Method and composition for inducing hematopoietic cell differentiation

Abstract

To provide a cultivation platform and a method for differentiating a pluripotent cell to a hematopoietic cell.SOLUTION: A production method for producing a hematopoietic cell comprises: a) acquiring an induction pluripotent stem cell (iPSC) including a genetic imprint; b) differentiation inducing the iPSC to a hematopoietic cell; (ii) bringing a mesoblast cell into contact with a composition including a BMP path activator, a bFGF, and a WNT path activator, for acquiring a mesoblast cell having a prospective fate established hematopoietic endothelial (HE) differentiation potency; the mesoblast cell being acquired in steps (i) and (ii) without performing an embryoid formation step; the hematopoietic cell including the prospective fate established hematopoietic endothelial cell, a hematopoietic stem cell / precursor cell(HSC), hematopoietic pluripotent precursor cell (MPP), pre-T cell precursor cell, pre-NK cell precursor cell, T cell precursor cell, NK cell precursor cell, T cell, NK cell, NKT cell, or B cell; the hematopoietic cell holding a genetic imprint included in the iPSC, and the hematopoietic cell having improved therapeutic characteristics.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 251,016, filed November 4, 2015; International Application No. PCT / US16 / 14918, filed January 26, 2016; and U.S. Provisional Patent Application No. 62 / 337,093, filed May 16, 2015, the disclosures of which are incorporated herein by reference in their entireties.

[0002] The present invention relates generally to compositions and methods for producing cells of all hematopoietic lineages from pluripotent stem cells, particularly 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] Human induced pluripotent stem cell (hiPSC) technology is a highly promising and potentially limitless source of hematopoietic cells with therapeutic potential for the treatment of numerous hematologic and non-hematologic malignancies, including cancer. To expand the potential of hiPSC and genome-modified hiPSC technologies as a homogeneous source of hematopoietic cell therapies, it is essential to be able to efficiently and reproducibly generate hematopoietic stem and progenitor cells (HSCs) as well as immune effector populations, including diverse subsets of T, B, NKT, and NK lymphoid cells and their progenitors.

[0004] In vitro derivation of HSCs capable of giving rise to lymphocytes is complicated by the presence of at least two spatiotemporally distinct waves of blood cell formation during embryonic development: primary and secondary hematopoiesis. Primary hematopoiesis begins in the extraembryonic yolk sac and gives rise to a transitional and limited hematopoietic repertoire that mainly contains embryonic erythroid and myeloid cells (but not HSCs). Only after the secondary hematopoietic wave occurs do early HSCs emerge from specialized endothelial progenitor cells within arterial duct structures, termed hemogenic endothelium (HE). Committed HE then undergo endothelial hematopoietic conversion to give rise to HSCs, which then eventually migrate to the bone marrow, where they sustain multilineage hematopoiesis, including T, B, NKT, and NK lymphoid cells, throughout the organism's life. Thus, the generation of HSCs and lymphoid effector cells from pluripotent stem cells relies on the ability to accurately recapitulate the intricate early embryonic hematopoietic developmental steps toward a secondary program via appropriately designed and validated methods and compositions.

[0005] Only a limited number of studies have described the induction of differentiation of hiPSCs into fate-committed HE in vitro. The main obstacle to utilizing hiPSCs for therapeutic purposes is that such cells must first be co-cultured with mouse- or human-derived stromal cells in the presence of an undefined serum-containing medium to maintain pluripotency and induce differentiation. In addition, existing protocols employ a strategy consisting of culturing iPSCs to form embryoid bodies (EBs), which are heterogeneous cell aggregates containing various differentiated cells, including ectodermal, mesodermal, and endodermal cells. These procedures require the aggregation of pluripotent cells, for example, by spinning to form aggregates, by allowing cells to adhere and aggregate in wells, or by passively gathering to form aggregates in suspension culture. The formed EBs are maintained in a differentiation-inducing culture system for a period of time, typically 7-10 days, to allow for appropriate differentiation, after which the EBs are transferred to adherent culture for further maturation or dissociated into single cells for cell type sorting for progression to the next differentiation step (Kennedy et al., Cell Reports 2012:1722-1735; Knorr, et al., Stem Cells Translational Medicine 2013 (2):274-283). For example, Kennedy et al. teach EB generation for iPSC differentiation, where pluripotent cells are treated with collagenase and trypsin to detach the cells and form small clumps, which are then cultured to form EBs. Although EB formation has been shown to promote differentiation of pluripotent stem cells, the need for aggregate formation and subsequent EB formation is laborious, cell numbers are only minimally expanded during this process, and the cellular contents within the three-dimensional EB aggregates are inconsistently and unevenly exposed to factors in the medium, resulting in a heterogeneous cell product at different stages of differentiation, posing major obstacles to the scalability and reproducibility of the manufacturing process, which needs to be efficient and streamlined.

[0006] Thus, there is a need for methods and compositions for differentiating stem cells to definitive hematopoiesis without reliance on co-culture or serum-containing media and without the need to form embryoid body aggregates as an intermediate. Summary of the Invention

[0007] The present invention relates generally to cell culture conditions, media, culture platforms and methods for the proliferation and differentiation of stem cells towards a hematopoietic cell fate.

[0008] In particular, the present invention provides methods and compositions for generating hematopoietic cell lineages through committed hemogenic endothelium (HE) derived from pluripotent stem cells, including hiPSCs, in a scalable and monolayer culture platform, without the need for EB formation, under serum / feeder-free conditions. The cells that can be differentiated according to the methods of the present invention range from pluripotent stem cells, to committed progenitor cells to specific highly differentiated cell types, and transdifferentiated cells, which are cells of various lineages that have transitioned directly to a hematopoietic fate without passing through a pluripotent intermediate. Similarly, the cells resulting from stem cell differentiation range from pluripotent stem cells or pluripotent progenitor cells to highly differentiated stem cells and all intervening hematopoietic cell lineages.

[0009] The present invention provides compositions and methods 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 without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with committed hemogenic endothelial (HE) differentiation potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and, optionally, a ROCK inhibitor, and / or a WNT pathway activator differentiates the mesodermal cells with committed HE differentiation potential into committed HE cells, which also proliferate during differentiation.

[0010] The methods provided herein for obtaining hematopoietic cells are superior to EB-mediated pluripotent stem cell differentiation because EB formation results in moderate to minimal cell proliferation, does not allow for monolayer culture, which is important for many applications requiring uniform proliferation and differentiation of cells within a population, and is laborious and less efficient.

[0011] Provided herein is a monolayer differentiation platform that promotes differentiation towards committed hemogenic endothelium resulting in induction of hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells and NK cells. The demonstrated monolayer differentiation strategy combined with enhanced differentiation efficiency and mass expansion allows for the delivery of therapeutically meaningful numbers of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. Furthermore, the present invention discloses that monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that are capable of in vitro differentiation, ex vivo regulation, and long-term hematopoietic self-renewal, reconstitution and engraftment in vivo. As used herein, iPSC-derived hematopoietic lineage cells include, but are not limited to, committed hemogenic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem cells and progenitors, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0012] One aspect of the present invention provides a culture platform for obtaining hematopoietic cells derived from pluripotent stem cells, the culture platform comprising: (i) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells capable of differentiation into committed hemogenic endothelium; (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells capable of differentiation into committed hemogenic endothelium; and (iii) a medium comprising a BMP activator, and optionally bFGF, and suitable for differentiation and proliferation of mesodermal cells from pluripotent stem cells. In some embodiments, the pluripotent stem cells of the culture platform are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the culture platform further comprises: (iv) a 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 proliferation of pluripotent stem cells.

[0013] In some embodiments of the above culture platform, the culture platform further comprises an additional medium: (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not comprising one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, and suitable for differentiation of pluripotent stem cell-derived pre-T cell progenitor cells into T cell progenitors or T cells; or (ii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, and suitable for differentiation of pluripotent stem cell-derived committed hemogenic endothelial cells into pre-T cell progenitor cells; these additional media are suitable for the generation of pluripotent stem cell-derived T lineage cells.

[0014] In some embodiments of the above culture platform, the culture platform may further comprise an additional medium: (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, and suitable for differentiation of pluripotent stem cell-derived pre-NK cell precursors into NK cell precursors or NK cells; or (ii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator; and suitable for differentiation of pluripotent stem cell-derived committed hemogenic endothelium into pre-NK cell precursors; these additional media are suitable for the generation of pluripotent stem cell-derived NK lineage cells.

[0015] In one embodiment, the culture platform provided above further comprises: (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of BMP activators, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but not a ROCK inhibitor, and suitable for differentiation of pluripotent stem cell-derived pre-HSCs into hematopoietic multipotent progenitor cells; (ii) a medium 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, Flt3L, and IL11, and suitable for differentiation of pluripotent stem cell-derived committed hemogenic endothelium into pre-HSCs; these media enable the generation of hematopoietic multipotent progenitor cells derived from pluripotent stem cells.

[0016] Another aspect of the present invention provides a composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, the composition comprising one or more of the following: (i) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; optionally, a Wnt pathway activator; and a pluripotent stem cell-derived mesoderm cell having a differentiation potential into a committed hemogenic endothelium, the composition comprising a pluripotent stem cell-derived mesoderm cell having a differentiation potential into a committed hemogenic endothelium, the composition optionally not comprising a TGFβ receptor / ALK inhibitor, and ... (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not a TGFβ receptor / ALK inhibitor; and suitable for differentiation and proliferation of mesodermal cells having the ability to differentiate into committed hemogenic endothelium from pluripotent stem cell-derived mesodermal cells; and (iii) a medium comprising a BMP activator, and optionally bFGF; and iPSCs, and suitable for differentiation and proliferation of mesodermal cells from pluripotent stem cell-derived mesodermal cells.

[0017] In some embodiments of the composition for differentiation and proliferation of hematopoietic cells derived from pluripotent stem cells, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in the hematopoietic cells derived therefrom.

[0018] In some embodiments of the composition for differentiation and proliferation of hematopoietic cells derived from pluripotent stem cells, the composition (vi) comprises a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and does not comprise a TGFβ receptor / ALK inhibitor; and comprises a pluripotent stem cell; and comprises an additional medium, such as a medium, suitable for seeding and proliferation of the pluripotent stem cell. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC. In some embodiments, the iPSC comprises one or more genetic imprints, and the one or more genetic imprints contained in the iPSC are retained in the hematopoietic cells derived therefrom.

[0019] In some embodiments of the above compositions for differentiation and proliferation of pluripotent stem cell-derived hematopoietic cells, the compositions further comprise: (i) a medium 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, TPO, a BMP activator, and a ROCK inhibitor; and comprising a pluripotent stem cell-derived pre-T cell progenitor cell and suitable for differentiation of said pluripotent stem cell-derived pre-T cell progenitor cell into a T cell progenitor cell or a T cell; or (ii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator; and comprising pluripotent stem cell-derived committed hemogenic endothelium and suitable for differentiation of said committed hemogenic endothelium into a pre-T cell progenitor cell. These additional media are suitable for the generation of pluripotent stem cell-derived T lineage cells.

[0020] In one embodiment of the above composition for differentiation and expansion of pluripotent stem cell-derived hematopoietic cells, the composition further comprises one or more media for generating hematopoietic multipotent progenitor cells derived from pluripotent stem cells, the media comprising: (i) one or more growth factors and cytokines selected from the group consisting of BMP activators, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but not a ROCK inhibitor, and comprising pluripotent stem cell-derived pre-HSCs and suitable for differentiation of the pre-HSCs into hematopoietic multipotent progenitor cells; and / or (ii) one or more growth factors and cytokines selected from the group consisting of BMP activators, ROCK inhibitors, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, and comprising pluripotent stem cell-derived committed hemogenic endothelium and suitable for differentiation of the committed hemogenic endothelium into pre-HSCs.

[0021] One aspect of the present invention provides a culture platform for generating pluripotent stem cell-derived T lineage cells, the culture platform comprising: (i) a culture medium comprising a BMP activator, and optionally bFGF, and suitable for differentiation and proliferation of pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells having fate-committed HE differentiation potential from said mesodermal cells; (iii) one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; optionally comprising a Wnt pathway activator; and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells having fate-committed HE differentiation potential from said mesodermal cells. a medium suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells having the potential to differentiate into hemogenic endothelium; (iv) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator; and a medium suitable for differentiation of said committed hemogenic endothelium into a pre-T cell precursor; and (v) a medium suitable for differentiation of said pre-T cell precursor into a T cell precursor or a T cell, 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, TPO, a BMP activator, and a ROCK inhibitor; and a medium suitable for differentiation of said pre-T cell precursor into a T cell precursor or a T cell.

[0022] In some embodiments of the above culture platform for generating pluripotent stem cell-derived T lineage cells, the culture platform further comprises (vi) a medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and growing pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs comprise one or more genetic imprints, and the one or more genetic imprints comprised in the iPSCs are retained in the pluripotent stem cell-derived T lineage cells differentiated therefrom.

[0023] Another aspect of the present invention provides a culture platform for generating pluripotent stem cell-derived NK cells, the culture platform comprising: (i) a culture medium comprising a BMP activator, and optionally bFGF, and suitable for differentiation and proliferation of mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells capable of differentiation into committed hemogenic endothelium from mesodermal cells; (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; optionally comprising a Wnt pathway activator; and optionally comprising a TGFβ receptor / ALK inhibitor. (iv) a medium suitable for differentiation of committed hemogenic endothelium from mesodermal cells having the potential to differentiate into committed hemogenic endothelium; (iv) a medium suitable for differentiation of committed hemogenic endothelium into pre-NK cell precursors, the medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7 and IL15, and optionally a BMP activator; and (v) a medium suitable for differentiation of pre-NK cell precursors into NK cell precursors or NK cells, the medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7 and IL15 ... VEGF, bFGF, TPO, a BMP activator and a ROCK inhibitor, the medium being suitable for differentiation of pre-NK cell precursors into NK cell precursors or NK cells.

[0024] In some embodiments of the above culture platform for generating pluripotent stem cell-derived NK cells, the culture platform further comprises (vi) a 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. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs comprise one or more genetic imprints, and the one or more genetic imprints comprised in the iPSCs are retained in the pluripotent stem cell-derived NK lineage cells differentiated therefrom.

[0025] Yet another aspect of the present invention provides a culture platform for generating pluripotent stem cell derived committed hemogenic endothelium (iHE), the culture platform comprising: (i) a culture medium comprising a BMP activator, and optionally bFGF, and suitable for differentiation and proliferation of mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells capable of differentiation into committed hemogenic endothelium from said pluripotent stem cell derived mesodermal cells; and (iii) a culture medium comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, IL11, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells capable of differentiation into committed hemogenic endothelium.

[0026] In some embodiments of the culture platform for generating pluripotent stem cell derived fate-committed hemogenic endothelium (iHE), the culture platform further comprises (iv) a 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 proliferation of pluripotent stem cells.

[0027] Yet another aspect of the present invention provides a culture platform for generating pluripotent stem cell-derived hematopoietic multipotent progenitor cells, the culture platform comprising: (i) a culture medium comprising a BMP activator, and optionally bFGF, and suitable for differentiation and proliferation of pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells capable of differentiation into committed hemogenic endothelium from said pluripotent stem cell-derived mesodermal cells; (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, and (iv) a medium comprising one or more growth factors and cytokines selected from the group consisting of a BMP activator, a ROCK inhibitor, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L and IL11, and suitable for differentiation of committed hemogenic endothelium into pre-HSC; and (v) a medium comprising one or more growth factors and cytokines selected from the group consisting of a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, and not comprising a ROCK inhibitor, and suitable for differentiation of pre-HSC into hematopoietic multipotent progenitor cells. In some embodiments, the culture platform further comprises: (vi) a 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 growing pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs comprise one or more genetic imprints, and the one or more genetic imprints comprised in the iPSCs are retained in the pluripotent stem cell-derived hematopoietic cells differentiated therefrom.

[0028] Another aspect of the present invention provides a method for inducing differentiation of a pluripotent stem cell into a committed hematopoietic cell, the method comprising: (i) contacting a pluripotent stem cell with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of a mesodermal cell from the pluripotent stem cell; (ii) contacting the mesodermal cell with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally no TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of a mesodermal cell having committed HE differentiation potential from the mesodermal cell; and (iii) contacting the mesodermal cell having committed HE differentiation potential. with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, and optionally no TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of committed hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having the ability to differentiate into committed hemogenic endothelium; and optionally exposing the pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells having hemogenic endothelium, and / or committed hemogenic endothelium to a low oxygen partial pressure of about 2% to about 10%.

[0029] In some embodiments of the method for inducing differentiation of pluripotent stem cells into hematopoietic cells, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not comprising 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 iPSCs comprise one or more genetic imprints, and the one or more genetic imprints comprised in the iPSCs are retained in the pluripotent stem cell-derived hematopoietic cells differentiated therefrom.

[0030] In some embodiments of the methods for directing differentiation of pluripotent stem cells into hematopoietic cells, the differentiation of the pluripotent stem cells into hematopoietic cells does not give rise to embryoid bodies and is in a monolayer culture form.

