiment, the iTC basal medium does not comprise fibronectin. In one embodiment, the BMP activator is BMP4. In one embodiment, the Notch pathway activator is a Notch ligand, including, but not limited to, Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells. In some embodiments, the culture medium comprises extracellular matrix proteins. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 4. In some embodiments, the culture medium is fully defined when Matrigel™ is substituted for Vitronectin. [Table 4]

[0173] One aspect of the present invention provides a culture medium for obtaining T cells from T cell precursors. In one embodiment, the culture medium comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IGF. In one embodiment, the culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7 and IGF does not comprise a BMP activator. In one embodiment, the culture medium comprises a combination of an iTC basal medium comprising a growth factor and cytokine selected from the group consisting of SCF, Flt3L, IL7, and IGF, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, and IL6, and one or more Notch pathway activators. In one embodiment, the iTC basal medium comprises a combination of IL2, IL3, IL6, and one or more Notch pathway activators. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and / or DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells. In some embodiments, the BMP activator comprises BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 5. In some embodiments, the culture medium is fully defined when Matrigel™ is substituted for Vitronectin. [Table 5]

[0174] One aspect of the present invention provides a culture medium for obtaining NK cell precursors from secondary HSCs. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and VEGF. In one embodiment, the culture medium comprises an iNK basal medium containing SCF, Flt3L, VEGF, a BMP activator, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15. In one embodiment, the iNK basal medium comprises a combination of IL2, IL3, IL6, and IL15. In one embodiment, the BMP activator is BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 6. In some embodiments, the culture medium is fully defined when Matrigel™ is substituted for Vitronectin. [Table 6]

[0175] One aspect of the present invention provides a culture medium for obtaining NK cells from NK cell precursors. In one embodiment, the culture medium comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, and IL7. In one embodiment, the culture medium comprises an iNK basal medium comprising SCF, Flt3L, IGF, IL7, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15, but does not include a BMP activator. In one embodiment, the iNK basal medium comprises a combination of IL2, IL3, IL6, and IL15. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 7. In some embodiments, the culture medium is fully defined when Matrigel™ is substituted for Vitronectin. In some embodiments, artificial antigens that stimulate NK proliferation, development, and maturation are introduced in the form of bead conjugations, cell membrane particles, or antigen-presenting cells. [Table 7]

[0176] Another aspect of the present invention is a culture platform for obtaining T cells, comprising: (i) a culture medium suitable for generating T cells from T cell precursors, comprising an iTC basal medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IGF, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, and IL6, and one or more Notch pathway activators, and no BMP activators; (ii) a culture medium suitable for generating T cell precursors from secondary HSCs, comprising an iTC basal medium and a BMP activator, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; (iii) VEGF, The present invention provides a culture platform comprising one or more of the following: (i) a culture medium suitable for generating secondary HSCs from hemogenic endothelium, the culture medium comprising an iHSC basal medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and a BMP activator, and not containing a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; (iv) a culture medium suitable for generating hemogenic endothelium from mesodermal cells, the culture medium comprising an iHSC basal medium containing a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and a BMP activator; and (v) a culture medium suitable for generating mesodermal cells from iPSCs, the culture medium comprising an iHSC basal medium containing a GSK3 inhibitor and a BMP activator. In some embodiments, the iPSCs are naive iPSCs.

[0177] In one embodiment, the culture platform for obtaining T cells comprises (i) an iTC basal medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, and IL7, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, and IL6, and one or more Notch pathway activators, and no BMP activators, wherein the culture platform is suitable for generating T cells from T cell precursors. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and iTC basal medium, wherein culture medium (ii) is suitable for generating T cell precursors from secondary HSCs. In one embodiment, the culture platform comprising culture media (i) and (ii) further comprises (iii) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium comprising a BMP activator, wherein the culture medium (iii) does not comprise a Wnt pathway activator and a TGFβ receptor / ALK inhibitor, and is suitable for generating secondary HSCs from hemogenic endothelium. In one embodiment, the culture platform comprising culture media (i), (ii), and (iii) further comprises (iv) a culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium, wherein the culture medium (iv) is suitable for generating secondary hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising culture media (i), (ii), (iii), and (iv) further comprises (v) a culture medium comprising a GSK3 inhibitor and an iHSC basal medium, wherein the culture medium (v) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0178] One aspect of the present invention is a culture platform for obtaining T cell precursors, comprising: (i) a culture medium suitable for generating T cell precursors from secondary HSCs, the culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and an iHSC basal medium; (ii) an iHSC basal medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and a BMP activator. (iii) a culture medium suitable for generating secondary hemogenic endothelium from mesodermal cells, comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium; and (iv) a culture medium suitable for generating mesodermal cells from iPSCs, comprising a GSK3 inhibitor and an iHSC basal medium. In some embodiments, the iPSCs are naive iPSCs.

[0179] In one embodiment, the culture platform for obtaining T cell precursors comprises (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and an iHSC basal medium, wherein the culture medium (i) is suitable for generating T cell precursors from secondary HSCs. In one embodiment, the culture platform comprising the culture medium (i) further comprises (ii) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium comprising a BMP activator, wherein the culture medium (ii) does not comprise a Wnt pathway activator or a TGFβ receptor / ALK inhibitor, and is suitable for generating HSCs from secondary hemogenic endothelium. In one embodiment, the culture platform comprising culture media (i) and (ii) further comprises (iii) a culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium, wherein the culture medium (iii) is suitable for generating secondary hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising culture media (i), (ii), and (iii) further comprises (iv) a culture medium comprising a GSK3 inhibitor and an iHSC basal medium, wherein the culture medium (iv) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0180] One aspect of the present invention provides a culture platform for obtaining secondary HSCs, the culture platform comprising one or more of the following: (i) a culture medium suitable for generating HSCs from secondary hemogenic endothelium, comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium containing a BMP activator, but not containing a Wnt pathway activator or a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for generating secondary hemogenic endothelium from mesodermal cells, comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium; or (iii) a culture medium suitable for generating mesodermal cells from iPSCs, comprising a GSK3 inhibitor and an iHSC basal medium. In some embodiments, the iPSCs are naive iPSCs.

[0181] In one embodiment, the culture platform for obtaining secondary HSCs comprises (i) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium comprising a BMP activator, wherein the culture medium (i) does not comprise a Wnt pathway activator and a TGFβ receptor / ALK inhibitor, and is suitable for generating HSCs from secondary hemogenic endothelium. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium, wherein the culture medium (ii) is suitable for generating secondary hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising the culture media (i) and (ii) further comprises (iii) a culture medium comprising a GSK3 inhibitor and an iHSC basal medium, wherein the culture medium (iii) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the resulting secondary HSCs are CD34+ HSCs. In some embodiments, the iPSCs are naive iPSCs.

[0182] Yet another aspect of the present invention provides a culture platform for obtaining hemogenic endothelium, the culture platform comprising one or more of: (i) a culture medium suitable for generating secondary hemogenic endothelium from mesodermal cells, the culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium containing a BMP activator; and (ii) a culture medium suitable for generating mesodermal cells from iPSCs, the culture medium comprising a GSK3 inhibitor and an iHSC basal medium. In some embodiments, the iPSCs are naive iPSCs.