[0031] In some embodiments of the above methods, the resulting pluripotent stem cell-derived committed hemogenic endothelial cells are CD34+. In some embodiments, the resulting hemogenic committed endothelial cells are CD34+CD43-. In some embodiments, the hemogenic committed endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the hemogenic committed endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the hemogenic committed endothelial cells are CD34+CD43-CD93-. In some embodiments, the hemogenic committed endothelial cells are CD34+CD93-.

[0032] In some embodiments of the above methods, the method further comprises: (i) contacting the pluripotent stem cell-derived committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, to induce differentiation of the committed hemogenic endothelium into a pre-T cell progenitor; and optionally (ii) contacting the pre-T cell progenitor 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, TPO, a BMP activator, and a ROCK inhibitor, to induce differentiation of the pre-T cell progenitor into a T cell progenitor or T cell. In some embodiments of the above methods, the pluripotent stem cell-derived T cell progenitor is CD34+CD45+CD7+. In some embodiments of the methods, the pluripotent stem cell-derived T cell progenitors are CD45+CD7+.

[0033] In some further embodiments of the above-described methods for directing differentiation of pluripotent stem cells towards hematopoietic cells, the method further comprises: (i) contacting the pluripotent stem cell-derived committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally, a BMP activator, to trigger differentiation of the committed hemogenic endothelium into a pre-NK cell progenitor; and optionally, (ii) contacting the pluripotent stem cell-derived pre-NK cell progenitor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to trigger differentiation of the pre-NK cell progenitor into an NK cell progenitor or an NK cell. In some embodiments, the pluripotent stem cell-derived NK progenitor cells are CD3-CD45+CD56+CD7+. In some embodiments, the pluripotent stem cell-derived NK cells are CD3-CD45+CD56+, and optionally further characterized as NKp46+, CD57+, and CD16+.

[0034] Another aspect of the present invention provides a method for generating T lineage cells derived from pluripotent stem cells, the method comprising: (i) contacting a pluripotent stem cell with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of mesodermal cells from the pluripotent stem cell; (ii) contacting the mesodermal cell with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not including a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells with committed HE differentiation potential from the mesodermal cell; (iii) contacting the mesodermal cell with committed HE differentiation potential with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; and not including a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells with committed HE differentiation potential from the mesodermal cell. (iv) contacting the committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, to induce differentiation of the committed hemogenic endothelium into a pre-T cell precursor; and (v) contacting the pre-T cell precursor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to induce differentiation of the pre-T cell precursor into a T cell precursor or a T cell; and optionally, the plated pluripotent stem cells, mesodermal cells, mesodermal cells with committed HE differentiation potential, and / or committed hemogenic endothelium may be exposed to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, group II of the above methods further comprises contacting 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 pluripotent stem cells; and / or the 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 the pluripotent stem cells to a T cell lineage does not produce embryoid bodies and is in a monolayer culture format.

[0035] Yet another aspect of the present invention provides a method for generating pluripotent stem cell-derived NK lineage cells, the method comprising: (i) contacting a pluripotent stem cell with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of mesodermal cells from the pluripotent stem cell; (ii) contacting a mesodermal cell with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells with committed HE differentiation potential from the mesodermal cell. (iii) inducing differentiation and proliferation of cells; (iii) contacting mesodermal cells having committed HE differentiation potential with a composition comprising one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; a ROCK inhibitor; optionally a Wnt pathway activator; and optionally not a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of committed pluripotent stem cell-derived hemogenic endothelium from said pluripotent stem cell-derived mesodermal cells having committed HE differentiation potential. (iv) contacting the pluripotent stem cell-derived committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to induce differentiation of the pluripotent stem cell-derived committed hemogenic endothelium into pre-NK cell precursors; and (v) contacting the pluripotent stem cell-derived committed hemogenic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to induce differentiation of the pluripotent stem cell-derived committed hemogenic endothelium into pre-NK cell precursors. L15, but not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to induce differentiation of said pluripotent stem cell-derived pre-NK cell precursors into pluripotent stem cell-derived NK cell precursors or NK cells; and, optionally, exposing the seeded pluripotent stem cells, pluripotent stem cell-derived mesoderm cells, and / or committed hemogenic endothelium to a hypoxic partial pressure of about 2% to about 10%.In some embodiments, the method for generating Group II pluripotent stem cell-derived NK lineage cells further comprises contacting 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 pluripotent stem cell-derived NK lineage cells does not produce embryoid bodies and is in a monolayer culture format.

[0036] Another aspect of the invention provides a method for generating pluripotent stem cell derived committed hemogenic endothelium, the method comprising: (i) contacting iPSCs with a composition comprising a BMP activator, and optionally bFGF, to induce differentiation and proliferation 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, and optionally not a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of pluripotent stem cell derived mesodermal cells having committed HE differentiation potential from the pluripotent stem cell derived mesodermal cells; (iii) contacting the pluripotent stem cell derived mesodermal cells having committed HE differentiation potential with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of pluripotent stem cell derived mesodermal cells having committed HE differentiation potential from the pluripotent stem cell derived mesodermal cells; the composition comprising one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; a ROCK inhibitor; and optionally a Wnt pathway activator, and optionally no TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of pluripotent stem cell-derived committed hemogenic endothelium from the pluripotent stem cell-derived mesodermal cells having committed HE differentiation potential; and optionally exposing the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, and / or committed hemogenic endothelium to a low oxygen partial pressure of about 2% to about 10%. In some embodiments, the method for generating pluripotent stem cell-derived committed hemogenic endothelium further comprises contacting 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; and / or the iPSCs are naive iPSCs. In some embodiments, the iPSCs comprise one or more genetic imprints, and the one or more genetic imprints comprised in the iPSCs are retained in the pluripotent stem cell-derived committed hemogenic endothelial cells differentiated therefrom. In some embodiments, the method for differentiating iPSCs into committed hemogenic endothelial cells does not produce embryoid bodies and is in a monolayer culture format.

[0037] Another aspect of the invention provides a method for generating pluripotent stem cell-derived hematopoietic multipotent progenitor cells, the method comprising: (i) contacting iPSCs with a composition comprising a BMP activator, and optionally bFGF, to induce differentiation and proliferation 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 including a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of said committed HE mesodermal cells from said mesodermal cells; (iii) contacting the committed HE mesodermal cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, but not including a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of committed HE mesodermal cells from said mesodermal cells. (iv) contacting the committed hemogenic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of BMP activators, ROCK inhibitors, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L and IL11 to induce differentiation of the committed hemogenic endothelium into pre-HSCs; and (v) contacting the pre-HSCs with BMP activators to induce differentiation and proliferation of the committed hemogenic endothelium into pre-HSCs. and contacting the pre-HSC with a composition comprising one or more growth factors and cytokines selected from the group consisting of a activating agent, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but not including a ROCK inhibitor, to induce differentiation of the pre-HSC into a hematopoietic multipotent progenitor cell; and optionally exposing the seeded pluripotent stem cells, mesoderm cells, and / or committed hemogenic endothelium to a hypoxic pressure of about 2% to about 10%. In some embodiments, the method for generating pluripotent stem cell-derived hematopoietic multipotent progenitor cells further comprises contacting the pluripotent stem cells 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 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 iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in the pluripotent stem cell-derived hematopoietic multipotent progenitor cells differentiated therefrom. In some embodiments, the differentiation of the pluripotent stem cells into hematopoietic multipotent progenitor cells using the above method does not produce embryoid bodies and is in a monolayer culture format.

[0038] A further aspect of the present invention provides compositions comprising one or more cell populations generated from the culture platform disclosed herein: (i) pluripotent stem cell-derived CD34+ committed hemogenic endothelium (iCD34) that has the ability to differentiate into multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, and B cells, and that are CD34+CD43-; (ii) pluripotent stem cell-derived committed hemogenic endothelium (iHE) that is CD34+ and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) pluripotent stem cell-derived committed HSC that are CD34+CD45+; (iv) CD34+CD4 (v) pluripotent stem cell-derived hematopoietic multipotent progenitor cells that are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells that are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) pluripotent stem cell-derived NK cell progenitor cells that are CD45+CD56+CD7+; (viii) pluripotent stem cell-derived NK cells that are CD3-CD45+CD56+ and optionally further characterized by NKp46+, CD57+, and CD16+; (ix) pluripotent stem cell-derived NKT cells that are CD45+Va24Ja18+CD3+; and (x) pluripotent stem cell-derived B cells that are CD45+CD19+.

[0039] Yet another aspect of the present invention provides one or more cell lines or clonal cells generated using the methods disclosed herein: (i) multipotent stem cell-derived CD34+ committed hemogenic endothelium (iCD34) that has the potential to differentiate into multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NK cells, and NKT cells and that are CD34+CD43-; (ii) multipotent stem cell-derived committed hemogenic endothelium (iHE) that is CD34+ and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) multipotent stem cell-derived committed HSC that is CD34+CD45+; (iv) CD34+CD45+, (v) pluripotent stem cell-derived hematopoietic multipotent progenitor cells (iMPP); (v) pluripotent stem cell-derived T cell progenitors that are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells that are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) pluripotent stem cell-derived NK cell progenitors that are CD45+CD56+CD7+; (viii) pluripotent stem cell-derived NK cells that are CD3-CD45+CD56+ and optionally further characterized as NKp46+, CD57+, and CD16+; (ix) pluripotent stem cell-derived NKT cells that are CD45+Va24Ja18+CD3+; and (x) pluripotent stem cell-derived B cells that are CD45+CD19+.

[0040] Another aspect of the invention provides a method of promoting hematopoietic self-renewal, reconstitution and engraftment using one or more of the cell populations, cell lines or clonal cells generated using the disclosed methods: (i) multipotent stem cell-derived CD34+ committed hemogenic endothelium (iCD34) that has the potential to differentiate into multipotent progenitors, T cell progenitors, NK cell progenitors, T cells, NK cells and NKT cells and that are CD34+CD43-; (ii) multipotent stem cell-derived committed hemogenic endothelium (iHE) that is CD34+ and at least one of CD43-, CD93-, CXCR4-, CD73- and CXCR4-CD73-; (iii) multipotent stem cell-derived committed HSC that is CD34+CD45+; (i (v) pluripotent stem cell-derived hematopoietic multipotent progenitor cells that are CD34+CD45+; (v) pluripotent stem cell-derived T cell progenitor cells that are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells that are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) pluripotent stem cell-derived NK cell progenitor cells that are CD45+CD56+CD7+; (viii) pluripotent stem cell-derived NK cells that are CD3-CD45+CD56+ and optionally further characterized as NKp46+, CD57+, and CD16+; (ix) pluripotent stem cell-derived NKT cells that are CD45+Va24Ja18+CD3+; and (x) pluripotent stem cell-derived B cells that are CD45+CD19+.

[0041] A further aspect of the present invention provides a method for generating hematopoietic cells with enhanced therapeutic properties, comprising obtaining iPSCs comprising one or more genetic imprints; and inducing differentiation of the iPSCs into hematopoietic cells. The differentiation step further comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP pathway 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 committed hemogenic endothelial (HE) differentiation potential capable of giving rise to hematopoietic cells. Preferably, the mesodermal cells and mesodermal cells with committed HE differentiation potential are obtained in step (i) and step (ii) without an embryoid body formation step, and the obtained hematopoietic cells include committed hemogenic endothelial cells, hematopoietic stem / progenitor cells (HSCs), hematopoietic multipotent progenitor cells (MPPs), pre-T cell precursors, pre-NK cell precursors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, or B cells. Furthermore, the hematopoietic cells retain the genetic imprint contained in the iPSCs for differentiation induction.

[0042] In some embodiments, the differentiation induction step of the above method comprises: (i) contacting the committed HE mesodermal cells with a composition comprising bFGF and a ROCK inhibitor to obtain committed HE cells; (ii) contacting the committed 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, Flt3L, and IL11 to obtain hematopoietic multipotent progenitor cells (MPPs); (iii) contacting the committed HE cells with one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7 to obtain hematopoietic multipotent progenitor cells (MPPs). and optionally one or more of a BMP activator, a ROCK inhibitor, TPO, VEGF, and bFGF to obtain a pre-T cell precursor, a T cell precursor, and / or a T cell; or (iv) contacting the committed HE cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, and IL15, and optionally one or more of a BMP activator, a ROCK inhibitor, VEGF, and bFGF to obtain a pre-NK cell precursor, a NK cell precursor, and / or a NK cell.

[0043] One approach to obtain iPSCs containing one or more genetic imprints can be to introduce one or more genetic imprints into iPSCs by gene editing during or after reprogramming non-pluripotent cells into iPSCs, which includes one or more genetically modified modalities and is introduced through intragenomic insertion, deletion or replacement in the genome of the iPSCs. Another approach can be to (i) introduce one or more genetic imprints into iPSCs by obtaining source-specific immune cells that are donor-specific, disease-specific or therapeutic response-specific and exhibit retainable therapeutic properties; (ii) reprogramming the source-specific immune cells into iPSCs; and optionally (iii) introduce additional genetic imprints into the iPSCs of step (ii) by gene editing during or after reprogramming the source-specific immune cells into iPSCs. With respect to the genetically modified modalities contained in the source cells, iPSCs and or iPSC-derived cells, they may include one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or their derived cells.

[0044] In some embodiments, the genetically modified modality is (i) deletion or reduction in expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) deletion or reduction in expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2AThe present invention includes one or more of the following: introduction or increase in expression of a surface triggering receptor for a R, CAR, TCR, or bispecific or multispecific engager. In some embodiments, the above genetically modified modalities are retained in one or more of committed HE cells derived from pluripotent stem cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, and B cells. In some embodiments, the bispecific or multispecific engager is specific for one or more lymphoid cell surface receptors and specific for one or more tumor-specific antigens on the surface of tumor cells. In certain embodiments, the surface triggering receptor is universal in hematopoietic cells including T cells, NK cells, NKT cells, macrophages, and neutrophils. In certain embodiments, the universal surface triggering receptor comprises an anti-epitope and a costimulatory domain, and the anti-epitope is specific for the bispecific or multispecific engager. In some embodiments, the costimulatory domain comprises IL2. In some embodiments, the lymphoid cell surface receptors include one or more of the following surface expressed modified modalities: CD3, CD16, CD64, and CD89; while the tumor-specific antigens include one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In some embodiments, the therapeutic properties of the source-specific immune cells include one or more of: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) resistance to the intratumoral microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0045] Yet another aspect of the invention provides hematopoietic cells having enhanced therapeutic properties and containing the same genetic imprint contained in the pluripotent stem cell from which they were derived. In some embodiments, the genetic imprint of the pluripotent stem cell comprises (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions, or substitutions in the genome of the pluripotent cell during or after reprogramming of the non-pluripotent cell into an iPSC; or (ii) one or more retainable therapeutic properties of a source-specific immune cell that is donor-specific, disease-specific, or therapeutic response-specific, from which the pluripotent cell was reprogrammed. In some embodiments, the genetically modified modalities include one or more of the following: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or their derived cells.

[0046] In some embodiments, the genetically modified modality is (i) deletion or reduction in expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) deletion or reduction in expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2AIn some embodiments, the hematopoietic cells include one or more of: introduction or increase in expression of a surface triggering receptor for a R, CAR, TCR, or bispecific or multispecific engager. In certain embodiments, the surface triggering receptor is universal in hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils. In certain embodiments, the universal surface triggering receptor comprises an anti-epitope and a costimulatory domain, and the anti-epitope is specific for the bispecific or multispecific engager. In some embodiments, the costimulatory domain comprises IL2. In some embodiments, the hematopoietic cells further comprise therapeutic properties of source-specific immune cells associated with one or more of the following: (i) expression of an antigen targeting receptor; (ii) HLA presentation or lack thereof; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune regulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0047] In certain embodiments, the bispecific or multispecific engager is specific for the universal surface triggering receptor and for one or more tumor-specific antigens on the surface of tumor cells. In some embodiments, the tumor-specific antigens include one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In certain embodiments, the therapeutic properties of the source-specific immune cells include one or more of: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0048] Certain embodiments include introducing one or more genetic imprints into the iPSCs after seeding and proliferation by contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor.

[0049] In certain embodiments, the surface triggering receptor is universal in hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils, hi certain embodiments, the universal surface triggering receptor comprises an anti-epitope and a costimulatory domain, and the anti-epitope is specific for the bispecific or multispecific engager.

[0050] In some embodiments, the costimulatory domain comprises IL2.

[0051] In certain embodiments, the bispecific or multispecific engager is specific for one or more tumor-specific antigens on the surface of tumor cells.

[0052] In some embodiments, the hematopoietic cells comprise committed hemogenic endothelial cells, hematopoietic stem or progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell precursors, pre-NK cell precursors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, the T cells comprise regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem). In certain embodiments, the NK cells comprise adaptive NK cells.

[0053] In certain embodiments, the hematopoietic cells comprise one or more bispecific or multispecific engagers for a surface receptor. In some embodiments, the bispecific or multispecific engager is a) hematopoietic cell type specific and specific for a surface receptor comprising CD3, CD16, CD64, or CD89; or b) hematopoietic cell type independent and specific for a universal surface triggering receptor.

[0054] Certain aspects relate to methods of treating a subject in need of cell therapy, comprising administering a therapeutically sufficient number of T cell progenitors resulting from induced pluripotent stem cell (iPSC) differentiation, in some embodiments, the subject (i) is a candidate for bone marrow or stem cell transplantation or has undergone chemotherapy or radiation therapy; (ii) has undergone myeloablative or non-myeolablative chemotherapy or radiation therapy; (iii) has a hyperproliferative disease of the hematopoietic system or cancer; (iv) has a solid tumor; or (v) has a viral infection or a disease associated with a viral infection; and in vivo, the administered T cell progenitors activate the thymus to reconstitute T cells. In some embodiments, the method of treating a subject in need of cell therapy further comprises administering a pharmaceutical composition comprising a bispecific or multispecific engager that is a) specific for an effector cell type and specific for a surface receptor comprising CD3, CD16, CD64, or CD89; or b) independent of effector cell type and specific for a universal surface triggering receptor contained in the T progenitor cells and T cells derived therefrom. In some embodiments, the genetic imprint from the source-specific immune cells comprises one or more of: (i) expression of an antigen targeting receptor; (ii) HLA presentation or lack thereof; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0055] Also provided are pharmaceutical compositions comprising a pharma- ceutically acceptable medium and one or more of the hematopoietic cells with enhanced therapeutic properties produced using the methods described herein. These hematopoietic cells with enhanced therapeutic properties include committed hemogenic endothelial cells, hematopoietic stem or progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell precursors, pre-NK cell precursors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, or B cells. In some embodiments, the T cells include regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem). In some embodiments, the NK cells include adaptive NK cells. Further provided are therapeutic uses of the above pharmaceutical compositions by introducing the composition into a subject suitable for adoptive cell therapy having an autoimmune disorder; a hematopoietic tumor; a solid tumor; cancer; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0056] Another aspect of the invention provides an antibody composition comprising one or more antibodies specific for at least one marker selected from the following: CCR10, CD164, CD95, CD144, CD166, lymphotoxin beta receptor, CD252, CD55, CD40, CD46, CD340, CD119, CD106, CD66a / c / e, CD49d, CD45RB, DLL4, CD107a, CD116, CD324, CD123, CD49f, CD200, CD71, CD172a, CD21, CD184, CD263, CD221, Notch4, MSC, CD97, CD319, CD69, CD 338, podoplanin, CD111, CD304, CD326, CD257, CD100, CD32, CD253, CD79b, CD33, CD83, GARP, CD183, CD357, CD31, CD165, CD102, CD146, CD49c, CD13, CD58, integrin α9β1, CD51, CD10, CD202b, CD141, CD49a, CD9, CD201, CD47, CD262, CD109, CD39, CD317, CD143, integrin β5, CD105, CD155, SSEA-4, and CD93; said markers for identifying committed hemogenic endothelium. In one embodiment, the antibody composition comprises an antibody specific for CD93; cells bound to the antibody specific for CD93 have one or more of a CD34+ phenotype, a CD43- phenotype, a CD73- phenotype, a CXCR4- phenotype, and a CD73-CXCR4- phenotype.

[0057] Yet another aspect of the present invention provides a method for identifying committed hemogenic endothelium in the differentiation of pluripotent stem cells, the method comprising: (i) obtaining a differentiating cell population; (ii) introducing a CD93-specific antibody into the cell population; and (iii) identifying a cell population having a CD93 expression level of less than 1%. In some embodiments, the differentiating cell population comprises CD34+ cells or CD34+CD43- cells. In some embodiments, the method further comprises isolating CD34+ cells from the differentiating cell population; or isolating CD34+CD43- cells from the differentiating cell population.

[0058] Further provided is a method for producing a pluripotent stem cell-derived committed hemogenic endothelium, comprising culturing a pluripotent stem cell-derived mesodermal cell having committed hemogenic endothelial (HE) differentiation potential in a medium containing a Wnt pathway agonist to obtain committed HE cells. Obtaining committed HE in the presence of a Wnt pathway activator increases the number and percentage of HE in a cell population; improves HE differentiation potential; and / or improves HE cellularity, compared to culturing without a Wnt pathway activator. In some embodiments, the medium further comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC. In some embodiments, the iPSC comprises one or more of the above genetic imprints, and the genetic imprints are retained in the committed HE derived from the pluripotent stem cell.

[0059] Certain aspects of the invention are directed to antigen-specific iPSCs or derived hematopoietic cells produced by the methods described herein. Certain aspects of the invention relate to compositions comprising antigen-specific iPSCs or derived hematopoietic cells produced by the methods described herein. Some aspects of the invention relate to pharmaceutical compositions comprising antigen-specific iPSCs or derived hematopoietic cells produced by the methods described herein and a pharma- ceutically acceptable medium.

[0060] Also provided herein are methods of generating antigen-specific induced pluripotent stem cells (iPSCs) and derived hematopoietic cells, the methods comprising: (i) isolating primary antigen-specific T cells from a selected source that is donor-specific, disease-specific, or treatment response-specific; (ii) reprogramming the primary antigen-specific T cells to obtain pluripotent stem cells; and (iii) inducing the differentiation of the pluripotent stem cells into hematopoietic cells. In some embodiments, the differentiation step comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP pathway 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 capable of differentiation into committed hemogenic endothelium (HE) without the formation of embryoid bodies. In some embodiments, the isolated primary antigen-specific T cells are enriched by (i) co-culturing the primary antigen-specific T cells with tumor cells, non-transformed cells, dendritic cells, thymic epithelial cells, endothelial cells, or artificial antigen-presenting cells, particles or peptides in plasma that express the antigen of interest; or sorting the primary antigen-specific T cells with a T cell receptor-specific binding agent specific for the antigen of interest. In some embodiments, the primary antigen-specific T cells or enriched primary antigen-specific T cells can be modulated with a transcription factor or small molecule to rejuvenate the cells.

[0061] Certain aspects of the invention are directed to antigen-specific iPSCs or derived hematopoietic cells produced by the methods described herein. Certain aspects of the invention relate to compositions comprising antigen-specific iPSCs or derived hematopoietic cells produced by the methods described herein. Some aspects of the invention relate to pharmaceutical compositions comprising antigen-specific iPSCs or derived hematopoietic cells produced by the methods described herein and a pharma- ceutically acceptable medium.

[0062] In some embodiments, the antigen-specific pluripotent stem cells obtained from the above method can be further genetically modified during or after reprogramming to include a genetic imprint comprising one or more genetically modified modalities via genomic insertion, deletion, or replacement. In some embodiments, the genetically modified modalities include one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates; cell surface proteins conveying secondary or tertiary antigen specificity; or proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or derived cells. In some embodiments, the genetically modified modality is (i) deletion or reduction in expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) deletion or reduction in expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2AIn some embodiments, the bispecific or multispecific engager is hematopoietic cell type specific and specific for a surface receptor selected from CD3, CD16, CD64, or CD89. In some embodiments, the surface triggering receptor is universal in hematopoietic cells including T cells, NK cells, NKT cells, macrophages, and neutrophils. In some embodiments, the bispecific or multispecific engager is hematopoietic cell type independent and capable of binding to hematopoietic cells that contain a matching universal surface triggering receptor. In some embodiments, the universal surface triggering receptor comprises an anti-epitope and a costimulatory domain, and the anti-epitope is specific for an epitope comprised in the bispecific or multispecific engager. In some embodiments, the costimulatory domain of the universal surface triggering receptor comprises all or a portion of IL2 for enhanced conotical or non-conotical cell activation and / or effector cell function. In some embodiments, the bispecific or multispecific universal engager is specific for one or more tumor-specific antigens on the surface of tumor cells. In some embodiments, the tumor-specific antigens comprise one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In some embodiments, the bispecific or multispecific engager is specific for the universal surface triggering receptor and for one or more tumor-specific antigens on the surface of tumor cells.

[0063] In some embodiments, the antigen-specific iPSC-derived hematopoietic cells comprise committed hemogenic endothelial cells (HE), hematopoietic stem or progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell progenitors, pre-NK cell progenitors, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils.

[0064] A further aspect of the present invention provides a method for determining the clonality of induced pluripotent stem cells and their derived cells. The method typically comprises reprogramming a mature source T cell or B cell to obtain an induced pluripotent stem cell (iPSC); and detecting the presence of a specific V(D)J gene rearrangement in the iPSC or hematopoietic cells derived therefrom that is identical to that contained in the mature T cell or B cell from which the iPSC was obtained. In some embodiments, the method further comprises isolating an iPSC or hematopoietic cell that contains a V(D)J gene rearrangement identical to that of the mature source T cell or B cell. In some embodiments, the method comprises, prior to reprogramming the source cell, obtaining a mature source T cell or B cell for reprogramming; and determining a V(D)J gene rearrangement contained in an immunoglobulin (Ig) or T cell receptor (TCR) specific for the mature source T cell or B cell.

[0065] Yet another aspect of the invention provides a method of tracking adoptive cells in vivo in cell therapy, the method comprising obtaining a blood, tissue, or tumor biopsy sample from a subject receiving adoptive cells for therapy, isolating effector cells in the sample; and determining a V(D)J gene rearrangement of the effector cells; the adoptive cells are derived from pluripotent stem cells reprogrammed from mature source T cells or B cells; the mature source T cells or B cells comprise a particular V(D)J gene rearrangement; and the presence of the same V(D)J gene rearrangement from the mature source T cells or B cells indicates homing, persistence and / or expansion of the adoptive cells.

[0066] Also provided is a pharmaceutical composition comprising a pharma- ceutically acceptable medium and one or more of the antigen-specific hematopoietic cells produced using the above method, the antigen-specific hematopoietic cells including committed hemogenic endothelial cells, hematopoietic stem or progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell precursors, pre-NK cell precursors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, or B cells. In some embodiments, the T cells include regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem). In some embodiments, the NK cells include adaptive NK cells. Further provided is a therapeutic use of the pharmaceutical composition by introducing the composition into a subject having an autoimmune disorder; hematopoietic malignancy; solid tumor; cancer; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus, suitable for adoptive cell therapy.

[0067] In summary, the present invention provides methods and compositions that allow for the direct differentiation of pluripotent stem cells in monolayers without embryoid body formation from pluripotent stem cells, thereby achieving differentiation and proliferation of mesodermal cells, fate-committed HE, and fate-committed HSCs from which other hematopoietic cells can be derived with very high levels of efficiency in a scalable and reliable manner. [Brief description of the drawings]

[0068] [Figure 1] Figure 1 shows a schematic of the multi-step culture process for hematopoietic differentiation of induced pluripotent stem cells (iPSCs) into committed hemogenic endothelial (iHE) and multipotent progenitor cells (iMPPs), which can be converted to a fully defined culture medium in which vitronectin is replaced with Matrigel™.

[0069] [Diagram 2]Figure 2 shows a schematic of the multi-step culture process for hematopoietic differentiation of induced pluripotent stem cells into T cell progenitor (ipro-T) and fully differentiated T (iT) cells, where the culture medium can be converted to a fully defined one in which vitronectin is replaced with Matrigel™.

[0070] [Diagram 3] Figure 3 shows a schematic of the multi-step culture process for hematopoietic differentiation of induced pluripotent stem cells into NK cell progenitors (ipro-NK) and fully differentiated NK (iNK) cells, where the culture medium can be converted to a fully defined one in which vitronectin is replaced with Matrigel™.

[0071] [Figure 4A] Figures 4A-C show flow cytometry profiles demonstrating the emergence of iHE over 10 days, as well as the yield of iCD34 and iHE cells per iPSC differentiation. Calculations are based on snapshots of representative and non-optimized cultures. [Figure 4B] Same as above. [Figure 4C] Same as above.

[0072] [Figure 5A] 5A-5D show modifications to the protocol, including seeding density and growth factor titration, to improve day 10 HE yield. A) Seeding density on day 0 affects day 10 HE population. B) Concentration of BMP4 on days 2-6 affects day 10 HE population. C) Concentration of CHIR on day 3.75 affects day 10 HE population. D) Seeding density on day 6 affects day 10 HE population. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above.

[0073] [Figure 6A]Figures 6A-B show that day 10 HE represents definitive hematopoiesis that is multipotent and dependent on the Notch signaling pathway. A) Morphological changes in the MPP assay over 7 days accompanied by flow cytometry profiles of nascent CD45 hematopoietic cells. B) iPSC-derived CD34+ cells give rise to Notch-dependent fate-committed CD45+ cells during the iMPP assay. [Figure 6B] Same as above.

[0074] [Figure 7A] Figures 7A-B show the effect of differentiation under hypoxic conditions on the generation of iHE and iMPP hematopoietic progenitors. A) Monolayer differentiation under hypoxia 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 the iMPP assay. [Figure 7B] Same as above.

[0075] [Figure 8-1] Figures 8A-D show the ability of sorted day 10 iCD34 cells to maintain their cryopreservation potential and pan-hematopoietic and lymphoid differentiation potential. A) Cryopreserved day 10 iCD34 cells survive upon thawing and display a phenotype similar to fresh iCD34+ cells. B) Viability of cryopreserved day 10 selected CD34+ cells immediately after thawing. C) Cryopreserved day 10 selected iCD34+ cells survive during an iMPP assay to give rise to CD45+ hematopoietic cells. D) Cryopreserved day 10 selected iCD34+ cells survive to give rise to iT and iNK lymphoid progenitors. [Figure 8-2] Same as above.

[0076] [Figure 9A]Figures 9A-B show that day 10 differentiation cultures can be shipped overnight at ambient temperature without losing HE differentiation potential. A) Day 7 cultures were either maintained in the incubator (control) or processed for overnight shipping and reintroduced into the incubator for an additional 2 days. Both day 10 cultures were then analyzed for the presence of iCD34 and iHE cells. For overnight shipping cultures, T-flasks contained 30% medium + 70% base or 100% medium. B) Calculation of cell number. [Figure 9B] Same as above.

[0077] [Figure 10-1] Figure 10A-C show early CD34+CD7+ T cell progenitors and mature CD4+ and CD8+ T cell subsets derived from hiPSCs using a CD45+CD56-gating strategy. A) Early T cell lineage markers mark the presence of ipro-T cells defined by CD34+ / CD7+. B) Mature T cell markers mark the presence of mature T cells defined by CD4+ or CD8+ cells. C) Five-day T cell differentiation comparing the differentiation potential of cord blood-derived CD34+ cells and iCD34+ cells to give rise to ipro-T cells. [Figure 10-2] Same as above.

[0078] [Figure 11A] Figure 11A-C show early CD56+CD7+CD161+ NK cell progenitors and mature CD56+CD16+CD8+ NK cell subsets derived from hiPSCs using a CD45+ gating strategy. A) Early NK lineage markers mark the presence of ipro-NK cells defined by CD7 and CD56. B) Mature NK lineage markers mark the presence of mature NK cells defined by CD57, CD16, CD94 and CD56. C) Five-day NK cell differentiation comparing the differentiation potential of cord blood-derived CD34+ cells and iCD34+ cells to give rise to ipro-NK cells. [Figure 11B] Same as above. [Figure 11C] Same as above.

[0079] [Figure 12A] Figures 12A-C show that the monolayer hiPSC hematopoietic differentiation platform allows for a scalable expansion strategy not seen during EB formation. A. hiPSCs were aggregated to form embryoid bodies and differentiated for 14 days and then analyzed for CD34 and CD43 expression. B. hiPSCs were plated in monolayer, differentiated for 8 days and then analyzed for CD34, CD43, CXCR4 and CD73. C. CD34 positive cells were counted and plotted over time for both monolayer and EB-mediated hematopoietic differentiation. [Figure 12B] Same as above. [Figure 12C] Same as above.

[0080] [Figure 13] Figure 13 illustrates a schematic of a scalable expansion strategy of a monolayer hiPSC hematopoietic differentiation platform for off-the-shelf iNK and iT cell production. Calculations are based on snapshots of representative and not optimized cultures.

[0081] [Figure 14] FIG. 14 shows that hiPSC-derived CD34 positive cells have immunoregulatory properties through the suppression of CD3+ T cell survival.

[0082] [Figure 15] FIG. 15 shows mature CD4+ and CD8+ T cell subsets derived from hiPSCs 30 days after HE isolation using a CD45+CD56-gating strategy.

[0083] [Figure 16] FIG. 16 shows that feeder-based suspension culture supports the maturation of iCD34-derived NK cells.

[0084] [Figure 17] FIG. 17 shows that iCD34-derived iNK, like peripheral blood NK cells, can secrete proinflammatory cytokines in response to cytokine stimulation.

[0085] [Figure 18] FIG. 18 shows that stromal-free differentiation of pro-NK cells from cord blood CD34 positive cells is more rapid than conventional stromal-based differentiation platforms using a CD45+ gating strategy.

[0086] [Figure 19] Stromal-free differentiation of iPSC-derived iCD34+ cells towards NK cells. Plate-bound DLL4 supports differentiation of CD56+CD7+CD161+ NK cell progenitors but not CD11b+ myeloid cells.

[0087] [Figure 20] FIG. 20 shows stromal-free differentiation of UCB CD34+ cells towards T cells.

[0088] [Figure 21] FIG. 21 shows stromal-free differentiation of iPSC-derived iCD34+ cells towards T cells.