[0183] In one embodiment, the culture platform for obtaining hemogenic endothelium comprises (i) a culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium comprising a BMP activator, wherein the culture medium (i) is suitable for generating secondary hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising the culture medium (i) further comprises (ii) a culture medium comprising a GSK3 inhibitor and an iHSC basal medium, wherein the culture medium (ii) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0184] A further aspect of the present invention is a culture platform for obtaining NK cells, comprising: (i) a culture medium suitable for generating NK cells from NK cell precursors, comprising an iNK basal medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IGF, and IL7, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15, and no BMP activators; (ii) a culture medium suitable for generating NK cell precursors from secondary HSCs, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and VEGF, a BMP activator, and an iNK basal medium; (iii) a culture medium suitable for generating NK cell precursors from secondary HSCs, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and VEGF, a BMP activator, and an iNK basal medium; The present invention provides a culture platform comprising one or more of the following: (i) a culture medium suitable for generating HSCs from secondary hemogenic endothelium, the culture medium comprising one or more growth factors and cytokines selected from the group consisting of F, Flt3L, IL15, IL3, IL6, IGF, and TPO, and a BMP activator, and not comprising a Wnt pathway activator and a TGFβ receptor / ALK inhibitor; (iv) a culture medium suitable for generating secondary hemogenic endothelium from mesodermal cells, the culture medium comprising an iHSC basal medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and a BMP activator; and (v) a culture medium suitable for generating mesodermal cells from iPSCs, the culture medium comprising a GSK3 inhibitor and an iHSC basal medium. In some embodiments, the iPSCs are naive iPSCs.

[0185] One embodiment of a culture platform for obtaining NK cells comprises (i) a culture medium comprising an iNK basal medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15, wherein the culture medium (i) does not contain a BMP activator and is suitable for generating NK cells from NK cell precursors. In one embodiment, the culture platform comprising the culture medium (i) further comprises (ii) a culture medium comprising an iNK basal medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and VEGF, a BMP activator, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15, wherein the culture medium (ii) is suitable for generating NK cell precursors from secondary HSCs. In one embodiment, the culture platform comprising culture media (i) and (ii) further comprises (iii) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium comprising a BMP activator, wherein the culture medium (iii) does not comprise a Wnt pathway activator and a TGFβ receptor / ALK inhibitor, and is suitable for generating HSCs from secondary hemogenic endothelium. In one embodiment, the culture platform comprising culture media (i), (ii), and (iii) further comprises (iv) a culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium, wherein the culture medium (iv) is suitable for generating secondary hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising culture media (i), (ii), (iii), and (iv) further comprises (v) a culture medium comprising a GSK3 inhibitor and an iHSC basal medium, wherein the culture medium (v) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0186] Another aspect of the present invention is a culture platform for obtaining NK cell precursors, comprising: (i) a culture medium suitable for generating NK cell precursors from secondary HSCs, comprising an iNK basal medium containing a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, and one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15; and (ii) one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. (iii) a culture medium suitable for generating secondary hemogenic endothelium from mesodermal cells, comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and iHSC basal medium; and (iv) a culture medium suitable for generating mesodermal cells from iPSCs, comprising a GSK3 inhibitor and iHSC basal medium. In some embodiments, the iPSCs are naive iPSCs.

[0187] One embodiment of a culture platform for obtaining NK cell precursors comprises (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and VEGF, and an iNK basal medium comprising one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15, wherein culture medium (i) is suitable for generating NK cell precursors from secondary HSCs. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium comprising a BMP activator, wherein culture medium (ii) does not comprise a Wnt pathway activator or a TGFβ receptor / ALK inhibitor, and is suitable for generating HSCs from secondary hemogenic endothelium. In one embodiment, the culture platform comprising culture media (i) and (ii) further comprises (iii) a culture medium comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium, wherein the culture medium (iii) is suitable for generating secondary hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising culture media (i), (ii), and (iii) further comprises (iv) a culture medium comprising a GSK3 inhibitor and an iHSC basal medium, wherein the culture medium (v) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0188] One embodiment of a culture platform for obtaining NK cell precursors includes one or more artificial antigens in the form of bead conjugations, cell membrane particles, and / or antigen-presenting cells to stimulate NK proliferation, development, and maturation.

[0189] Yet another aspect of the present invention provides a culture platform for generating secondary CD34+ cells, the culture platform comprising one or more of: (i) a culture medium suitable for generating HSCs from secondary hemogenic endothelium, comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and an iHSC basal medium containing a BMP activator, but not a Wnt pathway activator or a TGFβ receptor / ALK inhibitor; (ii) a culture medium suitable for generating secondary hemogenic endothelium from mesodermal cells, comprising a GSK3 inhibitor, optionally a TGFβ receptor / ALK inhibitor, and an iHSC basal medium; and (iii) a culture medium suitable for generating mesodermal cells from pluripotent stem cells, including iPSCs, comprising a GSK3 inhibitor and an iHSC basal medium. In some embodiments, the iPSCs are naive iPSCs.

[0190] II. iCD34 Platform A further aspect of the present invention provides a culture platform for obtaining secondary hemogenic endothelium using pluripotent stem cells. As used herein, secondary hemogenic endothelium refers to a hematopoietic cell population committed to definitive hematopoiesis with the ability to generate all hematopoietic cells, including but not limited to secondary HSCs, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, mature T cells, and / or NK cells.

[0191] In one embodiment, a culture platform for obtaining secondary hemogenic endothelium using pluripotent stem cells, including iPSCs, includes seeding with a medium containing MEKi, GSKi, and ROCKi. In some embodiments, the seeding medium does not contain or is essentially free of TGFβ receptor / ALK inhibitors. In one embodiment, the combination of small molecules used in seeding the culture medium of the present invention is shown in Table 9 as fate maintenance medium (FMM). The medium components can be present in the medium in amounts within the concentration ranges shown in Table 9. In one embodiment, the iPSCs used to obtain secondary hemogenic endothelium were cell lines generated using fate reprogramming medium (FRM) and further maintained in FMM to establish and sustain a ground state or naive state of the iPSC cell line suitable for stage-specific differentiation as disclosed herein. The ground state or naive iPSCs obtained in this manner are suitable for cryopreservation. In the present invention, maintained iPSC cell lines or clonal iPSCs can be seeded onto FMM for subsequent differentiation into secondary hemogenic endothelium. [Table 9]

[0192] One aspect of the present invention provides a culture medium for differentiation and expansion of mesodermal pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, the culture medium comprises a CD34 basal medium containing a BMP activator, optionally bFGF, and a small molecule in the combinations shown in Table 10. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises a small molecule, growth factor, and / or cytokine in the concentration ranges shown in Table 10. In some embodiments, the culture medium is fully defined when Matrigel™ is substituted for Vitronectin. [Table 10]

[0193] In one embodiment, the above culture medium for mesodermal differentiation and expansion from pluripotent stem cells further comprises between 0.2 and 50 ng of bFGF.

[0194] One aspect of the present invention provides a culture medium for obtaining mesodermal cells with secondary hematopoietic endothelial potential from pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, the culture medium comprises a BMP activator, a GSK3 inhibitor, and bFGF. In one embodiment, to achieve secondary hematopoietic endothelial potential, the culture medium containing the GSK3 inhibitor is applied only after mesodermal cell specialization. In one embodiment, the culture medium containing a BMP activator, a GSK3 inhibitor, and bFGF further comprises a CD34 basal medium containing a small molecule in the combination shown in Table 11. In one embodiment, the culture medium does not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the culture medium contains an extracellular matrix protein. In other embodiments, the culture medium herein contains a small molecule, growth factor, and / or cytokine in the concentration ranges shown in Table 11. In some embodiments, the culture medium is fully composition-defined when Matrigel™ is substituted for Vitronectin. [Table 11]

[0195] One aspect of the present invention provides a culture medium for obtaining secondary hemogenic endothelium from mesodermal cells. In one embodiment, the culture medium comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In one embodiment, the culture medium comprises a CD34 basal medium containing VEGF, bFGF, SCF, IL6, IL11, a ROCK inhibitor, and a small molecule in the combinations shown in Table 12. In one embodiment, the culture medium containing VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor does not contain IGF1 and / or EPO. In other embodiments, the culture medium herein comprises a small molecule, growth factor, and / or cytokine in the concentration ranges shown in Table 12. [Table 12]

[0196] One aspect of the present invention provides a culture platform for obtaining multipotent progenitor (MPP) cells from secondary hemogenic endothelium. The MPPs can be further differentiated into myeloid cells, including neutrophil precursors. In one embodiment, the culture platform comprises (i) a culture medium suitable for differentiating secondary hemogenic endothelium into pre-HSCs (Table 13), comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11. In another embodiment, the culture platform comprising a culture medium for differentiating secondary hemogenic endothelium into pre-HSCs further comprises (ii) a culture medium suitable for differentiating pre-HSCs into multipotent progenitors, comprising a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but excluding a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium herein comprises a small molecule, growth factor, and / or cytokine in the concentration ranges shown in Table 13. [Table 13]

[0197] One aspect of the present invention provides a culture platform for generating T cell precursors or T cells from secondary hemogenic endothelium. In one embodiment, the culture platform includes: (i) a medium suitable for differentiating secondary hemogenic endothelium into pre-T cell precursors (pre-proT), comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7; and (ii) a medium suitable for differentiating pre-T cell precursors into T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor (Table 14). In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture media herein comprise small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 14. [Table 14]

[0198] One aspect of the present invention provides a culture platform for generating NK cell precursors or NK cells from secondary hemogenic endothelium. In one embodiment, the culture platform comprises: (i) a medium suitable for differentiating secondary hemogenic endothelium into pre-NK cell precursors (pre-proNK), comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15; and (ii) a medium suitable for differentiating pre-NK cell precursors into NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, but excluding one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture media herein comprise small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 15. [Table 15]

[0199] Another aspect of the present invention is a culture platform for obtaining T cell precursors or T cells, comprising: (i) a medium suitable for differentiating pre-T cell precursors into T cell precursors or T cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and free of or essentially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; (ii) a medium suitable for differentiating secondary hemogenic endothelium into pre-T cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7; (iii) a medium suitable for differentiating secondary hemogenic endothelium into pre-T cell precursors, comprising a ROCK inhibitor, bFGF, VFGF, and IL7; The present invention provides a culture platform comprising one or more of the following: (i) a culture medium suitable for differentiation and expansion of secondary hematopoietic endothelium from mesodermal cells, comprising one or more growth factors and cytokines selected from the group consisting of EGF, SCF, IL6, and IL11; (iv) a culture medium suitable for obtaining the potential of secondary hematopoietic endothelium in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor; (v) a culture medium suitable for generating and expanding mesodermal cells from iPSCs, comprising a BMP activator and optionally bFGF; and (vi) a culture medium suitable for seeding and expanding naive iPSCs, comprising a MEKi, a GSKi, and a ROCKi, and not containing or essentially not containing a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above culture media do not contain or essentially not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0200] In one embodiment, a culture platform for generating T cell precursors or T cells comprises (i) a medium suitable for differentiating pre-T cell precursors into T cell precursors or T cells, comprising SCF, Flt3L, and IL7, and not comprising or essentially not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In another embodiment, a culture platform for obtaining T cell precursors or T cells, wherein the culture platform comprising medium (i) further comprises (ii) a medium suitable for differentiating secondary hemogenic endothelium into pre-T cell precursors, comprising BMP activators, ROCK inhibitors, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, and IL7. In another embodiment, the culture platform for obtaining T cell precursors or T cells, comprising media (i) and (ii), further comprises (iii) a culture medium suitable for differentiation and expansion of secondary hemogenic endothelium from mesodermal cells, comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11. In yet another embodiment, the culture platform for obtaining T cell precursors or T cells, comprising media (i), (ii), and (iii), further comprises (iv) a culture medium suitable for obtaining secondary hemogenic endothelium potential in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor. In yet another embodiment, the culture platform for obtaining T cell precursors or T cells, comprising media (i), (ii), (iii), and (iv), further comprises (v) a culture medium suitable for generating and expanding mesodermal cells from iPSCs, comprising a BMP activator and, optionally, bFGF.In another embodiment, a culture platform for obtaining T cell precursors or T cells, the culture platform comprising media (i), (ii), (iii), (iv), and (v), further comprises (vi) a culture medium suitable for seeding and expanding naive iPSCs, comprising an MEKi, a GSKi, and a ROCKi, and not containing or essentially not containing a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above media are not containing or essentially not containing a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, a Notch factor is used in the culture platform for generating T cell precursors or T cells. In some embodiments, Notch factors, including Jag1, Jag2, DLL-1, DLL-3, and DLL-4, can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides displayed by cells.

[0201] Another aspect of the present invention is a culture platform for obtaining NK cell precursors or NK cells, comprising: (i) a medium suitable for differentiating pre-NK cell precursors into NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising or essentially comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; (ii) a medium suitable for differentiating secondary hematopoietic endothelium into pre-NK cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15; (iii) a medium suitable for differentiating secondary hematopoietic endothelium into pre-NK cell precursors; (iv) a culture medium suitable for differentiation and expansion of secondary hemogenic endothelium from mesodermal cells, comprising an OCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; (v) a culture medium suitable for obtaining the potential of secondary hemogenic endothelium in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor; (v) a culture medium suitable for generating and expanding mesodermal cells from iPSCs, comprising a BMP activator and optionally bFGF; or (vi) a culture medium suitable for seeding and expanding naive iPSCs, comprising an MEKi, a GSKi, and a ROCKi, and not containing or essentially not containing a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above culture media do not contain or essentially not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.In some embodiments, NK maturation is achieved using one or more artificial antigens that stimulate NK proliferation, development, and maturation, and that are introduced in the form of bead conjugations, cell membrane particles, and / or antigen-presenting cells.