[0089] [Figure 22] FIG. 22 shows the reconstitution and engraftment of hiPSC-derived iCD34+ cells.

[0090] [Figure 23]Figures 23A-B show that iPSC-derived NK cells secrete proinflammatory cytokines in response to cell stimulation and have cytotoxic function comparable to peripheral blood NK cells and cord blood NK cells. A. iPSC-derived NK cells (iNK) or peripheral blood-derived NK cells (pbNK) were exposed to unstimulated (US) or stimulation with a 1:1 ratio of feeder cells for 4 hours, then harvested, stained for CD45, CD56, and TNF-α, and analyzed by flow cytometry. B. The cytotoxic function of iNK cells or cord blood-derived (CBNK) cells at effector to target cell ratios of 1:1, 3:1, and 10:1 was assessed every 2 hours for 90 hours.

[0091] [Figure 24] FIG. 24 shows the reprogramming of CAR-T cells into iPSCs (TiPSCs) that retain the same genetic imprint of the source T cells by PCR analysis of CAR (FTV106) integration in iPSCs derived from FTV106-transduced T cells.

[0092] [Diagram 25] Figure 25 shows the cell surface antibody screening of hiPSC-derived CD34+ cells using a CD34+CD43- gating strategy, and the workflow of hiPSC-derived CD34+ antibody screening, with representative results of three separate classifications shown. Class I markers include cell surface proteins with less than 1% expression on hiPSC-derived CD34+ cells. Class II markers include cell surface proteins with 1%-99% expression on CD34+ cells. Class III markers include cell surface proteins with greater than 99% expression on CD34+ cells.

[0093] [Figure 26-1] FIG. 26 shows anti-human antibodies used to analyze cell surface protein expression on hiPSC-derived CD34+ cells. [Figure 26-2] Same as above.

[0094] [Figure 27] FIG. 27 shows the expression of the CD93 marker within the CD34+ population; and the expression of CXCR4 and CD73 in the CD34+CD93− and CD34+CD93+ fractions of CD34+ cells.

[0095] [Figure 28] Figures 28A-B show that committed hematopoietic differentiation potential of the CD34+ population is enriched in the CD93- fraction. A. After 10 days of iNK cell differentiation from the CD34+CD93- population, absolute numbers of CD45+ hematopoietic cells and CD45+CD7+ lymphoid progenitors were assessed. B. After an additional 10 days of iNK differentiation, cultures were assessed for the presence of iNK cells by expression of the CD56 and NKp30 markers.

[0096] [Figure 29] Figures 29A-B show that modulation of WNT signaling improves iCD34 cell yield. A. Day 6 cell cultures were differentiated in the presence of the GSK3 inhibitor CHIR99021, Wnt agonists, or the WNT inhibitor IWP2. B. CHIR99021 increased the number and percentage of CD34+CD43-CD93-HE phenotype.

[0097] [Diagram 30] Figures 30A-B show that modulation of WNT signaling improves total hematopoietic and lymphoid output from iCD34 cells. A. CHIR99021 increased the production of CD45+ cells from conditioned HE. B. CHIR99021 increased the production of NK progenitor cells from conditioned HE.

[0098] [Diagram 31] FIG. 31 shows that deletion of B2-microglobulin (B2M) in iPSCs resulted in lack of expression of HLA class I genes.

[0099] [Figure 32-1]Figures 32A-D show that HLA class I null iPSCs can differentiate into iCD34HE and further into all hematopoietic and lymphoid progenitors. A. B2M- / - iPSCs and wild-type iPSCs were differentiated for 10 days to generate HE. B. B2M- / - iPSCs can differentiate into CD34+HE at a frequency similar to wild-type controls. C. B2M- / -HE can generate all CD45+ hematopoietic progenitors with the same efficiency as wild-type HE. D. B2M- / - iPSC-derived HE cells can generate iNK progenitors with the same efficiency as wild-type HE. [Figure 32-2] Same as above.

[0100] [Diagram 33] FIG. 33 shows that CD34+CD43-HE differentiated from B2M- / - iPSCs remain HLA class I null.

[0101] [Figure 34A] Figures 34A-B show that modulation of HLA class I on iPSCs increases persistence of iPSCs in immunocompetent recipients. A. Transfected HLA-modified iPSCs express HLA-E on the cell surface and maintain a pluripotent phenotype. B. Luciferin imaging of teratomas 72 hours after injection of B2M- / -HLAE iPSCs showing increased persistence compared to wild-type iPSCs in vivo. [Figure 34B] Same as above.

[0102] [Diagram 35] Figure 35 shows that HLA-E expression is retained in both day 10 (CD34+CD43-HE cells) and day 17 (CD45+ all hematopoietic progenitor cells) differentiated cells derived from B2M- / -HLA-E iPSCs.

[0103] [Figure 36-1]Figures 36A-C show that HLA class I conditioned iPSCs can generate functional CD34+ HE. A. B2M- / -HLA-E iPSCs can differentiate into CD34+ HE at a frequency similar to wild type controls. B. B2M- / -HLA-E iPSCs can differentiate into CD34+ HE in numbers similar to wild type controls. C. B2M- / -HLA-E HE can generate CD45+ total hematopoietic progenitor cells with efficiency similar to wild type HE. [Figure 36-2] Same as above.

[0104] [Figure 37A] Figures 37A-B show that iPSCs genetically modified to express the high affinity CD16 receptor and the 41BBL costimulatory molecule retain expression throughout differentiation into iCD34 cells. A. Undifferentiated cells at day 0. B. Differentiated cells at day 10. [Figure 37B] Same as above.

[0105] [Figure 38A] Figures 38A-B show that iPSCs genetically modified to express CD19 chimeric antigen receptor (CAR) and truncated LNGFR cell surface marker as a common identifier for CAR retain expression through differentiation into iCD34 cells. A. Undifferentiated cells at day 0. B. Differentiated cells at day 10. [Figure 38B] Same as above.

[0106] [Figure 39] Figure 39 shows cell type-specific expression of the CAR driven by the endogenous TCR promoter and knockout of TCR expression and function by locus-specific insertion of the CAR.

[0107] [Diagram 40]Figures 40A-B show an off-the-shelf targeting strategy to engage the entire effector repertoire of T cells targeting cancer and other diseases. A. Illustrated iPSC-derived hematopoietic effector cells with their respective lineage-specific triggering molecules capable of binding to engagers that recognize specific target cells. B. Illustrated modification of a universal engager (a specific and lineage-independent triggering molecule with a specific anti-epitope binding) that is ubiquitously expressed on all derived hematopoietic cells.

[0108] [Diagram 41] FIG. 41 shows that iPSCs genetically modified to express the HACD16 receptor retain expression through differentiation into day 20 iNK cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] The present invention generally relates to methods and compositions for differentiating stem cells toward a pre-defined fate into committed hematopoietic cells. More specifically, the present invention provides a multi-step differentiation platform where iPSCs or iPSC-derived cells at various developmental stages can be induced to adopt a pre-defined hematopoietic phenotype ranging from pre-defined hemogenic endothelium to fully differentiated hematopoietic cells 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 adopt a pre-defined hematopoietic fate (e.g., CD34+ committed hematopoietic stem cells). Alternatively, the present methods and compositions generate pre-defined hemogenic endothelium (HE) from naive iPSCs in a scalable manner by avoiding the formation of EBs or clumps. A.Definition

[0110] 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. The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0111] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0112] It should be understood that the term "and / or" means either one or both of the alternatives.

[0113] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs by about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about ±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.

[0114] 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 compared to 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0115] 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 a medium, refer to a composition that is free of a particular substance or source thereof, e.g., free of 95%, free of 96%, free of 97%, free of 98%, free of 99%, or undetectable when measured by routine methods. The term "free of" or "essentially free of" a particular component or substance in a composition 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 concentration that is functionally inactive. The same meaning applies to the term "absence of," which refers to the absence of a particular substance or source of a composition.

[0116] As used herein, the term "appreciable" refers to a quantity, level, value, number, frequency, proportion, size, magnitude, amount, weight or length range, or event that is readily detectable by one or more standard methods. The terms "not-appreciable" and "not appreciable" and their synonyms refer to a quantity, level, value, number, frequency, proportion, size, magnitude, amount, weight or length range, or event that is not readily detectable or undetectable by standard methods. In one embodiment, an event is not detectable if it occurs with a probability of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001% or less.

[0117] Throughout this specification, unless the context requires otherwise, the words "comprising" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of other steps or elements or groups of steps or elements. In certain embodiments, the terms "comprising," "having," "containing," and "comprising" are used interchangeably.

[0118] "Consisting of" means inclusive and limited to everything listed before the phrase "consisting of." Thus, the phrase "consisting of" implies that the listed elements are required or mandatory, and that no other elements may be present.

[0119] "Consisting essentially of" is meant to include all elements that are recited in the phrase and are limited to other elements that do not intervene or contribute to the operation or function specified in the disclosure of the recited elements. That is, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the operation or function of the recited elements.

[0120] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included 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.

[0121] The term "ex vivo" generally refers to activities carried out outside an organism, such as experiments or measurements made in or on living tissue in an artificial environment outside the organism, preferably with minimal differences from natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues obtained from an organism and cultured, usually under sterile conditions, in laboratory equipment, typically for a few hours or up to about 24 hours, but up to 48 or 72 hours depending on the circumstances. In some embodiments, such tissues or cells can be harvested, frozen, and then thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues that last longer than a few days are typically considered "in vitro", although in some embodiments the term may be used interchangeably with ex vivo.

[0122] The term "in vivo" generally refers to activities carried out within an organism.

[0123] As used herein, the term "primitive streak" refers to the early embryonic structure where gastrulation or the formation of the three germ layers (mesoderm, endoderm and ectoderm) begins.

[0124] As used herein, the term "mesoderm" refers to one of three germ layers that emerge during early embryogenesis and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0125] As used herein, the terms "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process termed endothelial-to-hematopoietic transition. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm, through hemangioblasts, to committed hemogenic endothelium and hematopoietic progenitor cells.

[0126] The term "hematopoietic stem cell," or "definitive hematopoietic stem cell," as used herein, refers to a CD34+ stem cell that can give rise to both mature myeloid cell types and mature lymphoid cell types, including T cells, natural killer cells, and B cells.

[0127] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potency" or "increasing developmental potency" refers to a method of increasing the capacity of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-initialized state. That is, a cell that is less differentiated than the same cell in a non-initialized state is a reprogrammed cell.

[0128] As used herein, the term "differentiation" is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, a blood cell or a muscle cell. A differentiated or differentiation-induced cell is a cell that has achieved a more specialized ("committed") position within a cell lineage. The term "committed" as applied to the process of differentiation refers to a cell that has progressed down a differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a particular 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.

[0129] As used herein, the term "differentiation marker gene" or "differentiation gene" refers to a gene whose expression indicates that cell differentiation is occurring in a cell, such as a pluripotent cell. Differentiation marker genes include, but are not limited to, the following genes: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH 11, 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, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), ZIC1, GATA1, GATA2, HDAC4, HDAC5, HDAC7, HDAC9, NOTCH1, NOTCH2, NOTCH4, PAX5, RBPJ, RUNX1, STAT1 and STAT3.

[0130] As used herein, the terms "differentiation marker gene profile" or "differentiation gene profile," "differentiation gene expression profile," "differentiation gene expression signature," "differentiation gene expression panel," "differentiation gene panel," or "differentiation gene signature" refer to the expression or expression levels of multiple differentiation marker genes.

[0131] As used herein, the term "potency" refers to the sum of all developmental options that a cell can attain (i.e., developmental potential). The continuum of potential includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and well-differentiated cells.

[0132] As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or soma (i.e., embryo proper). For example, an embryonic stem cell is 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 that ranges from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0133] As used herein, the term "induced pluripotent stem cell" or iPSC refers to stem cells generated from differentiated mature neonatal or embryonic cells that are induced or transformed, i.e., reprogrammed, into cells capable of differentiating into tissues of all three germ layers or layers (mesoderm, endoderm, and ectoderm). Generated iPSC does not refer to the cell as it exists in nature. As used herein, iPSC encompasses genomically modified iPSCs or iPSCs reprogrammed from immune cells of a preferential donor or patient, i.e., unique genetic imprints are contained in the iPSCs, and / or derived lymphoid effector cells.

[0134] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to a preferential therapeutic property in a source cell or iPSC and can be retained in iPSCs derived from the source cell and / or hematopoietic cells derived from the iPSC. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and iPSCs derived from a source cell can be further differentiated into specific cell types, including any hematopoietic cell. Depending on the context, iPSCs derived from a source cell and differentiated cells therefrom may be collectively referred to as "derived cells." As used herein, genetic imprints that confer preferential therapeutic properties are incorporated into iPSCs through donor-specific, disease-specific, or treatment response-specific reprogramming of selected source cells, or through the introduction of genetic modification patterns into iPSCs using genome editing. In the context of source cells obtained from a specifically selected donor situation, disease situation or treatment situation, the genetic imprints contributing to the preferential therapeutic properties may include any situation-specific genetic or epigenetic modifications that manifest a retainable phenotype, i.e., preferential therapeutic properties, inherited by the derived cells of the selected source cells, regardless of whether the underlying molecular phenomenon has been identified. Donor-specific, disease-specific, or treatment response-specific source cells may include genetic imprints that can be retained in iPSCs and derived hematopoietic cells, including, but not limited to, those from a given single-specific TCR, such as virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, such as those homozygous for a point mutation that codes for high affinity CD16 receptor in the selected donor; and selected HLA-matched donor cells that exhibit a given HLA requirement, i.e., a population-increased haplotype.As used herein, preferential therapeutic properties include improved engraftment, improved trafficking, improved homing, improved viability, improved self-renewal, improved persistence, improved control and regulation of immune responses, improved survival, and improved cytotoxicity of derived cells. Preferential therapeutic properties may also be related to expression of antigen targeting receptors; HLA presentation or lack thereof; tolerance to the tumor microenvironment; induction of bystander immune cells and immune regulation; improved on-target specificity with reduced off-tumor effects; and resistance to treatments such as chemotherapy.

[0135] The term "enhanced therapeutic properties" as used herein refers to enhanced therapeutic properties of cells when compared to typical immune cells of the same normal cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties when compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of immune responses, survival, and cytotoxicity. Therapeutic properties of immune cells are also manifested by: antigen targeting receptor expression; HLA presentation or lack thereof; resistance to intratumoral microenvironment; induction of bystander immune cells and immune regulation; improved target specificity with reduced extratumoral effects; resistance to treatments such as chemotherapy.

[0136] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, capable of forming a link between an immune cell, e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil, and a tumor cell; and activating the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0137] As used herein, the term "surface triggering receptor" refers to a receptor that can trigger or initiate an immune response, such as a cytotoxic response. The surface triggering receptor may be engineered and expressed on effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, the surface triggering receptor promotes binding between effector cells and specific target cells (e.g., tumor cells) by bispecific or multispecific antibodies, regardless of the effector cell's natural receptor and cell type. Using this approach, iPSCs containing universal surface triggering receptors may be generated, and then the iPSCs may be differentiated into a population of various effector cell types that express the universal surface triggering receptor. By "universal" it is meant that the surface triggering receptor can be expressed on and activate any effector cell, regardless of cell type, and that all effector cells expressing the universal receptor can be coupled or linked to an engager with the same epitope recognizable by the surface triggering receptor, regardless of the tumor-binding specificity of the engager. In some embodiments, engagers with the same tumor targeting specificity are used to couple with the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificity are used to couple with the universal surface triggering receptor. Thus, binding one or more effector cell types can kill one particular type of tumor cell in some cases, and can kill more than one type of tumor in other cases. Surface triggering receptors usually contain a costimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the engager. The bispecific engager is specific for an anti-epitope of a surface triggering receptor at one end and a tumor antigen at the other end.

[0138] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxic or otherwise adverse effects of a cell therapy. In some cases, expression of the safety switch protein is conditionally controlled to address safety concerns regarding transplanted engineered cells that have permanently incorporated into their genome a gene encoding the safety switch protein. This conditional control can be variable and can include control via small molecule mediated post-translational activation and tissue-specific and / or temporal transcriptional control. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and / or antibody mediated depletion. In some cases, the safety switch protein is activated by an exogenous molecule (e.g., a prodrug) that, when activated, causes apoptosis and / or cell death of the therapeutic cell. Examples of safety-switch proteins include, but are not limited to, caspase 9, thymidine kinase, cytosine deaminase, B-cell CD20, suicide genes such as modified EGFR, and any combination of these. In this strategy, a prodrug administered in the event of an adverse event is activated by the suicide gene product and kills the transduced cell.

[0139] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative, therapeutic, or symptomatic properties against a disease and can be administered to ameliorate, alleviate, mitigate, reverse, or reduce the severity of the disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the occurrence of a disease or to reduce the severity of such a disease or pathological condition if it does occur. Pharmaceutically active proteins include whole proteins or peptides or pharmaceutical active fragments thereof. Pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides are also included. The term pharmaceutical active proteins also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharma- ceutical active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0140] As used herein, the term "signal transduction molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases intercellular communication. Signal transduction refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along a pathway that ultimately initiates biochemical events in cells. Signal transduction pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signal transduction, tyrosine kinase receptor signal transduction, integrin signal transduction, toll gate signal transduction, ligand-gated ion channel signal transduction, ERK / MAPK signal transduction pathway, Wnt signal transduction pathway, cAMP-dependent pathway, and IP3 / DAG signal transduction pathway.

[0141] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, genetically incorporated into cells to promote antigen and / or epitope specificity, including but not limited to: i) antigen specificity, in the case of unique chimeric antigen receptors (CARs) or T cell receptors (TCRs); ii) engager specificity, in the case of monoclonal antibodies or bispecific engagers; iii) transformed cell targeting; iv) cancer stem cell targeting; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0142] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Examples of pharmaceutical active proteins or peptides include, but are not limited to, antibodies or fragments thereof, growth factors, and / or cytokines.

[0143] As used herein, the term "signal transduction molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases intercellular communication. Signal transduction refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along a pathway that ultimately initiates biochemical events in cells. Signal transduction pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signal transduction, tyrosine kinase receptor signal transduction, integrin signal transduction, toll gate signal transduction, ligand-gated ion channel signal transduction, ERK / MAPK signal transduction pathway, Wnt signal transduction pathway, cAMP-dependent pathway, and IP3 / DAG signal transduction pathway.

[0144] As used herein, the term "specific" may be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0145] As used herein, the term "adoptive cell therapy," as used herein, refers to cell-based immunotherapy involving the transfusion of autologous or allogeneic lymphocytes, which may or may not be genetically modified and which may be T cells or B cells that have been expanded ex vivo prior to transfusion.

[0146] A "therapeutically sufficient amount," as used herein, includes within its meaning a non-toxic, but sufficient and / or effective amount of a particular therapeutic and / or pharmaceutical composition that is believed to produce a desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, reduce, and / or ameliorate at least one symptom associated with the disease or condition of the subject being treated.

[0147] As used herein, the term "embryonic stem cell" refers to the spontaneously occurring pluripotent stem cell of the inner cell mass of embryonic blastocyst. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers (ectoderm, endoderm and mesoderm) during development. Embryonic stem cells do not contribute to extraembryonic membranes or placenta, i.e., they are not pluripotent.

[0148] 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, but not all three, germ layers (ectoderm, mesoderm, and endoderm). Thus, a multipotent cell may 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" refers to a cell being capable of forming many cell types in a given lineage, but not cells of other lineages. For example, a multipotent hematopoietic cell can form a wide variety of blood cells (red blood cells, white blood cells, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to a state of a cell that is less than totipotent or pluripotent in its developmental potential.

[0149] Differentiation of pluripotent stem cells requires changes in the culture system, such as changing stimuli in the medium or the physical state of the cells. The most common strategies utilize the formation of embryoid bodies (EBs) as a common and critical intermediate to initiate lineage-specific differentiation. EBs are three-dimensional clusters that have been shown to mimic embryonic development by giving 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 further processed to continue differentiation, typically for days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multi-layered cell cluster, typically achieved by one of several methods, including settling the pluripotent cells into droplets and settling the cells into "U"-bottom well plates, or by mechanical agitation. To promote EB development, pluripotent stem cell aggregates require additional differentiation cues, as aggregates maintained in pluripotent culture maintenance medium do not form proper EBs. Thus, pluripotent stem cell aggregates need to be transferred to differentiation medium that provides optimal lineage-directed induction cues. EB-based pluripotent stem cell medium typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm) with moderate proliferation within the EB cell clumps. Although known to promote cell differentiation, EBs produce heterogeneous cells with inconsistent differentiation states due to inconsistent cell exposure to environmental differentiation cues within the three-dimensional structure. Moreover, EBs are laborious to generate and maintain. Moreover, cell differentiation via EBs is accompanied by moderate cell proliferation, which also contributes to low differentiation efficiency.

[0150] In comparison, "aggregate formation" can be used to expand cell populations derived from pluripotent stem cells, unlike "EB formation". For example, during aggregate-based pluripotent stem cell expansion, the medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates by forming larger aggregates, but these aggregates can be periodically mechanically or enzymatically separated into smaller aggregates to maintain cell proliferation and increase cell number in culture. Unlike EB culture, cells cultured in aggregates in maintenance medium maintain markers of pluripotency. Pluripotent stem cell aggregates require additional differentiation cues to induce differentiation.

[0151] As used herein, "monolayer differentiation" refers to a differentiation method that is different from differentiation through three-dimensional multi-layered cell clusters, i.e., "EB formation". Monolayer differentiation does not require EB formation to induce differentiation, although other advantages are disclosed herein. Since monolayer culture does not mimic embryonic development such as EB formation, differentiation toward specific lineages is considered minimal compared to differentiation into all three germ layers in EBs.

[0152] Pluripotency can be determined, in part, by assessing the pluripotent properties of the cells, 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 cell lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation consisting of the three somatic cell lineages, and (vi) formation of embryoid bodies consisting of cells from the three somatic cell lineages.

[0153] Two types of pluripotency have been described above: "primed" or "metastable" pluripotency, which resembles the epiblast stem cells (EpiSCs) of late blastocysts, and "naive" or "ground" pluripotency, which resembles the inner cell mass of early / pre-implantation blastocysts. Both pluripotent states exhibit the characteristics described above, but the naive or ground state further exhibits (i) preinactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single cell culture, (iii) a global decrease in DNA methylation, (iv) decreased localization of H3K27me3 repressive chromatin marks on developmentally regulated gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard methodologies for reprogramming cells, in which an exogenous pluripotent gene is induced into a somatic cell, expressed, and then silenced or removed from the resulting pluripotent cell, are generally seen to have the pluripotency characteristics of the primed state. Under standard pluripotent cell culture conditions, such cells will remain in the primed state unless expression of the exogenous transgene is maintained, which is what is characteristic of the ground state observed.

[0154] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small, spherical shape with a high nucleus-to-cytoplasm ratio, prominent presence of nucleoli, and typical intercellular spaces.

[0155] As used herein, "feeder cells" or "feeders" are terms describing one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, since the feeder cells provide growth factors and nutrients for the support of the second cell type. Feeder cells may be 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 can typically be inactivated by irradiation or treatment with mitotic inhibitors such as mitomycin to prevent the feeder cells 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 leukemia cells. Without limiting the above, one particular 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, various feeder cells can be used in part to maintain pluripotency, direct differentiation to certain lineages, and promote maturation into specialized cell types such as effector cells.

[0156] As used herein, a "feeder-free (FF)" environment refers to an environment, such as a cell culture or medium, that is essentially free of feeder cells and / or has not been preconditioned by the growth of feeder cells. A "preconditioned" medium refers to a medium that is harvested after the growth of feeder cells in the medium for a period of time, e.g., at least one day. A preconditioned medium contains numerous mediators, including growth factors and cytokines, secreted by the feeder cells grown in the medium.

[0157] As used herein, the term "subject" refers to any animal, and preferably refers to a human patient, livestock, or other domesticated animal.

[0158] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, 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.

[0159] "Adhere" refers to the attachment of cells to a container, e.g., cells attach to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate medium. Certain classes of cells will not be maintained or grow in culture unless they adhere to a cell culture container. Certain classes of cells ("non-adherent cells") will be maintained and / or grow in culture without attachment.

[0160] "Culture" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (or in some cases simply "medium"), "supplement" and "medium supplement" refer to nutritional components that foster cell cultures.

[0161] "Cultivate" refers to maintaining, propagating (growing) and / or differentiating cells outside a tissue or body, such as in a sterile plastic (or coated plastic) cell culture dish or flask. Cultivation may utilize a medium as a source of nutrients, hormones and / or other factors that help propagate and / or maintain the cells.

[0162] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or a surface (e.g., a culture dish surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that are aggregated in a tube can be dissociated from each other, such as by enzymatic or mechanical dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture dish or other surface. Thus, dissociation may involve breaking cellular interactions with the extracellular matrix (ECM) and the culture substrate (e.g., the culture surface) or breaking the ECM between cells.

[0163] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells can also be differentiated from stem cells or progenitor cells. T cells can be CD3+ cells. T cells can be any type of T cell, and can be T cells at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, γδ T cells, etc. Further types of helper T cells include cells such as Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (Tcm cells), stem cell memory T cells (Tscm cells), and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express T cell receptors (TCRs) or chimeric antigen receptors (CARs).

[0164] As used herein, the term "naive T cells" or Tn refers to mature T cells that, unlike activated or memory T cells, have not encountered their cognate antigen in the periphery. Naive T cells are usually characterized by cell surface expression of L-selectin (CD62L); absence of activation markers CD25, CD44 or CD69; and absence of memory CD45RO isoforms. Naive T cells also express functional IL-7 receptors, consisting of IL-7 receptor-α (CD127) subunits and common gamma chain (CD132) subunits. In the naive state, T cells are considered to be quiescent and non-dividing, and require the common gamma chain cytokines IL-7 and IL-15 for homeostatic survival.

[0165] As used herein, the term "central memory T cells" or Tcm refers to a subgroup or subpopulation of T cells that have lower expression or pro-apoptotic signaling genes (e.g., Bid, Bnip3 and Bad) and higher expression of genes associated with trafficking to secondary lymphoid organs (e.g., CD62L, CXCR3, CCR7) compared to effector memory T cells, or Tcm.

[0166] As used herein, the term "stem memory T cell," or "stem cell memory T cell," or Tscm, refers to a subgroup or subpopulation of T cells that are capable of self-renewal, generating Tcm, Tems and Teffs (effector T cells), and express CD27 as well as lymphoid homing molecules such as CCR7 and CD62L, properties important for mediating long-lasting immunity.

[0167] As used herein, the term "NK cells" or "natural killer cells" refers to a peripheral blood lymphocyte subset defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). The NK cells provided herein can also be differentiated from stem or progenitor cells. As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to an NK cell subset that is phenotypically CD3- and CD56+, expresses NKG2C and CD57, and optionally CD16, but lacks expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated CD56+ NK cell subpopulation comprises expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and DNAM-1. CD56+ may be dim or brightly expressed.

[0168] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are currently recognized. Invariant or type I NKT cells express a very limited TCR repertoire, a standard α chain (Vα24-Jα18 in humans) coupled with a limited range of β chains (Vβ11 in humans). The second NKT cell population is called non-classical or non-invariant type II NKT cells, which show a more heterogeneous use of TCRαβ. Type I NKT cells are currently considered suitable for immunotherapy. Acquired or invariant (Type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, αGalCer, CD161, and CD56. The NKT cells provided herein can also be differentiated from stem or progenitor cells.

[0169] As used herein, the terms "B lymphocytes" or "B cells" are used interchangeably and refer to a subset of lymphocytes characterized by lack of T cell receptor (CD3) and expression of CD19 or CD20, the B cell receptor (BCR, Ig) containing immunoglobulin heavy and light chains. As provided herein, B cells can also be derived from stem or progenitor cells via differentiation induction. B cells include any subtype of B cells and can be at any stage of development, including but not limited to pro-B cells, pre-B cells, naive B cells, B-1 B cells, B-2 B cells, marginal zone B cells, follicular B cells, memory B cells, plasmablastic cells, plasma cells, and regulatory B cells. B. Overview

[0170] The present invention generally relates to a multi-step process for differentiating naive pluripotent cells into non-pluripotent cells, or into partially differentiated cells, including mesodermal cells, committed hemogenic endothelium, committed hematopoietic stem or progenitor cells, CD34+ cells, multipotent progenitor cells (MPPs) (differentiable to myeloid lineage, including neutrophil progenitors), T cell progenitors, NK cell progenitors; or into fully differentiated terminal hematopoietic cells, such as, for example, 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 produced using the disclosed methods.

[0171] Unlike the methods used in the art, the present invention avoided the formation of EBs in iPSC differentiation. As provided, hematopoietic lineage cells derived from iPSCs were obtained by maintaining their pluripotent ground or naive state by seeding clonal iPSC cells in TGFβ-free medium, differentiating the clonal iPSCs in a monolayer format without EB formation, and utilizing a stepwise strategy of applying appropriate combinations of small molecule chemicals, growth factors and cytokines at the early and middle stages of differentiation. Thus, the present invention allows direct transfer of expanded clonal iPSCs to adherent culture in monolayer form for immediate differentiation without the need for EB formation from iPSCs.

[0172] That is, the present invention provides a culture platform that allows for highly efficient differentiation of stem cells into definitive hematopoiesis 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 direct differentiation of iPSCs in monolayer culture without the need for EBs or clumps from iPSCs. C. Culture Platform

[0173] Existing methods for culturing pluripotent cells rely heavily on feeder cells or media preconditioned with feeder cells and containing fetal bovine serum. However, such environments may be inappropriate for producing cells for clinical and therapeutic use. For example, exposure to animal components may pose serious risks of immune rejection and transmission of unidentified pathogens to treated patients, and may reactivate intraanimal retroviruses, so cells cultured in such contaminated environments are generally considered inappropriate for human cell transplantation. Culture systems using animal-free media, such as the feeder-free environment contemplated herein, facilitate the production of clinical grade cell lines, particularly HSC lines, T cell lines, B cell lines, NKT cell lines, or NK cell lines derived from hESCs, hiPSCs, and pluripotent stem cells.

[0174] In certain embodiments, the feeder-free environment is essentially free of human feeder cells and is not preconditioned by feeder cells, including but not limited to mouse fetal fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells. The feeder-free culture medium is suitable for use in culturing pluripotent cells, reprogramming cells, single cell culture, dissociation, and passaging of pluripotent cells, cell sorting of pluripotent cells, generating ground state pluripotent cells, maintaining ground state pluripotency, and inducing pluripotent cell differentiation. In certain embodiments, the feeder-free environment is used to induce pluripotency, improve the efficiency of reprogramming, increase or maintain the differentiation potential of cells, and / or induce differentiation. Furthermore, in certain embodiments, the feeder-free environment is substantially free of cytokines and growth factors, including bFGF.

[0175] In some aspects of the invention, one or more of the above iPSC differentiation steps may be performed under feeder-free conditions. Such feeder-free conditions may be in the form of, but not limited to, monolayer and suspension culture. In one embodiment of the invention, differentiation of pluripotent cells into mesodermal cells is performed under monolayer feeder-free conditions. In another embodiment of the invention, differentiation of mesodermal cells into committed hemogenic endothelial cells is performed under monolayer feeder-free conditions. In yet another embodiment of the invention, differentiation of committed hemogenic endothelial cells into hematopoietic stem cells is performed under monolayer feeder-free conditions. In one embodiment of the invention, differentiation of committed hematopoietic stem cells into multipotent progenitor cells, T cell progenitor cells or NK cell progenitor cells is performed under suspension feeder-free conditions or under monolayer feeder-free conditions followed by suspension feeder-free conditions. In another embodiment of the invention, differentiation of T cell precursor cells into fully differentiated T cells or differentiation of NK cell precursor cells into fully differentiated NK cells is carried out under suspension feeder-free conditions or under monolayer feeder-free followed by suspension feeder-free conditions.

[0176] Any suitable vessel or cell culture vessel may be used as a support for cell culture in basal medium and / or cell culture additives. In some embodiments, cell attachment is promoted by coating the surface of the culture vessel with an adhesion-promoting matrix / substratum (e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.), although in certain embodiments, the effect of the cell culture media and additives disclosed herein may be enhanced. Substrates suitable for culturing and passaging cells are known in the art and include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombospondin, mixtures of matrices produced by natural cell lines, such as Matrigel™, and synthetic or artificial surfaces, such as polyamine monolayers and carboxy-terminated monolayers. In some embodiments, providing feeder-free conditions includes culturing cells on a matrix-coated surface. In one embodiment, the culture platform contemplated herein includes a matrix / substrate that includes Matrigel™ or vitronectin. In some embodiments of the cultures, Matrigel™ is used, so the cultures are fully defined.

[0177] In some embodiments of the present invention, one or more of the above differentiation steps may be performed under serum-free conditions. Examples of commercially available serum-free media suitable for cell attachment and / or cell induction include mTeSR™1, TeSR™2 or StemSpan™ from Stem Cell Technologies (Vancouver, Canada), Primate ES / iPS Cell Medium from ReproCELL (Boston, Massachusetts), StemPro™-34 from Invitrogen (Carlsbad, California), StemPro™ hESC SFM from Invitrogen, and X-VIVO™ from Lonza (Basel, Switzerland).

[0178] In further embodiments, one or more of the media of the culture platform are feeder-free environments, and are optionally substantially free of cytokines and / or growth factors. In other embodiments, the cell culture media contain additives such as serum, extracts, growth factors, hormones, cytokines, and the like. In general, 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 can 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 various mammalian cells; KOSR (Knockout serum replacement); L-glutamic acid (L-glut); NEAA (non-essential amino acids). Other media additives can include, but are not limited to, MTG, ITS, βME, antioxidants (e.g., ascorbic acid). In some embodiments, the media 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 (e.g., IL-1 to IL-18), various colony-stimulating factors (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), and other cytokines that act on stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines may be obtained commercially, for example from R&D Systems, Inc. (Minneapolis, Minn.), and may be natural or recombinant.In some other embodiments, the media of the present invention comprises one or more of the following: bone morphogenetic protein (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 one or more cytokines from leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors / mitogens and cytokines are stage-specific and / or cell type-specific, at concentrations determined experimentally or guided by established cytokine fields.