[0202] In one embodiment, the culture platform for generating NK cell precursors or NK cells comprises (i) a medium suitable for differentiating pre-NK cell precursors into NK cell precursors or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not including or essentially not including one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, NK maturation is achieved using one or more artificial antigens that stimulate NK proliferation, development, and maturation, introduced in the form of bead conjugation, cell membrane particles, and / or antigen-presenting cells. In another embodiment, the culture platform for obtaining NK cell precursors or NK cells comprises medium (i), which further comprises (ii) a medium suitable for differentiating secondary hemogenic endothelium into pre-NK cell precursors, comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15. In another embodiment, the culture platform for obtaining NK cell precursors or NK cells comprises medium (i) and (ii), which further comprises (iii) a culture medium suitable for differentiation and expansion of secondary hemogenic endothelium from mesodermal cells, comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11. In yet another embodiment, the culture platform for obtaining NK cell precursors or NK cells, comprising media (i), (ii), and (iii), further comprises (iv) a culture medium suitable for obtaining secondary hemogenic endothelial potential in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor.In yet another embodiment, the culture platform for obtaining NK cell precursors or NK cells, comprising media (i), (ii), (iii), and (iv), further comprises (v) a culture medium suitable for generating and expanding mesodermal cells from iPSCs, comprising a BMP activator and, optionally, bFGF. In another embodiment, the culture platform for obtaining NK cell precursors or NK cells, comprising media (i), (ii), (iii), (iv), and (v), further comprises (vi) a culture medium suitable for seeding and expanding naive iPSCs, comprising an MEKi, a GSKi, and a ROCKi, and not containing or essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above media are not containing or essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0203] One aspect of the present invention provides a culture platform for generating secondary hemogenic endothelium, the culture platform comprising one or more of: (i) a culture medium for differentiation and expansion of secondary hemogenic endothelium from mesodermal cells, the culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11; (ii) a culture medium for obtaining secondary hematopoietic potential in mesodermal cells, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a culture medium for differentiation and expansion of mesodermal cells from naive iPSCs, the culture medium comprising a BMP activator and optionally bFGF; and (iv) a seeding and expansion culture of naive iPSCs comprising MEKi, GSKi, and ROCKi, the seeding culture not comprising a TGFβ receptor / ALK inhibitor. In some embodiments, the secondary hemogenic endothelium is CD34+. In some embodiments, all of the above media do not contain or are essentially free of TGFβ receptor / ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0204] In one embodiment, the culture platform for obtaining secondary hemogenic endothelium comprises (i) a culture medium suitable for differentiating and expanding secondary hemogenic endothelium from mesodermal cells, the culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, the culture medium (ii) being suitable for obtaining secondary hematopoietic potential in mesodermal cells. In another embodiment, the culture platform comprising culture media (i) and (ii) further comprises (iii) a culture medium comprising a BMP activator and, optionally, bFGF, the culture medium (iii) being suitable for differentiating and expanding mesodermal cells from naive iPSCs. In yet another embodiment, the culture platform comprising culture media (i), (ii), and (iii) further comprises (iv) a culture medium comprising an MEKi, a GSKi, and a ROCKi, wherein the culture medium (v) does not contain or is essentially free of a TGFβ receptor / ALK inhibitor, and the culture medium (v) is suitable for seeding and expanding naive iPSCs. In some embodiments, all of the above media do not contain or are essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0205] One aspect of the present invention is a culture platform for generating CD34+ secondary hemogenic endothelium, comprising: (i) a culture medium for differentiating and expanding secondary hemogenic endothelium from mesodermal cells, the culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, wherein the secondary hemogenic endothelium comprises CD34+ secondary hemogenic endothelium; and (ii) a culture medium for obtaining secondary hematopoietic potential in mesodermal cells, the culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, wherein the secondary hemogenic endothelium comprises CD34+ secondary hemogenic endothelium; The present invention provides a culture platform comprising one or more of: (i) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor; (ii) a culture medium for differentiating and expanding mesodermal cells from naive iPSCs, the culture medium comprising a BMP activator and, optionally, bFGF; and (iv) a seeding or expansion culture of naive iPSCs comprising MEKi, GSKi, and ROCKi, the seeding culture being free or essentially free of TGFβ receptor / ALK inhibitors. In some embodiments, all of the above media are free or essentially free of TGFβ receptor / ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0206] One aspect of the present invention provides a culture platform for generating mesodermal cells, the culture platform comprising one or more of: (i) a culture medium for differentiating and expanding mesodermal cells from naive iPSCs, the culture medium comprising a BMP activator and, optionally, bFGF; and (ii) a seeding or expansion culture of naive iPSCs comprising MEKi, GSKi, and ROCKi, the seeding culture being free or essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above media are free or essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, the culture platform for generating mesodermal cells may further comprise (iii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, for obtaining secondary hematopoietic potential in the mesodermal cells. In some embodiments, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor does not comprise a TGFβ receptor / ALK inhibitor.

[0207] C. Methods for Obtaining CD34+ Cells, Secondary Hematopoietic Endothelial, Multipotent Progenitors, T Cell Progenitors, or NK Cell Progenitors, T Cells, and / or NK Cells The present invention provides a method for generating secondary hematopoietic cells from pluripotent stem cells using a multi-stage culture platform containing one or more culture media. The method is suitable for feeder-free conditions. The method is also suitable for monolayer culture and therefore does not require EB formation or aggregation intermediates to differentiate pluripotent stem cells, unlike methods known in the art. The provided method generates and simultaneously expands secondary hemogenic endothelial (iHE), secondary HSC (iHSC), and CD34+ HE (iCD34) cells derived from pluripotent stem cells, which can be further differentiated into multipotent progenitor cells (iMPP), natural killer cell precursors (ipro-NK), T cell precursors (ipro-T), mature NK cells (iNK), and T cells (iT). A further aspect of the present invention also provides a method for generating myeloid cells differentiated from pluripotent stem cell-derived CD34+ cells, HE, HSC, and / or MPP.

[0208] In one embodiment, the present invention provides a method for differentiating and expanding cells of the hematopoietic system from pluripotent cells in monolayer culture, comprising contacting the pluripotent cells with a BMP pathway activator and optionally bFGF, wherein pluripotent stem cell-derived mesodermal cells are obtained and expanded from the pluripotent stem cells without forming embryoid bodies, which are then placed in contact with a BMP pathway activator, bFGF, and a WNT pathway activator, wherein expanded mesodermal cells having secondary hemogenic endothelial (HE) potential are obtained from the pluripotent stem cells without forming embryoid bodies. Subsequent contact with bFGF and optionally a ROCK inhibitor and / or a WNT pathway activator causes the mesodermal cells having secondary HE potential to differentiate into secondary HE cells, which are also expanded during differentiation.

[0209] The provided method for obtaining hematopoietic cells is superior to EB-mediated differentiation of pluripotent stem cells because EB formation does not result in cell expansion, does not allow monolayer culture, and is cumbersome and inefficient. Additionally, the present invention also discloses that monolayer culture using the methods presented herein results in functional hematopoietic cells capable of long-term hematopoietic self-renewal, reconstitution, and engraftment in vivo.

[0210] As described in detail below, the present invention provides methods for obtaining hematopoietic cells from pluripotent cells via obtaining secondary HSCs or secondary hemogenic endothelium. In particular, the present invention provides methods for directing the differentiation of hematopoietic cells from pluripotent cells without forming EBs for differentiation.

[0211] I. iHSC Platform 1. Differentiating and expanding iHSCs from pluripotent stem cells, mesoderm derived from pluripotent stem cells, or HE (iHSC platform) One aspect of the present invention provides methods for generating and expanding secondary HSCs (iHSCs) using a multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are differentiated into mesodermal cells, followed by a second step in which the mesodermal cells are differentiated into hemogenic endothelium (HE) and expanded. In a third step, the HE cells are differentiated into secondary HSCs (iHSCs), which are also simultaneously expanded. The present invention also provides methods for generating secondary HSCs (iHSCs), comprising differentiating and expanding mesodermal cells derived from pluripotent stem cells into HE, and differentiating the HE into iHSCs. Alternatively, the present invention provides methods for generating and expanding secondary HSCs (iHSCs), comprising differentiating HE derived from pluripotent stem cells into iHSCs. In some embodiments of the above methods, the pluripotent stem cells comprise iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0212] In one embodiment of a method for producing secondary HSCs (iHSCs) from naive pluripotent stem cells, the method includes: (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent cells with a culture medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein, and expanding the differentiated mesodermal cells; (2) differentiating the mesodermal cells into secondary hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, and expanding the secondary HE cells; and (3) differentiating the secondary HE cells into secondary HSCs by contacting the secondary HE cells with a third culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and expanding the secondary HSCs. In some embodiments, the pluripotent stem cells comprise iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the sorting uses CD34-positive and CD43-negative. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In some embodiments, the above method comprises differentiating the mesodermal cells into secondary hemogenic endothelium by contacting the mesodermal cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and a TGFβ receptor inhibitor.

[0213] In one embodiment of a method for generating and expanding secondary HSCs (iHSCs) from mesodermal cells derived from pluripotent stem cells, the method includes: (1) differentiating the mesodermal cells into secondary HE cells by contacting the mesodermal cells with a medium containing a BMP pathway activator, a Wnt pathway activator, and optionally at least one TGFβ receptor inhibitor, thereby expanding the secondary HE cells; and (2) differentiating the HE cells into iHSCs by contacting the HE cells with a second medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

[0214] In one embodiment of a method for generating and expanding secondary HSCs (iHSCs) from pluripotent stem cell-derived HE, the method comprises contacting HE cells with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby differentiating and expanding iHSCs from the pluripotent stem cell-derived HE. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4.