[0179] In general, approaches to differentiate induced pluripotent cells include direct or indirect modulation of specific cellular pathways using polynucleotide-based, polypeptide-based 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 regulators. "Contacting," as used herein, can include 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 cell differentiation. Such contacting can occur, for example, by introducing the one or more agents into the cell in in vitro culture. That is, contacting can occur by introducing the one or more agents into the cell in nutrient cell culture medium. The cell can be maintained in the medium containing the one or more agents for a period of time sufficient for the cell to reach a 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 vector or vectors are introduced by retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system carrying an expression cassette, or mRNA.

[0180] In other embodiments, one or more of the stage-specific feeder-free serum-free media of the culture platforms described 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 comprise a small molecule inhibitor of the TGFβ / activin signaling pathway, including but not limited to a TGFβ receptor inhibitor or an ALK5 inhibitor.

[0181] The culture platform contemplated herein also provides numerous advantages by utilizing homogenous populations of industrial or clinical grade pluripotent cells with reduced spontaneous differentiation and / or ground-state pluripotency. In one embodiment, the homogenous iPSCs are maintained in a composition that includes a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, and does not include a TGFβ receptor / ALK inhibitor. As used herein, the term "homogenous" refers to a cell population 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 when each cell expresses one or more identical 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 other cells in the population.

[0182] In various embodiments, the cell culture medium of the culture platform for generating hematopoietic cell lineages through fate-committed hemogenic endothelium herein does not contain or is substantially free of inhibitors of the TGFβ / activin signaling pathway, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In one embodiment, the culture platform comprises a seeding medium for maintaining naive hiPSCs, comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor. Without wishing to be bound by a particular theory, the inventors have found that TGFβR / ALK5 inhibitors increase the efficiency of reprogramming, while hindering the long-term maintenance, quality, and uniformity of the pluripotent cell population. That is, while inhibition of TGFβ pathway signaling improved the efficiency of cell reprogramming, relief of this inhibition contributes to the subsequent maintenance of pluripotent cell populations in in vitro culture systems, particularly in systems using feeder cell-free enzymatic passaging of single cells, where spontaneous differentiation is reduced and a homogeneous pluripotent population that maintains a "basal" or "naive" pluripotent state is preferred. As used herein, the term "long-term", measured by but not limited to passage number, often means at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more passages. As defined, "passage" refers to the act of distributing and seeding cells onto multiple cell culture surfaces or vessels when the cells have expanded to a desired extent. Additionally, as disclosed herein, by culturing metastable pluripotent cells in medium containing a GSK3 inhibitor and a MEK inhibitor, and optionally a ROCK inhibitor, but not containing a TGFβR / ALK5 inhibitor, the transitional pluripotent cells achieve reduced spontaneous differentiation and / or achieve ground state pluripotency.

[0183] To obtain hematopoietic cells by differentiating iPSCs without forming EB intermediates, it is also important to achieve basal or naïve pluripotency of iPSCs. In addition, the efficiency of differentiation of naïve iPSCs into committed HE is also greatly affected by the use of monolayer culture that does not allow the formation of EBs and their clumps. In some embodiments, the culture platform comprises a medium that contains a ROCK inhibitor and is free or essentially free of a TGFβR / ALK5 inhibitor. In some other embodiments, the culture platform comprises a medium that contains a GSK3 inhibitor but does not contain a TGFβR / ALK5 inhibitor and promotes the generation of committed HE cells and / or committed HSC cells by using the culture platform provided herein. 1. TGFβ receptor / ALK inhibitor

[0184] TGFβ receptor (e.g., ALK5) inhibitors may include antibodies against TGFβ receptors (e.g., ALK5), dominant-negative mutants of TGFβ receptors (e.g., ALK5), and antisense nucleic acids that suppress the expression of TGFβ receptors (e.g., ALK5). 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) and available commercially, e.g., from Tocris Bioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine; Wnt3a / BIO (see, e.g., Dalton, et al., Molecular Pharmacology 62(1):65-74 (2002)), which are incorporated by reference herein. al., see WO 2008 / 094597; 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):85-90 (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., those listed in Stiefl, et al., WO2008 / 006583, incorporated herein by reference). Additionally, while "ALK5 inhibitors" are not intended to include non-specific kinase inhibitors, "ALK5 inhibitors" should be understood to include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as, for example, SB-431542 (see, e.g., Inman, et al., J. Mol. Pharmacol. 62(1): 65-74 (2002)). Without intending to limit the scope of the present invention, ALK5 inhibitors are believed to affect the mesenchymal-epithelial transition / transition (MET) process. The TGFβ / activin pathway is a driver of epithelial-mesenchymal transition (EMT). Thus, inhibition of the TGFβ / activin pathway may promote the MET (i.e., reprogramming) process.

[0185] From the data showing the effect of ALK5 inhibition, it is believed that the inhibition of TGFβ / activin pathway will give the same ALK5 inhibition effect.That is, any inhibitor of TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or instead of the ALK5 inhibitor described in each subsection 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 of SMAD2 / 3 and SMAD4, and activators / agonists of SMAD6 and SMAD7.In addition, the following classifications are merely for organizational 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 classifications.

[0186] TGFβ receptor (TGFβR) inhibitors may include antibodies against TGFβ receptors, dominant negative mutants of TGFβ receptors, and siRNAs or antisense nucleic acids targeting TGFβ receptors. 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); lerdelimumb (CAT-152); metelimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580 276;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-transduced 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)).

[0187] Inhibitors of SMAD2 / 3 phosphorylation may include antibodies against SMAD2 or SMAD3, dominant negative mutants of SMAD2 or SMAD3, and antisense nucleic acids targeting SMAD2 or SMAD3. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (Morin) (see, for example, Wrzesinski, supra; Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); and Kataoka, et al., EP1992360, which are incorporated herein by reference).

[0188] Inhibitors of SMAD2 / 3 and SMAD4 interaction may include antibodies against SMAD2, SMAD3 and / or smad4, dominant negative mutants of SMAD2, SMAD3 and / or smad4, and antisense nucleic acids targeting SMAD2, SMAD3 and / or smad4. Specific examples of inhibitors of SMAD2 / 3 and bSMAD4 interaction include, but are not limited to, 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 (2006)).

[0189] SMAD6 and SMAD7 activators / agonists include, but are not limited to, antibodies against dominant negative mutants of SMAD6 or SMAD7, and antibodies against antisense nucleic acids targeting SMAD6 or SMAD7. 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). 2. WNT pathway agonists

[0190] As used herein, the terms "Wnt signal promoter," "Wnt pathway activator," "Wnt pathway activating agent," or "Wnt pathway agonist" refer to agonists of the Wnt signal pathway, including, but not limited to, one or more of Wntl, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wntl0b, 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: a Dkk polypeptide, a crescent polypeptide, a cerberus polypeptide, an axin polypeptide, a Frzb polypeptide, a T-cell factor polypeptide, or a dominant negative disheveled polypeptide.

[0191] Non-limiting examples of Wnt pathway agonists further 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, an organic small molecule that promotes Wnt / β-catenin signaling, an organic small molecule that inhibits expression or activity of a Wnt antagonist, an antisense oligonucleotide that inhibits expression of a Wnt antagonist, a ribozyme that inhibits expression of a Wnt antagonist, an RNAi construct, siRNA, or shRNA that inhibits 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.

[0192] Wnt pathway agonists further include GSK3 inhibitors, such as, for example, nucleic acids comprising a nucleotide sequence encoding a dominant negative GSK-3, GSK3α, or GSK3 polypeptide, polypeptides comprising the amino acid sequence of a dominant negative GSK-3, GSK3α, or GSK3 polypeptide, small organic molecules that bind to and inhibit expression or activity of GSK-3, GSK3α, or GSK3, RNAi constructs, siRNA, or shRNA that bind to and inhibit expression and / or activity of GSK-3, GSK3α, or GSK3, antisense oligonucleotides that bind to and inhibit expression of GSK-3, GSK3α, or GSK3, antibodies that bind to and inhibit expression and / or activity of GSK-3, GSK3α, or GSK3, ribozymes that bind to and inhibit expression of GSK-3, GSK3α, or GSK3, as well as any GSK-3-independent reagent that activates β-catenin target genes with effects similar to those of GSK-3 inhibition. 3.GSK3 inhibitors

[0193] GSK3 inhibitors are specific examples of Wnt pathway agonists suitable for use in the compositions contemplated herein, and may include, but are not limited to, polynucleotides, polypeptides, and small molecules.The GSK3 inhibitors contemplated herein may reduce GSK3α / β expression and / or GSK3α / β activity.The GSK3 inhibitors contemplated herein include, but are not limited to, anti-GSK3 antibodies, dominant negative GSK3 mutants, siRNAs, shRNAs, miRNAs, and antisense nucleic acids that target GSK3.

[0194] Other exemplary GSK3 inhibitors include Kenpaullone, l-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, TDZD-8, BIO, BIO-Acetoxime, (5-Methyl-lH-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopenadienylruthenium complex, TDZD-8. These include, but are not limited to, 4-benzyl-2-methyl-l,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(l-pyridyl)-[l,3,4]-oxadiazole, OTDZT, α-4-dibromoacetophenone, AR-AO 144-18, 3-(l-(3-hydroxypropyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, TWS119 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-l-(4,5-dibromo-thiophen-2-yl)-ethanone, GF109203X, RO318220, TDZD-8, TIBPO, and OTDZT.

[0195] In certain exemplary embodiments, said GSK3 inhibitor is CHIR99021, BIO, or Kenpaullone.

[0196] In a preferred embodiment, the GSK3 inhibitor is CHIR99021.

[0197] In another embodiment, the GSK3 inhibitor is BRD0705. 4. ERK / MEK Inhibitors

[0198] The ERK / MEK inhibitor suitable for use in the composition contemplated herein includes, but is not limited to, polynucleotide, polypeptide, and small molecule.The ERK / MEK inhibitor contemplated herein can reduce the expression of MEK or ERK and / or the activity of MEK or ERK.The exemplary MEK / ERK inhibitor contemplated herein includes, but is not limited to, anti-MEK or anti-ERK antibody, dominant negative MEK or ERK mutant, siRNA, shRNA, miRNA and antisense nucleic acid that target MEK or ERK.

[0199] Other exemplary ERK / MEK inhibitors include, but are not limited to, PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSKl 120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEAl 19, AZD6244, FR180204, and PTK787.

[0200] Further exemplary MEK / ERK inhibitors include compounds disclosed in WO 99 / 01426, WO 02 / 06213, WO 03 / 077914, WO 05 / 051301, and WO 2007 / 044084.

[0201] Further 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-ylmeethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo-2 -chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazol-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, 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-carbo 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (hereinafter, MEK inhibitor 2), 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide, and pharma- ceutical acceptable salts thereof.

[0202] In a preferred embodiment, the MEK / ERK inhibitor is PD98059. 5.ROCK Inhibitors

[0203] Rho-associated kinase (ROCK) is a serine-threonine kinase that acts as a downstream effector of Rho kinase (there are three isoforms: 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 can reduce ROCK expression and / or ROCK activity. Examples of ROCK inhibitors contemplated herein include, but are not limited to, anti-ROCK antibodies, dominant-negative ROCK mutants, siRNAs, shRNAs, miRNAs, and antisense nucleic acids that target ROCK.

[0204] 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 those ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated by reference in its entirety.

[0205] In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, or pyrintegrin.

[0206] In a preferred embodiment, the ROCK inhibitor is thiazovivin.

[0207] The content of these small molecules in the compositions and cell culture media contemplated herein can vary and may be optimized depending on the specific molecules and combinations used, the type of cells cultured in the medium, and the specific culture conditions, including the specific application. In one embodiment, the small molecules are present in the composition at a concentration sufficient to induce pluripotency, increase the efficiency of reprogramming, increase or maintain the differentiation potential of cells, or induce or maintain ground state pluripotency.

[0208] Another aspect of the present invention relates to Notch activators for use with the present invention. Notch includes 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. The Notch agonists bind to the Notch receptor and initiate or mediate signaling events associated with the Notch receptor, such as cleavage of the intracellular domain of Notch and translocation 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 ligand can be agarose beads, magnetic beads, and latex beads. In one embodiment, the Notch ligand peptide is bound / immobilized to beads. In another embodiment, the Notch ligand peptide is bound / immobilized to the surface of a polystyrene plate. In some embodiments, the immobilization of Notch ligand is non-covalent. In some embodiments, the Notch ligand peptide is provided by cells.

[0209] Yet another aspect of the present invention relates to BMP pathway activators, including those disclosed in the following publications, the disclosures of which are incorporated herein by reference: WO2014011540, WO2014062138, and WO2005117994. BMP pathway activators used in the present invention include, but are not limited to, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In one non-limiting embodiment of the present invention, the BMP pathway activator is BMP-4. BMPs are multifunctional cytokines and members of the transforming growth factor beta superfamily. Bone morphogenetic protein (BMP) receptors mediate BMP signaling through the activation of Smads. BBMP ligands bind to BMP receptors BMPRI and BMPRII. Phosphorylation of BMPRII is followed by activation of BMPRI. Phosphorylated BMPRI then phosphorylates receptor-activated Smad proteins (R-Smads), which bind to common mediator-Smads (co-Smads) and enter the nucleus to regulate gene expression. In one embodiment, the BMP pathway activator is BMP4.

[0210] The present invention provides compositions for obtaining hematopoietic cells from iPSCs via committed HSCs differentiated from iPSCs, or via committed hemogenic endothelium differentiated from iPSCs, each of which approaches does not result in EB formation from iPSCs for the desired cell differentiation. 6. Platform for hiPSC Differentiation I. iCD34 Platform

[0211] One embodiment of the present invention provides a culture platform for obtaining fate-committed hemogenic endothelium using pluripotent stem cells. As used herein, fate-committed hemogenic endothelium is a hematopoietic cell population committed to definitive hematopoiesis that has the potential to give rise to all hematopoietic cells, including but not limited to fate-committed HSC, hematopoietic multipotent progenitor (MPP), T cell progenitor, NK cell progenitor, T cell, NK cell, NKT cell, and / or B cell.

[0212] In one embodiment, the culture platform for obtaining fate-committed hemogenic endothelium using pluripotent stem cells including iPSCs comprises a seeding medium comprising MEKi, GSKi, and ROCKi. In some embodiments, the seeding medium is free or essentially free of TGFβ receptor / ALK inhibitors. In one embodiment, the combination of small molecules in the seeding medium of the present invention is shown in Table 9 as Fate Maintenance Medium (FMM). The components of the medium may be present in the medium in amounts within the concentration ranges shown in Table 1. In one embodiment, the iPSCs used to obtain fate-committed hemogenic endothelium were cell lines generated by the use of Fate Reprogramming Medium (FRM) and further maintained in FMM to establish and maintain the ground or naïve state of the iPSC cell line and suitable for stage-specific differentiation as described herein. The ground or naïve iPSCs thus obtained are suitable for cryopreservation. In the present invention, stored iPSC cell lines or clonal iPSCs can be seeded into FMM for subsequent differentiation into committed hemogenic endothelium. [Table 1]

[0213] One aspect of the present invention provides a medium for differentiation and proliferation of mesoderm from pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, the medium comprises CD34 basal medium containing a BMP activator, and optionally bFGF, and small molecules in the combinations shown in Table 2. In some embodiments, the medium comprises extracellular matrix proteins. In other embodiments, the medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 2. In some embodiments, the medium is fully defined, with vitronectin replaced by Matrigel™. [Table 2]

[0214] In one embodiment, the above-mentioned medium for differentiation and proliferation of mesoderm from pluripotent stem cells further contains 0.2 to 50 ng of bFGF.

[0215] One aspect of the present invention provides a medium for obtaining mesodermal cells capable of differentiation into committed hemogenic endothelium from pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, the medium comprises a BMP activator, a Wnt pathway activator, and bFGF. In one embodiment, the Wnt pathway activator is a GSK3 inhibitor. In one embodiment, the medium comprising a GSK3 inhibitor is applied only after the lineage commitment of mesodermal cells to obtain the differentiation potential into committed HE. In one embodiment, the medium comprising a BMP activator, a GSK3 inhibitor, and bFGF further comprises a CD34 basal medium comprising a small molecule in the combination shown in Table 3. In one embodiment, the medium does not comprise a TGFβ receptor / ALK inhibitor. In some embodiments, the medium comprises an extracellular matrix protein. In other embodiments, the medium herein comprises a small molecule, a growth factor, and / or a cytokine in the concentration range shown in Table 11. In some embodiments, the medium is fully defined, with vitronectin replaced by Matrigel™. [Table 3]

[0216] One aspect of the present invention provides a medium for obtaining committed hemogenic endothelium from mesodermal cells. In one embodiment, the 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 medium comprises a CD34 basal medium comprising VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor, and a small molecule in the combination shown in Table 4. In one embodiment, the 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 further comprises one or more of a Wnt pathway activator, IGF, and EPO. In some embodiments, the medium for generating committed HE from mesodermal cells comprises a ROCK inhibitor, a Wnt pathway activator, VEGF, bFGF, SCF, IL6, and IL11, and one or more of an IGF and EPO. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the Wnt pathway activator is CHIR99021. In one embodiment, the medium comprising VEGF, bFGF, SCF, IL6, IL11 and a ROCK inhibitor does not comprise one or more of a Wnt pathway activator, a TGFβ receptor / ALK inhibitor, IGF1, and EPO. In other embodiments, the medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 4. [Table 4]

[0217] One aspect of the present invention provides a culture platform for obtaining multipotent progenitor cells (MPPs) from committed hemogenic endothelium. The MPPs can be further differentiated into myeloid lineage, including neutrophil progenitors. In one embodiment, the culture platform comprises a medium (i) that includes 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, Flt3L, and IL11, and is suitable for differentiation of committed hemogenic endothelium into pre-HSCs (Table 5). In another embodiment, the culture platform comprising a medium for differentiating committed hemogenic endothelium into pre-HSCs further comprises (ii) a medium that includes a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and does not include a ROCK inhibitor, and is suitable for differentiation of committed hemogenic endothelium into multipotent progenitor cells. 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 medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 5. [Table 5] II. iNK / iT Platform

[0218] One aspect of the invention provides a culture platform for generating T cell precursors or T cells from committed hemogenic endothelium. In one embodiment, the culture platform comprises (i) a medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator, suitable for differentiation of committed hemogenic endothelium into pre-T cell precursors (pre-iproT), and / or (ii) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, not comprising one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, suitable for differentiation of the pre-T cell precursor into T cell precursors or T cells (Table 6). In some embodiments, the medium for differentiating committed HE into pre-iproT comprises a ROCK inhibitor, SCF, Flt3L, TPO, and IL7; and does not comprise a BMP activator. In some embodiments, the medium for differentiating pre-iproT into T cell precursors or T cells comprises SCF, Flt3L, and IL7. 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 medium herein comprises a small molecule, growth factor, and / or cytokine in the concentration ranges shown in Table 6. [Table 6]

[0219] One aspect of the invention provides a culture platform for generating NK cell precursors or NK cells from committed hemogenic endothelium. In one embodiment, the culture platform comprises: (i) a medium suitable for differentiation of committed hemogenic endothelium into pre-NK cell precursors (pre-iproNK), comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator; and (ii) a medium suitable for differentiation of the pre-NK cell precursor 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 one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor. In some embodiments, the medium for differentiating committed HE into pre-iproNK comprises a ROCK inhibitor, SCF, Flt3L, TPO, IL3, IL7, and IL15; and does not comprise a BMP activator. In some embodiments, the medium for differentiating pre-iproNK into NK cell precursors or NK cells comprises SCF, Flt3L, and IL15. 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 medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 7. [Table 7]

[0220] Another aspect of the invention provides a culture platform for obtaining T cell precursors or T cells, said culture platform comprising one or more of the following: (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and free or essentially free of one or more of VEGF, bFGF, BMP activators and ROCK inhibitors, and suitable for differentiation of pre-T cell precursors into T cell precursors or T cells; (ii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, and suitable for differentiation of committed hemogenic endothelial pre-T cell precursors. (iii) a medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, and suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells; (iv) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and suitable for obtaining differentiation potential of committed hemogenic endothelium in mesodermal cells; (v) a medium comprising a BMP activator, and optionally bFGF, and suitable for development and proliferation of mesodermal cells from iPSCs; and (vi) a medium comprising MEKi, GSKi, and ROCKi, and free or essentially free of TGFβ receptor / ALK inhibitors, and suitable for seeding and proliferation of naive iPSCs. 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.

[0221] In one embodiment, said culture platform for generating T cell progenitors or T cells comprises (i) a medium comprising SCF, Flt3L, and IL7, and free or essentially free of one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor, and suitable for differentiation of pre-T cell progenitors into T cell progenitors or T cells. In another embodiment, said culture platform for generating T cell progenitors or T cells comprising medium (i) further comprises (ii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator; and suitable for differentiation of committed hemogenic endothelial pre-T cell progenitors. In another embodiment, said culture platform for generating T cell progenitors or T cells comprising media (i) and (ii) further comprises (iii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; optionally a Wnt pathway activator, and suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells. In yet another embodiment, said culture platform for generating T cell progenitors or T cells comprising media (i), (ii) and (iii) further comprises (iv) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and suitable for obtaining differentiation potential of committed hemogenic endothelium in mesodermal cells. In yet another embodiment, said culture platform for generating T cell progenitors or T cells comprising media (i), (ii), (iii) and (iv) further comprises (v) a medium comprising a BMP activator, and optionally bFGF, and suitable for the generation and proliferation of mesodermal cells from iPSCs.In another embodiment, the culture platform for generating T cell progenitors or T cells, comprising media (i), (ii), (iii), (iv) and (v), further comprises (vi) a medium comprising MEKi, GSKi and ROCKi, and free or essentially free of TGFβ receptor / ALK inhibitors, and suitable for seeding and growing naive iPSCs. 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. In some embodiments, Notch factors are used in the culture platform for generating T cell progenitors or T cells. In some embodiments, Notch factors, including Jag1, Jag2, DLL-1, DLL-3 and DLL-4, can be introduced as soluble peptides, bead-bound peptides, surface-bound peptides, or cell-presented peptides.

[0222] Another aspect of the invention provides a culture platform for obtaining NK cell precursors or NK cells comprising one or more of the following: (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and free or essentially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors, and suitable for differentiation of pre-NK cell precursors into NK cell precursors or NK cells; (ii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator, and suitable for differentiation of committed hemogenic endothelial cells into pre-NK cell precursors. (iii) a medium suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells, comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator; (iv) a medium suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor; (v) a medium suitable for development and proliferation of mesodermal cells from iPSCs, comprising a BMP activator, and optionally bFGF; (vi) a medium suitable for seeding and proliferation of naive iPSCs, comprising MEKi, GSKi, and ROCKi, and 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.In some embodiments, NK maturation is carried out using one or more of the artificial antigens introduced in the form of bead conjugates, plasma membrane particles and / or antigen presenting cells to stimulate NK proliferation, development and maturation.

[0223] In one embodiment, said culture platform for generating NK cell precursors or NK cells comprises (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7 and IL15, and free or essentially free of one or more of VEGF, bFGF, BMP activators and ROCK inhibitors, and suitable for differentiation of pre-NK cell precursors into NK cell precursors or NK cells. In some embodiments, NK maturation is performed using one or more of artificial antigens for stimulating NK proliferation, development and maturation introduced in the form of bead conjugates, plasma membrane particles and / or antigen presenting cells. In another embodiment, said culture platform for generating NK cell precursors or NK cells comprising medium (i) further comprises (ii) a medium comprising one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7 and IL15; and optionally a BMP activator; and suitable for differentiation of committed hemogenic endothelium into pre-NK cell precursors. In another embodiment, said culture platform for generating NK cell precursors or NK cells comprising medium (i) and (ii) further comprises (iii) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6 and IL11; and optionally a Wnt pathway activator; and suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells. In yet another embodiment, said culture platform for generating NK cell precursors or NK cells comprising media (i), (ii) and (iii) further comprises (iv) a medium comprising a BMP activator, bFGF and a GSK3 inhibitor and suitable for obtaining committed hemogenic endothelial differentiation potential in mesodermal cells. In yet another embodiment, said culture platform for generating NK cell precursors or NK cells comprising media (i), (ii), (iii) and (iv) further comprises (v) a medium comprising a BMP activator, and optionally bFGF and suitable for generating and expanding mesodermal cells from iPSCs.In another embodiment, the culture platform for generating NK cell precursors or NK cells, comprising media (i), (ii), (iii), (iv) and (v), further comprises (vi) a medium comprising MEKi, GSKi and ROCKi, and free or essentially free of TGFβ receptor / ALK inhibitors, and suitable for seeding and growing naive iPSCs. 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.

[0224] One aspect of the present invention provides a culture platform for generating committed hemogenic endothelium, said culture platform comprising one or more of the following: (i) a medium for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells, comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; (ii) a medium for obtaining committed hematopoietic differentiation potential in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a medium for differentiation and proliferation of mesodermal cells from naïve iPSCs, comprising a BMP activator, and optionally bFGF; and (iv) a seeding and proliferation medium for naïve iPSCs, comprising MEKi, GSKi, and ROCKi, and wherein the seeding medium does not comprise a TGFβ receptor / ALK inhibitor. In some embodiments, said committed hemogenic endothelium is CD34+. 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.

[0225] In one embodiment, the culture platform for obtaining committed hemogenic endothelium comprises (i) a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; and suitable for differentiation and proliferation of committed hemogenic endothelium from mesodermal cells. In one embodiment, the culture platform comprising the medium (i) further comprises (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and suitable for obtaining committed hematopoietic differentiation potential in mesodermal cells. In another embodiment, the culture platform comprising the medium (i) and (ii) further comprises (iii) a medium comprising a BMP activator, and optionally bFGF, and suitable for differentiation and proliferation of mesodermal cells from naive iPSCs. In yet another embodiment, the culture platform comprising the media (i), (ii) and (iii) further comprises (iv), a medium comprising MEKi, GSKi and ROCKi, and not containing or essentially not containing TGFβ receptor / ALK inhibitor, and suitable for seeding and growing naive iPSCs. In some embodiments, all the above media are not containing or essentially not containing 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.

[0226] One embodiment of the present invention provides a culture platform for generating CD34+ fate-committed hemogenic endothelium, said culture platform comprising one or more of the following: (i) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, wherein the culture platform is adapted to differentiate and promote the differentiation of committed hemogenic endothelium from mesodermal cells, wherein the committed hemogenic endothelium comprises CD34+ fate-committed hemogenic endothelium. (ii) a medium for differentiation and proliferation of mesodermal cells comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a medium for differentiation and proliferation of mesodermal cells from naive iPSCs comprising a BMP activator, and optionally bFGF; and (iv) a seeding or proliferation medium for naive iPSCs comprising MEKi, GSKi, and ROCKi, and the seeding medium does not contain or is essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above media are free of 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.

[0227] One aspect of the present invention provides a culture platform for generating mesodermal cells, the culture platform comprising one or more of the following: (i) a medium for differentiation and proliferation of mesodermal cells from naive iPSCs, comprising a BMP activator, and optionally bFGF; and (ii) a seeding or proliferation medium for naive iPSCs, comprising MEKi, GSKi, and ROCKi, and the seeding medium does not contain or is 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. In some embodiments, the culture platform for generating mesodermal cells may further comprise (iii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and for obtaining committed hematopoietic differentiation potential in mesodermal cells, in some embodiments, the medium comprising a BMP activator, bFGF, and a GSK3 inhibitor does not comprise a TGFβ receptor / ALK inhibitor. C. Methods for Obtaining Committed Hemopoietic Endothelial, Multipotent Progenitor Cells, T Cell or NK Cell Progenitor Cells, T Cells and / or NK Cells

[0228] The present invention provides a method for generating pluripotent stem cell-derived committed hematopoietic cells using a multi-step culture platform comprising one or more 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 for differentiation of pluripotent stem cells, as compared to methods known in the art. The method provided generates and simultaneously expands pluripotent stem cell-derived committed hemogenic endothelial (iHE), CD34+HE (iCD34), which can be further differentiated into multipotent progenitor cells (iMPP), natural killer cell progenitor cells (ipro-NK), T cell progenitor cells (ipro-T), mature NK cells (iNK) and mature T cells (iT). Yet a further aspect of the present invention provides a method for generating differentiated myeloid cells from pluripotent stem cell-derived CD34+, HE, HSC, and / or MPP.

[0229] In one embodiment, the invention provides a method for differentiation and expansion of hematopoietic cells from pluripotent cells in monolayer culture, the method comprising contacting the pluripotent cells with a BMP pathway activator, and optionally bFGF, to obtain and expand pluripotent stem cell-derived mesodermal cells without formation of embryoid bodies from the pluripotent stem cells, and then contacting the pluripotent cells with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded pluripotent stem cell-derived mesodermal cells with committed hemogenic endothelial (HE) differentiation potential without formation of embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF, and optionally a ROCK inhibitor, and / or a WNT pathway activator, differentiates the mesodermal cells with committed HE differentiation potential into committed HE cells, which also proliferate during differentiation.

[0230] The provided method for obtaining hematopoietic cells is superior to EB-mediated pluripotent stem cell differentiation because EB formation does not induce cell proliferation, does not allow monolayer culture, and is laborious and less efficient.Furthermore, the present invention discloses that monolayer culture using the method provided herein results in functional hematopoietic cells capable of long-term hematopoietic self-renewal, reconstitution, and engraftment in vivo.

[0231] As described in detail below, the present invention provides a method for obtaining hematopoietic cells from pluripotent cells through the acquisition of fate-committed hemogenic endothelium. In particular, the present invention provides a method for inducing differentiation of hematopoietic cells from pluripotent cells without the formation of EBs for differentiation. I. iCD34 Platform 1. Derivation and proliferation of fate-committed iHE

[0232] One aspect of the invention provides a method for generating committed hemogenic endothelium (iHE) using an optimized multi-step process. In general, the method begins with a first step in which pluripotent stem cells are seeded and expanded. The pluripotent stem cells are then differentiated into mesodermal cells and expanded during this process. The expanded mesodermal population is then differentiated into a mesodermal population with the potential to differentiate into committed hemogenic endothelium, which is subsequently differentiated and expanded into committed hemogenic endothelium. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naïve iPSCs. The invention further provides a method for generating and expanding committed hemogenic endothelium (iHE), the method comprising differentiating and expanding pluripotent stem cell-derived mesodermal cells, and obtaining mesodermal cells with the potential to differentiate into iHE, which are then differentiated into iHE. Alternatively, the present invention provides a method for generating and expanding committed hemogenic endothelium, comprising differentiating pluripotent stem cell-derived mesodermal cells with committed HE differentiation potential into committed iHE. The method disclosed herein utilizes an optimized monolayer iCD34 culture platform in which EBs are not formed and which is free or essentially free of TGFβ receptor / ALK inhibitors.

[0233] In one embodiment of the method of generating committed hemogenic endothelium (iHE) from pluripotent stem cells, the method includes: (1) differentiating and expanding a mesoderm population from pluripotent stem cells by contacting the cells with a medium comprising a BMP activator, and optionally bFGF; (2) differentiating and expanding the mesoderm population by contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF, so that the mesoderm cells have a committed HE differentiation potential; (3) differentiating and expanding the mesoderm cells with a committed HE differentiation potential into committed HE cells by contacting the cells with a medium comprising a ROCK inhibitor; one or more of a growth factor and cytokine selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. 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 resulting iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34 positive. 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 other embodiments, the sorting uses CD34 positive, CD43 negative, CD93-. In some other embodiments, the sorting uses CD34 positive, CD93 negative. In some embodiments, the medium in the method is free or essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, contacting the cells with medium containing a GSK3 inhibitor occurs only after lineage commitment of the mesoderm cells to acquire committed HE differentiation potential.In some embodiments, the methods further comprise exposing the seeded iPSCs, and / or mesodermal cells to a low oxygen tension of about 2% to about 10%. In some embodiments, the methods further comprise seeding the pluripotent stem cells by contacting the pluripotent cells with a medium comprising a MEKi, a GSKi, and a ROCKi, and in which the pluripotent stem cells proliferate.

[0234] In one embodiment of the method of generating committed hemogenic endothelium (iHE) from seeded pluripotent stem cells, the method includes: (1) contacting the pluripotent stem cells with a medium comprising a BMP activator, and optionally bFGF, to differentiate and proliferate mesodermal cells from the pluripotent stem cells; (2) contacting the mesodermal cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF, to obtain mesodermal cells with committed iHE differentiation potential; (3) contacting the mesodermal cells with a medium comprising a ROCK inhibitor; one or more of a growth factor and cytokine selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator, to differentiate and proliferate committed HE cells from the mesodermal cells with iHE differentiation potential. 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 resulting iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the method further comprises sorting using CD34 positive. 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 sorting uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the sorting uses CD34 positive, CD93 negative. In some embodiments, the medium in the method is free or essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises exposing the seeded iPSCs, and / or mesoderm cells to a low oxygen tension of about 2% to about 10%.

[0235] In one embodiment of the method for generating committed hemogenic endothelium (iHE) from pluripotent stem cell-derived mesodermal 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 committed HE differentiation potential; (2) differentiating and expanding committed HE cells from mesodermal cells with committed HE differentiation potential by contacting mesodermal cells with a medium containing a ROCK inhibitor; one or more of a growth factor and cytokine selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. 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, CXCR4, and / or CD93. In some embodiments, the selection uses CD34 positive and CD43 negative. In some embodiments, the selection uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the selection uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In some embodiments, the selection uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the selection uses CD34 positive and CD93 negative. In some embodiments, the medium in the above method is free or essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises exposing the mesodermal cells to a hypoxic tension of about 2% to about 10%.