[0215] 2. Differentiating and expanding HE from pluripotent stem cells or mesoderm derived from pluripotent stem cells (iHSC platform) One aspect of the present invention provides a method for generating and expanding secondary hemogenic endothelium using a multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are differentiated into mesodermal cells, followed by a second step in which the mesodermal cells are expanded and differentiated into hemogenic endothelium. Starting pluripotent cells include, but are not limited to, ground-state induced pluripotent stem cells or naive induced pluripotent stem cells and embryonic stem cells. In some embodiments, the generated and expanded hemogenic endothelium is secondary hemogenic endothelium. In some embodiments, the generated secondary hemogenic endothelium is CD34-positive. Alternatively, the present invention provides a method for generating and expanding hemogenic endothelium by differentiating mesodermal cells derived from pluripotent stem cells into secondary hemogenic endothelium.

[0216] In one embodiment of a method for differentiating and expanding secondary hemogenic endothelium from pluripotent stem cells, the method includes: (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the cells with a first culture medium containing at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein; and (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second culture medium containing a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby expanding the secondary HE cells. In some embodiments, the HE cells obtained from the above method express CD34. In some embodiments, the above method includes a GSK3 inhibitor as a Wnt pathway activator. In some embodiments, the above method includes CHIR99021 or BIO as a GSK3 inhibitor. In some embodiments, the above method includes CHIR99021 as a GSK3 inhibitor. In some embodiments, the above methods include SB431542 or A83-01 as a TGFβ receptor inhibitor. In some embodiments, the above methods include SB431542 as a TGFβ receptor inhibitor.

[0217] In one embodiment of a method for differentiating and expanding hemogenic endothelium from mesodermal cells derived from pluripotent stem cells, the method comprises differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and optionally a TGFβ receptor inhibitor. In some embodiments, the secondary HE cells obtained from the above method express CD34.

[0218] 3. Differentiating and expanding mesodermal cells from iPSCs (iHSC platform) One aspect of the present invention provides a method for generating mesodermal cells from pluripotent stem cells, including but not limited to ground state induced pluripotent stem cells or naive induced pluripotent stem cells and embryonic stem cells.

[0219] In one embodiment of a method for generating and expanding mesodermal cells from pluripotent stem cells, the method comprises differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein. In some embodiments, the method comprises a GSK3 inhibitor as the Wnt pathway activator. In some embodiments, the method comprises CHIR99021 or BIO as the GSK3 inhibitor. In some embodiments, the method comprises CHIR99021 as the GSK3 inhibitor.

[0220] 4. Obtaining T cell precursors from pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, HE, or secondary HSCs (iHSC and iTC platforms) One aspect of the present invention provides a method for generating T cell precursors from pluripotent stem cells using a multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are differentiated into mesodermal cells and the mesodermal cells are expanded, followed by a second step in which hemogenic endothelium is differentiated and expanded. In a third step, HE cells are differentiated into secondary HSCs (iHSCs) and the iHSCs are expanded. In a fourth step, the iHSCs are differentiated into T cell precursors. The present invention also provides a method for generating T cell precursors, comprising differentiating mesodermal cells derived from pluripotent stem cells into HE, then differentiating the HE into iHSCs, and then differentiating the iHSCs into T cell precursors. The present invention further provides a method for generating T cell precursors, comprising differentiating HE derived from pluripotent stem cells into iHSCs, then differentiating the iHSCs into T cell precursors. Alternatively, the present invention provides a method for generating T cell precursors, comprising differentiating iHSCs derived from pluripotent stem cells into T cell precursors.

[0221] In one embodiment of a method for producing T cell precursors from pluripotent stem cells, the method includes: (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein, and expanding the mesodermal cells; (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally, at least one of a TGFβ receptor inhibitor, and expanding the HE cells; and (3) differentiating the HE cells into hemogenic endothelium by contacting the mesodermal cells with a second medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and optionally, a TGFβ receptor inhibitor, and expanding the HE cells. (3) differentiating the iHSCs into secondary HSCs by contacting the iHSCs with a third medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and expanding the iHSCs; and (4) differentiating the iHSCs into T cell precursors by contacting the iHSCs with a fourth medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the BMP activator is BMP4. In one embodiment, the Notch pathway activator is Jag1, Jag2, DLL-1, DLL-3, and DLL-4.In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells.

[0222] In one embodiment of a method for generating T cell precursors from mesodermal cells derived from pluripotent stem cells, the method includes: (1) contacting the mesodermal cells with a culture medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one TGFβ receptor inhibitor, thereby differentiating the mesodermal cells into HE cells, and expanding the HE; (2) contacting the HE cells with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby differentiating the resulting HE cells into iHSCs and expanding the iHSCs; and (3) contacting the iHSCs with a third culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators, thereby differentiating the iHSCs into T cell precursors. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the sorting uses CD34-positive and CD43-negative. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to a surface, or peptides presented by cells.

[0223] In one embodiment of a method for generating T cell precursors from pluripotent stem cells derived from HE, the method includes: (1) differentiating the HE into iHSCs by contacting the HE cells with a medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs; and (2) differentiating the iHSCs into T cell precursors by contacting the iHSCs with a second medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathways. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD73 negativity. In other embodiments, the sorting uses CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity.

[0224] In one embodiment of a method for generating T cell precursors from pluripotent stem cell-derived iHSCs, the method comprises differentiating the iHSCs into T cell precursors by contacting the iHSCs with a medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the method further comprises sorting and obtaining the pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells.

[0225] 5. Obtaining T cells from pluripotent stem cells, mesodermal cells derived from pluripotent stem cells, HE, iHSC, or T cell precursors (iHSC and iTC platforms)

[0226] One aspect of the present invention provides a method for generating T cells from pluripotent stem cells using a multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are differentiated into mesodermal cells, resulting in the expansion of the mesodermal cells as they differentiate; then, in a second step, the mesodermal cells are differentiated into hemogenic endothelium, resulting in the expansion of HE cells. In a third step, the HE cells are differentiated into secondary HSCs (iHSCs), resulting in the expansion of iHSCs. In a fourth step, the iHSCs are differentiated into T cell precursors (ipro-T). In a fifth step, the iHSCs are differentiated into T cells. The present invention also provides a method for generating T cells, comprising the steps of differentiating mesodermal cells derived from pluripotent stem cells into HE, differentiating the HE into iHSCs, differentiating the iHSCs into T cell precursors, and then differentiating the T cell precursors into T cells. The present invention further provides a method for generating T cells, the method comprising differentiating HE derived from pluripotent stem cells into iHSCs, differentiating the iHSCs into T cell precursors, and differentiating the T cell precursors into T cells. Alternatively, the present invention provides a method for generating T cells, the method comprising differentiating iHSC derived from pluripotent stem cells into T cell precursors and differentiating the T cell precursors into T cells. Furthermore, the present invention provides a method for generating T cells, the method comprising differentiating T cell precursors derived from pluripotent stem cells into T cells.

[0227] In one embodiment of a method for producing T cells from pluripotent stem cells, the method includes the steps of: (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein, thereby expanding the mesodermal cells; (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby expanding the HE cells; and (3) differentiating the HE cells into hemogenic endothelium by contacting the BMP activator and one or more growth factors selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. (3) differentiating the HE cells into iHSCs by contacting them with a third medium containing one or more growth factors and cytokines selected from the group consisting of BMP activators, SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators, thereby expanding the iHSCs; (4) differentiating the iHSCs into T cell precursors by contacting them with a fourth medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators; and (5) differentiating the T cell precursors into T cells by contacting them with a fifth medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the method further comprises sorting using CD34 positive. In some embodiments, the sorting step uses CD34 positive and CD43 negative. In some embodiments, the sorting step uses CD34 positive, CD43 negative, and CD73 negative.In some other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the BMP activator is BMP4. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells.

[0228] In one embodiment of a method for generating T cells from mesodermal cells derived from pluripotent stem cells, the method includes the steps of: (1) contacting the mesodermal cells with a medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby differentiating the mesodermal cells into HE cells, thereby expanding the secondary HE cells; and (2) contacting the HE cells with a second medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby differentiating the resulting HE cells into iHSCs, wherein the iHSCs are (3) differentiating the iHSCs into T cell precursors by contacting the iHSCs with a third culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators; and (4) differentiating the T cell precursors into T cells by contacting the T cell precursors with a fourth culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting step uses CD34 positive and CD43 negative. In some embodiments, the sorting step uses CD34 positive, CD43 negative, and CD73 negative. In other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4.In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells.

[0229] In one embodiment of a method for generating T cells from pluripotent stem cell-derived HE, the method includes the steps of: (1) differentiating the pluripotent stem cell-derived HE into iHSCs by contacting the HE cells with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs; and (2) differentiating the iHSCs from a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6. (2) differentiating the iHSCs into T cell precursors by contacting them with a second medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators; and (3) differentiating the T cell precursors into T cells by contacting them with a third medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHSCs using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34-positive. In some embodiments, the sorting step uses CD34-positive and CD43-negative. In some embodiments, the sorting step uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting step uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, or peptides presented by cells.

[0230] In one embodiment of a method for generating T cells from iHSCs derived from pluripotent stem cells, the method includes: (1) contacting the iHSCs with a medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators, thereby differentiating the iHSCs into T cell precursors, thereby expanding the iHSCs; and (2) contacting the T cell precursors with a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators, thereby differentiating the T cell precursors into T cells. In some embodiments, the method further includes sorting and obtaining pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the method further includes sorting using CD34 positivity. In some embodiments, the sorting step uses CD34 positive and CD43 negative. In some embodiments, the sorting step uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to a surface, or peptides presented by cells.

[0231] In one embodiment of a method for generating T cells from T cell precursors derived from pluripotent stem cells, the method comprises differentiating the T cell precursors into T cells by contacting the T cell precursors with a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators.

[0232] 6. Obtaining NK cell precursors from pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, HE, or iHSC (iHSC and iNK platforms) One aspect of the present invention provides a method for generating NK cell precursors from pluripotent stem cells using a multi-step process. Generally, the method begins in a first step, where pluripotent stem cells are differentiated into mesodermal cells, resulting in the expansion of mesodermal cells; then in a second step, the mesodermal cells are differentiated into hemogenic endothelium, resulting in the expansion of HE cells. In a third step, the HE cells are differentiated into secondary HSCs, resulting in the expansion of secondary HSCs. In a fourth step, iHSCs are differentiated into NK cell precursors. The present invention also provides a method for generating NK cell precursors, comprising differentiating mesodermal cells derived from pluripotent stem cells into HE, then differentiating the HE into iHSCs, and then differentiating the iHSCs into NK cell precursors. The present invention further provides a method for generating NK cell precursors, comprising differentiating HE derived from pluripotent stem cells into iHSCs, then differentiating the iHSCs into NK cell precursors. Alternatively, the present invention provides a method for generating NK cell precursors, the method comprising differentiating iHSCs derived from pluripotent stem cells into NK cell precursors.

[0233] In one embodiment of a method for producing NK cell precursors from pluripotent stem cells, the method includes the steps of: (1) contacting the pluripotent stem cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein, thereby differentiating the pluripotent stem cells into mesodermal cells, thereby expanding the mesodermal cells; and (2) contacting the mesodermal cells with a second medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby differentiating the mesodermal cells into hemogenic endothelium, thereby expanding the HE cells. (3) differentiating the HE cells into iHSCs by contacting the HE cells with a third culture medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs; and (4) differentiating the iHSCs into NK cell precursors by contacting the iHSCs with a fourth culture medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting step uses CD34 positive and CD43 negative. In some embodiments, the sorting step uses CD34 positive, CD43 negative, and CD73 negative. In other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

[0234] In one embodiment of a method for generating NK cell precursors from mesodermal cells derived from pluripotent stem cells, the method includes the steps of: (1) differentiating the mesodermal cells derived from pluripotent stem cells into HE cells by contacting the mesodermal cells with a medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby expanding the HE cells; and (2) differentiating the HE cells from pluripotent stem cells by contacting the mesodermal cells with a medium comprising a BMP activator, and VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TP. (2) differentiating the obtained HE cells into iHSCs by contacting them with a second culture medium containing one or more growth factors and cytokines selected from the group consisting of BMP activators and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15, thereby expanding the iHSCs; and (3) differentiating the iHSCs into NK cell precursors by contacting the iHSCs with a third culture medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34-positive. In some embodiments, the sorting step uses CD34-positive and CD43-negative. In some embodiments, the sorting step uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

[0235] In one embodiment of a method for generating NK cell precursors from pluripotent stem cell-derived HE, the method includes: (1) differentiating the pluripotent stem cell-derived HE into iHSCs by contacting the HE cells with a medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs; and (2) differentiating the iHSCs into NK cell precursors by contacting the iHSCs with a second medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD73 negativity. In other embodiments, the sorting uses CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity.

[0236] In one embodiment of a method for generating NK cell precursors from pluripotent stem cell-derived iHSCs, the method comprises differentiating the iHSCs into NK cell precursors by contacting the iHSCs with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the method further comprises sorting and obtaining pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the method further comprises sorting using CD34-positive cells. In some embodiments, the sorting step uses CD34-positive cells and CD43-negative cells. In some embodiments, the sorting step uses CD34-positive cells, CD43-negative cells, and CD73-negative cells. In other embodiments, the sorting step uses CD34-positive cells, CD43-negative cells, CD73-negative cells, and CXCR4-negative cells.

[0237] 7. Obtaining NK cells from pluripotent stem cells, mesodermal cells derived from pluripotent stem cells, HE, iHSC, or NK cell precursors (iHSC and iNK platforms) One aspect of the present invention provides a method for generating NK cells from pluripotent stem cells using a multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are differentiated into mesodermal cells, resulting in the expansion of mesodermal cells; then, in a second step, the mesodermal cells are differentiated into hemogenic endothelium, resulting in the expansion of HE cells. In a third step, the HE cells are differentiated into secondary HSCs (iHSCs), resulting in the expansion of HSCs. In a fourth step, the iHSCs are differentiated into NK cell precursors (ipro-NK). In a fifth step, the iHSCs are differentiated into NK cells. The present invention also provides a method for generating NK cells, comprising the steps of differentiating mesodermal cells derived from pluripotent stem cells into HE, differentiating the HE into iHSCs, differentiating the iHSCs into NK cell precursors, and then differentiating the NK cell precursors into NK cells. The present invention further provides a method for generating NK cells, comprising the steps of differentiating pluripotent stem cell-derived HEs into iHSCs, differentiating the iHSCs into NK cell precursors, and differentiating the NK cell precursors into NK cells. Alternatively, the present invention provides a method for generating NK cells, comprising the steps of differentiating pluripotent stem cell-derived iHSCs into NK cell precursors and differentiating the pluripotent stem cell-derived NK cell precursors into NK cells. Furthermore, the present invention provides a method for generating NK cells, comprising the step of differentiating pluripotent stem cell-derived NK cell precursors into NK cells.

[0238] In one embodiment of a method for producing NK cells from pluripotent stem cells, the method includes the steps of: (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and an extracellular matrix protein, thereby expanding the mesodermal cells; (2) differentiating the mesodermal cells into secondary hemogenic endothelium by contacting the mesodermal cells with a second medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby expanding the HE cells; and (3) differentiating the HE cells into secondary hemogenic endothelium by contacting the BMP activator and a second medium comprising at least one of a BMP activator and a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby expanding the HE cells. (3) differentiating the HE cells into iHSCs by contacting them with a third medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15, thereby expanding the iHSCs; (4) differentiating the iHSCs into NK cell precursors by contacting them with a fourth medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15; and (5) differentiating the NK cell precursors into NK cells by contacting them with a fifth medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the method includes differentiating mesodermal cells into secondary hemogenic endothelium by contacting the mesodermal cells with a medium containing at least one of a BMP pathway activator, a Wnt pathway activator, and a TGFβ receptor inhibitor. In one embodiment, the BMP activator is BMP4.

[0239] In one embodiment of a method for generating NK cells from mesodermal cells derived from pluripotent stem cells, the method includes the steps of: (1) differentiating the mesodermal cells derived from pluripotent stem cells into HE cells by contacting the mesodermal cells with a medium comprising a BMP pathway activator, a Wnt pathway activator, and optionally at least one of a TGFβ receptor inhibitor, thereby expanding the mesodermal cells; and (2) contacting the HE cells with a second medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the resulting HE cells. (3) differentiating the iHSCs into NK cell precursors by contacting the iHSCs with a third medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and (4) differentiating the NK cell precursors into NK cells by contacting the NK cell precursors with a fourth medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting step uses CD34 positive and CD43 negative. In some embodiments, the sorting step uses CD34 positive, CD43 negative, and CD73 negative. In other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.In some embodiments, the method includes differentiating mesodermal cells into secondary hemogenic endothelium by contacting the mesodermal cells with a medium comprising at least one of a BMP pathway activator, a Wnt pathway activator, and a TGFβ receptor inhibitor.

[0240] In one embodiment of a method for generating NK cells from pluripotent stem cell-derived HE, the method includes the steps of: (1) differentiating the pluripotent stem cell-derived secondary HE into iHSCs by contacting the HE cells with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs; and (2) differentiating the iHSCs from the pluripotent stem cells with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, thereby expanding the iHSCs. (2) differentiating the iHSCs into NK cell precursors by contacting them with a second medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15; and (3) differentiating the NK cell precursors into NK cells by contacting them with a third medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the iHSCs obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34-positive. In some embodiments, the sorting step uses CD34-positive and CD43-negative. In some embodiments, the sorting step uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting step uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

[0241] In one embodiment of a method for generating NK cells from iHSCs derived from pluripotent stem cells, the method comprises: (1) differentiating the iHSCs into NK cell precursors by contacting the iHSCs with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and (2) differentiating the NK cell precursors into NK cells by contacting the NK cell precursors with a second medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the method further comprises sorting and obtaining the pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73, and / or CXCR4.

[0242] In one embodiment of a method for generating NK cells from NK cell precursors derived from pluripotent stem cells, the method includes differentiating the NK cell precursors into NK cells by contacting the NK cell precursors with a fifth medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15.

[0243] II. iCD34 Platform 1. Deriving and Expanding Secondary iHE (iCD34 Platform) One aspect of the present invention provides a method for generating secondary hemogenic endothelium (iHE) using an optimized multi-step process. Generally, the method begins with a first step of seeding and expanding pluripotent stem cells. In this step, the pluripotent stem cells are then differentiated into mesoderm cells, resulting in the expansion of mesoderm cells. The expanded mesoderm population is then differentiated into a mesoderm population with secondary hemogenic endothelial potential, and secondary hemogenic endothelium is then differentiated and expanded from mesoderm cells with secondary hemogenic endothelial potential. Alternatively, the present invention provides a method for generating secondary hemogenic endothelium (iHE), comprising the steps of differentiating and expanding mesoderm cells from pluripotent stem cells; and then differentiating and expanding secondary hemogenic endothelium (iHE) from mesoderm cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The present invention further provides a method for generating and expanding secondary hemogenic endothelium (iHE), comprising differentiating and expanding pluripotent stem cell-derived mesodermal cells to obtain mesodermal cells with secondary iHE potential, and then differentiating these into iHE. Alternatively, the present invention provides a method for generating and expanding secondary hemogenic endothelium, comprising differentiating pluripotent stem cell-derived mesodermal cells into iHE. The methods disclosed herein utilize an optimized monolayer iCD34 culture platform that does not involve EB formation, and that is free of or essentially free of TGFβ receptor / ALK inhibitors.

[0244] In one embodiment of a method for producing secondary hemogenic endothelium (iHE) from pluripotent stem cells, the method includes the steps of: (1) differentiating and expanding a mesoderm population from pluripotent stem cells by contacting the cells with a medium containing a BMP activator and, optionally, bFGF; (2) differentiating and expanding the mesoderm population by contacting the cells with a medium containing a BMP activator, a Wnt pathway activator, and bFGF to obtain secondary HE potential in the mesoderm cells; and (3) differentiating and expanding the mesoderm cells with secondary HE potential into secondary HE cells by contacting the cells with a medium containing a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the sorting step uses CD34-positive and CD43-negative. In some embodiments, the sorting step uses CD34-positive, CD43-negative, and CD73-negative. In other embodiments, the sorting step uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In some embodiments, the medium in the method does not contain or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the cells are contacted with a culture medium containing a GSK3 inhibitor to achieve secondary HE potential only after mesodermal cell differentiation. In some embodiments, the method further comprises placing the seeded iPSCs and / or mesodermal cells under a hypoxic atmosphere of between about 2% and about 10%.In some embodiments, the above method further comprises the step of expanding the pluripotent stem cells upon plating the pluripotent stem cells by contacting the pluripotent cells with a medium comprising a MEKi, a GSKi, and a ROCKi.

[0245] In one embodiment of a method for generating secondary hemogenic endothelium (iHE) from seeded pluripotent stem cells, the method includes the steps of: (1) differentiating and expanding mesodermal cells from the pluripotent stem cells by contacting the pluripotent stem cells with a medium containing a BMP activator and, optionally, bFGF; (2) obtaining mesodermal cells with secondary iHE potential by contacting the mesodermal cells with a medium containing a BMP activator, a Wnt pathway activator, and bFGF; and (3) differentiating and expanding secondary HE cells from the mesodermal cells with iHE potential by contacting the mesodermal cells with a medium containing a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the above method further comprises sorting using CD34-positive. In some embodiments, the sorting step uses CD34-positive and CD43-negative. In some embodiments, the sorting step uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting step uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In some embodiments, the medium in the above method does not contain or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises placing the seeded iPSCs and / or mesoderm cells under a hypoxic atmosphere of between about 2% and about 10%.

[0246] In one embodiment of a method for generating secondary hemogenic endothelium (iHE) from mesodermal cells derived from pluripotent stem cells, the method includes: (1) contacting mesodermal cells with a medium containing a BMP activator, a Wnt pathway activator, and bFGF to obtain mesodermal cells with secondary HE potential; and (2) differentiating and expanding secondary HE cells from mesodermal cells with secondary HE potential by contacting the mesodermal cells with a medium containing a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the sorting step uses CD34-positive and CD43-negative. In some embodiments, the sorting step uses CD34-positive, CD43-negative, and CD73-negative. In other embodiments, the sorting step uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In some embodiments, the medium in the above method does not contain or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises placing the mesodermal cells under a hypoxic tension of between about 2% and about 10%.

[0247] In one embodiment of a method for obtaining secondary hemogenic endothelial (iHE) potential in mesodermal cells derived from pluripotent stem cells, the method includes contacting the mesodermal cells with a medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11, thereby expanding the mesodermal cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, and / or CXCR4. In some embodiments, the medium in the above method does not contain or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes placing the mesodermal cells under a hypoxic condition of between about 2% and about 10%.

[0248] 2. Deriving and expanding pluripotent stem cell-derived mesodermal cells with secondary hemogenic endothelial potential (iCD34 platform) One aspect of the present invention provides a method for generating mesodermal cells derived from pluripotent stem cells using an optimized multi-step process. Generally, the method begins with a first step of seeding pluripotent stem cells. The seeded pluripotent stem cells are then allowed to develop into mesoderm. In a third step, the mesoderm is further differentiated into mesodermal cells with secondary hematopoietic endothelial potential. Alternatively, the present invention provides a method for generating mesodermal cells derived from pluripotent stem cells, the method comprising differentiating seeded pluripotent stem cells into mesoderm, and differentiating the mesoderm into mesodermal cells with secondary hematopoietic potential. The present invention also provides a method for generating mesodermal cells derived from pluripotent stem cells with secondary hematopoietic potential, the method comprising differentiating the mesoderm derived from pluripotent stem cells into mesodermal cells with secondary hematopoietic endothelial potential. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that is free of, or essentially free of, TGFβ receptor / ALK inhibitors.

[0249] In one embodiment of a method for obtaining secondary hemogenic endothelial potential in mesodermal cells derived from pluripotent stem cells, the method includes: (1) differentiating and expanding mesodermal cells from the pluripotent stem cells by contacting the pluripotent stem cells with a medium comprising a BMP activator and, optionally, bFGF; and (2) obtaining secondary hemogenic endothelial potential in the mesodermal cells by contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the medium in the above method does not contain or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes placing the pluripotent stem cells under a low oxygen tension of between about 2% and about 10%. In some embodiments, the above method further comprises the step of plating the pluripotent stem cells by contacting the cells with a medium comprising a MEKi, a GSKi, and a ROCKi.

[0250] In one embodiment of a method for generating pluripotent stem cell-derived mesodermal cells with secondary hemogenic endothelial potential from pluripotent stem cell-derived mesoderm, the method comprises differentiating the mesoderm into mesodermal cells with secondary hemogenic endothelial potential by contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the medium in the above method does not comprise or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises placing the mesodermal cells under a hypoxic tension of between about 2% and about 10%.

[0251] 3. Derivation and expansion of mesoderm from pluripotent stem cells One aspect of the present invention provides a method for generating mesoderm from pluripotent stem cells using an optimized multi-step process. Generally, the method begins with seeding pluripotent stem cells in a first step, followed by differentiating the seeded cells into mesoderm in a second step. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that is free of or essentially free of TGFβ receptor / ALK inhibitors.

[0252] In one embodiment of a method for producing pluripotent stem cell-derived mesoderm from pluripotent cells, the method comprises differentiating and expanding mesoderm cells from the plated pluripotent stem cells by contacting the cells with a medium comprising a BMP activator and, optionally, bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the medium in the above method does not comprise or is essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the above method further comprises placing the plated iPSCs under a low oxygen tension of between about 2% and about 10%. In some embodiments, the above method further comprises plating and expanding the iPSCs by contacting the pluripotent cells with a medium comprising an MEKi, a GSKi, and a ROCK.

[0253] 4. Deriving Hematopoietic Multipotent Progenitors (iMPPs) (iCD34 Platform and iMPP Platform) One aspect of the present invention provides a method for generating pluripotent stem cell-derived multipotent progenitors (iMPPs) using an optimized multistep process. Generally, the method begins with a first step of seeding pluripotent stem cells. The seeded cells are expanded and differentiated into mesodermal cells. The mesoderm is expanded and differentiated into mesodermal cells with secondary hemogenic endothelial potential, which are then differentiated into secondary hemogenic endothelium. HE cells are expanded and differentiated into pre-HSCs, which are then expanded and differentiated into multipotent progenitors capable of differentiating into myeloid cells, including neutrophil precursors. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived multipotent progenitors (iMPPs), comprising the steps of differentiating seeded pluripotent cells into mesoderm, differentiating the mesoderm into mesodermal cells with secondary hemogenic endothelial potential, and then differentiating the mesodermal cells into secondary iHEs, which are then differentiated into iMPPs. The present invention further provides a method for generating iMPPs from pluripotent stem cells, comprising differentiating mesoderm from the pluripotent stem cells into mesodermal cells with secondary hematopoietic endothelial potential, then...

Claims

1. 1. A method for differentiating cells derived from umbilical cord blood, the method comprising: (a) enriching a population of cord blood cells for CD34+ cells to produce an enriched cell population; and (b) culturing the enriched cell population in a first culture medium comprising IL7, SCF, and Flt3L to obtain differentiated cells. wherein the differentiated cells comprise (i) T lineage cells, including pre-T cell precursors, or (ii) NK lineage cells, including pre-NK cell precursors.

2. The method of claim 1 , wherein the first culture medium comprises a ROCK inhibitor.

3. The method described in claim 1, wherein the differentiated cells include T lineage cells that include the pre-T cell precursors.

4. 4. The method of claim 3, further comprising culturing the pre-T cell precursors in a second culture medium comprising SCF, Flt3L, and IL7 to obtain T cell precursors or T cells, wherein the second culture medium does not comprise one or more of VEGF, bFGF, BMP4, or ROCK inhibitor.

5. 4. The method of claim 3, wherein the first culture medium further comprises one or more of bFGF, DLL4, VEGF, or a BMP activator.

6. The method described in claim 1, wherein the differentiated cells include NK lineage cells that include the pre-NK cell precursors.

7. 7. The method of claim 6, further comprising culturing the pre-NK cell precursors in a second culture medium comprising one or both of IL15 and IL3 to obtain NK cell precursors or NK cells, wherein the second culture medium does not comprise one or more of VEGF, bFGF, BMP4, or a ROCK inhibitor.

8. 7. The method of claim 6, wherein the first culture medium further comprises one or more of IL15, IL3, and DLL4.

9. 9. The method of any one of claims 1 to 8, wherein the first culture medium, the second culture medium, or both, do not contain stromal cells.

10. 1. A composition for differentiating umbilical cord blood cells, the composition comprising: (a) an enriched population of CD34+ cells derived from umbilical cord blood; and (b) a first culture medium containing IL7, SCF, and Flt3L; wherein the composition is suitable for differentiating the CD34+ cells to obtain differentiated cells comprising (i) T lineage cells comprising pre-T cell precursors or (ii) NK lineage cells comprising pre-NK cell precursors.

11. The composition of claim 10 , wherein the first culture medium comprises a ROCK inhibitor.

12. The composition described in claim 10, wherein the differentiated cells include T lineage cells that include the pre-T cell precursors.

13. 13. The composition of claim 12, wherein the first culture medium further comprises one or more of bFGF, DLL4, VEGF, or a BMP activator.

14. The composition described in claim 10, wherein the differentiated cells include NK lineage cells that include the pre-NK cell precursors.

15. 15. The composition of claim 14, wherein the first culture medium further comprises one or more of IL15, IL3, and DLL4.

16. The composition of claim 10 , wherein the composition does not include stromal cells.

17. 17. A differentiation platform comprising the composition of any one of claims 10 to 16, further comprising a second culture medium for culturing cells obtained from culturing the CD34+ cells in the first culture medium.

18. 18. The differentiation platform of claim 17, wherein the second culture medium (i) comprises SCF, Flt3L, and IL7, (ii) does not comprise one or more of VEGF, bFGF, BMP4, or ROCK inhibitor, and (iii) is suitable for differentiating pre-T cell precursors to obtain T cell precursors or T cells.

19. 18. The differentiation platform of claim 17, wherein the second culture medium (i) comprises one or both of IL15 and IL3, (ii) does not comprise one or more of VEGF, bFGF, BMP4, or a ROCK inhibitor, and (iii) is suitable for differentiating pre-NK cell precursors to obtain NK cell precursors or NK cells.

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