[0236] In one embodiment of the method of obtaining committed hemogenic endothelial (iHE) differentiation potential in pluripotent stem cell-derived mesodermal cells, the method comprises contacting mesodermal cells with and expanding the mesodermal cells in a medium comprising a ROCK inhibitor; one or more of a growth factor and cytokine selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. 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, CXCR4, and / or CD93. In some embodiments, the medium in the above method is free or 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 comprises exposing the mesodermal cells to a low oxygen tension of about 2% to about 10%. 2. Derivation and expansion of pluripotent stem cell-derived mesodermal cells capable of differentiation into committed hemogenic endothelium

[0237] One aspect of the present invention provides a method for producing pluripotent stem cell-derived mesodermal cells using an optimized multi-step process. In general, the method begins with seeding pluripotent stem cells. The seeded pluripotent stem cells are then developed into mesoderm and further differentiated into mesodermal cells with a committed hematopoietic endothelial potential. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived mesodermal cells, comprising differentiating the seeded pluripotent stem cells into mesoderm and then differentiating the mesoderm into mesodermal cells with a committed hematopoietic potential. The present invention further provides a method for producing pluripotent stem cell-derived mesodermal cells with a committed HE potential, comprising differentiating the pluripotent stem cell-derived mesoderm into mesodermal cells with a committed 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 or essentially free of TGFβ receptor / ALK inhibitors.

[0238] In one embodiment of the method for obtaining a committed hemogenic endothelial differentiation potential in mesodermal cells derived from pluripotent stem cells, the method includes: (1) contacting the pluripotent stem cells with a medium comprising a BMP activator, and optionally bFGF, thereby differentiating and expanding mesodermal cells from the pluripotent stem cells; and (2) contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF, thereby obtaining a committed hemogenic endothelial differentiation potential in the mesodermal cells. 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 is free or essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes exposing the pluripotent stem cells to a low oxygen tension of about 2% to about 10%. In some embodiments, the above methods further comprise seeding the pluripotent stem cells by contacting the cells with a medium comprising a MEKi, a GSKi, and a ROCKi.

[0239] In one embodiment of the method of generating pluripotent stem cell-derived mesodermal cells capable of differentiation into committed hemogenic endothelium from pluripotent stem cell-derived mesoderm, the method comprises differentiating the mesoderm into mesodermal cells capable of differentiation into committed hemogenic endothelium 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 method is free or 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 method further comprises exposing the mesodermal cells to a low oxygen tension of about 2% to about 10%. 3. Derivation and Expansion of Mesoderm from Pluripotent Stem Cells

[0240] One aspect of the present invention provides a method for generating pluripotent stem cell-derived mesoderm using an optimized multi-step process. In general, the method begins with a first step in which pluripotent stem cells are seeded, and the seeded cells are then differentiated into mesoderm in a second step. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The method disclosed herein utilizes an optimized iCD34 culture platform that is free or essentially free of TGFβ receptor / ALK inhibitors.

[0241] In one embodiment of the method of generating pluripotent stem cell-derived mesoderm from pluripotent cells, the method comprises differentiating and expanding mesoderm cells from the seeded 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 method is free or essentially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the method further comprises exposing the seeded iPSCs to a low oxygen tension of about 2% to about 10%. In some embodiments, the method further comprises seeding and expanding the iPSCs by contacting the pluripotent cells with a medium comprising a MEKi, a GSKi, and a ROCKi. 4. Derivation of hematopoietic multipotent progenitor cells (iMPPs)

[0242] One aspect of the present invention provides a method for generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs) using an optimized multi-step process. In general, the method begins with seeding of pluripotent stem cells. The seeded cells are expanded and differentiated into mesodermal cells. The mesoderm is expanded and differentiated into mesodermal cells with a fate-committed hemogenic endothelium potential, which are then differentiated into fate-committed hemogenic endothelium. The fate-committed HE cells are expanded and differentiated into pre-HSCs, which are then differentiated into multipotent progenitor cells capable of differentiating into myeloid cells, including neutrophil progenitors. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs), comprising differentiating seeded pluripotent cells into mesoderm, differentiating the mesoderm into mesodermal cells with a fate-committed hemogenic endothelium potential, then differentiating the mesodermal cells into fate-committed iHEs, which are then differentiated into iMPPs. The present invention further provides a method for producing pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium, then differentiating said mesoderm cells into committed iHE, then differentiating them into iMPPs. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived mesoderm cells into committed iHE, then differentiating them into iMPPs. Furthermore, the present invention provides a method for producing pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived iHE into iMPPs. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized monolayer iCD34 culture platform that does not form EBs and does not contain or is essentially free of TGFβ receptor / ALK inhibitors.

[0243] In one embodiment of the method of generating hematopoietic multipotent progenitor cells (iMPPs) from pluripotent stem cells, the method comprises: (1) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the pluripotent cells with a medium comprising a BMP activator, and optionally bFGF; (2) obtaining in the mesodermal cells the ability to differentiate into committed hemogenic endothelium by contacting the mesodermal cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (3) administering to the mesodermal cells a medium comprising a growth factor and cytokine selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11. and optionally a Wnt pathway activator; and (4) differentiating and expanding said committed HE cells from said committed hemogenic endothelial capable mesodermal cells into iMPPs by contacting the cells with a medium comprising one or more of a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L and IL11, and optionally a ROCK inhibitor. In some embodiments, the above method further comprises seeding and expanding pluripotent stem cells by contacting the cells with a medium comprising a MEKi, a GSKi, and a ROCKi. In some embodiments, the method further comprises differentiating the committed HE cells into pre-HSCs by contacting the HE cells with a medium comprising a BMP activator, a ROCK inhibitor, and one or more of a growth factor and cytokine selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating the committed HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a medium comprising a BMP activator, and one or more of a growth factor and cytokine selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and which is free or essentially free of a ROCK inhibitor.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 above method further comprises exposing the seeded pluripotent stem cells, mesoderm, and / or mesoderm cells capable of differentiation into committed hemogenic endothelium to a low oxygen tension of about 2% to about 10%. 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, CXCR4, and / or CD93. 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 selection uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the selection uses CD34 positive, and CD93 negative. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin.

[0244] In one embodiment of the method of generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesoderm, the method includes: (1) obtaining a differentiation potential for committed hemogenic endothelium in the pluripotent stem cell-derived mesoderm cells by contacting the mesoderm cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (2) one or more of a growth factor and cytokine selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. (3) differentiating and expanding the committed HE cells from the mesodermal cells capable of differentiation into the committed hemogenic endothelium by contacting the cells with a medium comprising a activating agent; and (4) differentiating the committed HE cells into iMPPs by contacting the HE cells with a medium comprising a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-HSCs by contacting the HE cells with a medium 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, Flt3L, and IL11. In other embodiments, the method comprising differentiating the committed HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a medium comprising a BMP activator and one or more of the growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and which is free or essentially free of a ROCK inhibitor. In some embodiments, the medium in the above methods is free or essentially free of a TGFβ receptor inhibitor.In some embodiments, the above methods further comprise exposing mesoderm cells, and / or mesoderm cells having the potential to differentiate into committed hemogenic endothelium, to a low oxygen tension of about 2% to about 10%.

[0245] In one embodiment of the method of generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesodermal cells capable of differentiation into committed hemogenic endothelium, the method comprises: (1) differentiating and expanding committed HE cells from pluripotent stem cell-derived mesodermal cells capable of differentiation into committed hemogenic endothelium by contacting the cells with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11, a ROCK inhibitor, and optionally a Wnt pathway activator; and (2) differentiating the committed HE cells into iMPPs by contacting the HE cells with a medium comprising a BMP activator, and one or more of a growth factor and cytokine selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-HSCs by contacting the HE cells with a medium comprising a BMP activator, a ROCK inhibitor, and one or more of a growth factor and cytokine selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating the committed HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a medium comprising a BMP activator, and one or more of a growth factor and cytokine selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and not comprising or essentially not comprising a ROCK inhibitor. In some embodiments, the medium in the method does not comprise or is essentially not comprising a TGFβ receptor inhibitor. In some embodiments, the above method further comprises exposing the mesodermal cells capable of differentiating into committed hemogenic endothelium to a low oxygen tension of about 2% to about 10%.

[0246] In one embodiment of the method of generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs) from pluripotent stem cell-derived committed HE cells, the method further comprises differentiating the committed HE cells into iMPPs by contacting the HE cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of BMP activators, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-HSCs by contacting the HE cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of BMP activators, ROCK inhibitors, and TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating the committed HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a medium comprising a BMP activator and one or more of the growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and not comprising or essentially not comprising a ROCK inhibitor. In some embodiments, the medium in the above method is not comprising or essentially not comprising a TGFβ receptor inhibitor. In some embodiments, the above method further comprises exposing the mesodermal cells capable of differentiation into committed hemogenic endothelium to a hypoxic tension of about 2% to about 10%. 5. Obtaining pluripotent stem cell-derived T cell progenitors (ipro-T) or pluripotent stem cell-derived T cells - iCD34 platform and iT platform

[0247] One aspect of the invention provides a method of generating pluripotent stem cell-derived T cell progenitors (ipro-T) or pluripotent stem cell-derived T cells using an optimized multi-step process. In general, the method begins with pluripotent stem cells, from which mesodermal cells are differentiated and expanded. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naïve iPSCs. The mesoderm is then differentiated into committed hemogenic endothelial mesodermal cells. The committed hemogenic endothelial mesodermal cells are then differentiated into committed hemogenic endothelial cells while expanding in the medium. The committed HE cells can then be differentiated into pre-iproT, then into T cell progenitors (pro-T), and subsequently into T cells in the same medium. Alternatively, the present invention provides a method for producing a pluripotent stem cell-derived T cell precursor (ipro-T) or a pluripotent stem cell-derived T cell, comprising differentiating a seeded pluripotent stem cell into mesoderm, differentiating the mesoderm into a mesoderm cell capable of differentiation into committed hemogenic endothelium, then differentiating the mesoderm cell capable of differentiation into committed hemogenic endothelium into an iHE, then differentiating it into a T cell precursor or a T cell. The present invention further provides a method for producing a pluripotent stem cell-derived T cell precursor (ipro-T) or a pluripotent stem cell-derived T cell, comprising differentiating a pluripotent stem cell-derived mesoderm into a mesoderm cell capable of differentiation into committed hemogenic endothelium, then differentiating it into an iHE, then differentiating it into an ipro-T or a T cell. Alternatively, the present invention provides a method for producing a pluripotent stem cell-derived T cell precursor or a pluripotent stem cell-derived T cell, comprising differentiating a pluripotent stem cell-derived mesodermal cell capable of differentiation into a fate-committed hemogenic endothelium into an iHE, which is then differentiated into an ipro-T or a T cell. Furthermore, the present invention provides a method for producing a pluripotent stem cell-derived T cell precursor (ipro-T) or a pluripotent stem cell-derived T cell, comprising differentiating a pluripotent stem cell-derived iHE into an ipro-T or a T cell. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC.The methods disclosed herein utilize an optimized iCD34 culture platform that is free or essentially free of TGFβ receptor / ALK inhibitors. In some embodiments, Notch factors, including but not limited to Jag1, Jag2, DLL-1, DLL-3 and DLL-4, can be introduced as soluble peptides, bead-bound peptides, surface-bound peptides, or cell-presented peptides.

[0248] In one embodiment of the method of generating pluripotent stem cell-derived T cell progenitors (ipro-T) or pluripotent stem cell-derived T cells (iT) from pluripotent stem cells, the method comprises: (1) differentiating the seeded pluripotent stem cells into mesoderm by contacting the cells with a medium comprising a BMP activator, and optionally bFGF; (2) differentiating the mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium by contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (3) differentiating the mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium by contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (4) differentiating the mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium by contacting the cells with a medium comprising a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and (4) differentiating the committed HE cells into ipro-T or iT by contacting the cells with a medium comprising one or more of growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the method further comprises seeding and expanding pluripotent stem cells by contacting the cells with a medium comprising MEKi, GSKi, and ROCKi. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the committed HE cells into pre-iproT by contacting the HE cells with a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator.In other embodiments, the method comprising differentiating the committed HE cells into pre-iproT further comprises differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not comprising or essentially not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the method comprises the pluripotent stem cell-derived pro-T with one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides bound to beads, peptides bound to a surface, or peptides presented by the cells. In some embodiments, the method further comprises exposing the seeded pluripotent stem cells, mesoderm, and / or mesoderm cells capable of differentiation into committed hemogenic endothelium to a hypoxic tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the method is free or essentially free of a TGFβ receptor inhibitor.

[0249] In one embodiment of the method of generating pluripotent stem cell-derived T cell progenitors (ipro-T) or pluripotent stem cell-derived T cells from pluripotent stem cell-derived mesoderm, the method includes: (1) differentiating the mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium by contacting the cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (2) one or more of a growth factor and cytokine selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt activator. (3) differentiating the committed HE cells into ipro-T or iT by contacting the cells with a medium comprising a pathway activator; and (4) differentiating the committed HE cells into ipro-T or iT by contacting the cells with a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproT by contacting the HE cells with a medium comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator. In other embodiments, the method comprising differentiating the committed HE cells into pre-iproT further comprises differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not comprising or essentially not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method comprises differentiating the pluripotent stem cell-derived pro-T with one or more Notch factors.In some embodiments, the Notch factor is Jag1, Jag2, DLL-1, DLL-3 or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides bound to beads, peptides bound to a surface, or peptides presented by cells. In some embodiments, the above method further comprises exposing mesoderm cells having the potential to differentiate into mesoderm and / or committed hemogenic endothelium to a hypoxic tension of about 2% to about 10%. 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, CXCR4, and / or CD93. In some embodiments, the BMP activator of the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the above method is free or essentially free of a TGFβ receptor inhibitor.

[0250] In one embodiment of a method for generating a pluripotent stem cell-derived T cell precursor (ipro-T) or a pluripotent stem cell-derived T cell (iT) from a pluripotent stem cell-derived mesodermal cell capable of differentiation into committed hemogenic endothelium, the method comprises: (1) differentiating the mesodermal cell capable of differentiation into committed hemogenic endothelium into a committed HE cell by contacting the cell with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator; and (2) differentiating the committed HE cell into an ipro-T or iT by contacting the HE cell with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, and IL7; and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproT by contacting the HE cells with a medium comprising a ROCK inhibitor; one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator. In other embodiments, the method comprising differentiating the committed HE cells into pre-iproT further comprises differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, and IL7, and essentially free of or free of one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the method comprises a pluripotent stem cell-derived pro-T with one or more Notch factors. In some embodiments, the Notch factor is Jag1, Jag2, DLL-1, DLL-3, or DLL-4.In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides bound to beads, peptides bound to a surface, or peptides presented by cells. In some embodiments, the above method further comprises exposing the committed hemogenic endothelial mesodermal cells to a low oxygen tension of about 2% to about 10%. 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, CXCR4, and / or CD93. In some embodiments, the BMP activator of the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the above method is free or essentially free of a TGFβ receptor inhibitor.

[0251] In one embodiment of a method for generating pluripotent stem cell-derived T cell precursors (ipro-T) or pluripotent stem cell-derived T cells (iT) from pluripotent stem cell-derived HE cells, the method comprises differentiating the committed HE cells into ipro-T or T by contacting the committed HE cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproT by contacting the HE cells with a medium comprising a ROCK inhibitor; one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator. In other embodiments, the method comprising differentiating the committed HE cells into pre-iproT further comprises differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, and IL7, and not comprising or essentially not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the method comprises pluripotent stem cell-derived pro-T with one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides bound to beads, peptides bound to a surface, or peptides presented by the cells. In some embodiments, the method further comprises exposing the pluripotent stem cell-derived HE cells to a hypoxic tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above methods express CD34.In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the method is free or essentially free of TGFβ receptor inhibitors. 6. Obtaining pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells - iCD34 platform and iNK platform

[0252] One aspect of the invention provides a method for generating pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) using an optimized multi-step process. In general, the method begins with pluripotent stem cells, which in some embodiments are seeded. The pluripotent stem cells are developed into mesodermal cells, expanded, and then differentiated into mesodermal cells with the potential to differentiate into committed hemogenic endothelium. Committed hemogenic endothelium is then differentiated and expanded from the mesodermal cells with the potential to differentiate into committed hemogenic endothelium. The HE cells can be differentiated into pre-iproNK and then into NK cell precursors (pro-NK), and can be differentiated into NK cells sequentially in the same medium. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells, comprising differentiating seeded pluripotent stem cells into mesoderm, differentiating the mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium, then differentiating the mesoderm cells capable of differentiation into committed hemogenic endothelium into iHE, and then differentiating them into NK cell precursors. The present invention further provides a method for producing pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesoderm cells capable of differentiation into committed hemogenic endothelium, then differentiating the mesoderm cells capable of differentiation into committed hemogenic endothelium into iHE, and then differentiating them into ipro-NK or iNK. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived NK cell precursors or iNKs, comprising differentiating pluripotent stem cell-derived mesodermal cells capable of differentiation into committed hemogenic endothelium into iHEs, which are then differentiated into ipro-NK cells or iNKs. Furthermore, the present invention provides a method for producing pluripotent stem cell-derived NK cell precursors (ipro-NKs) or pluripotent stem cell-derived NK cells, comprising differentiating pluripotent stem cell-derived iHEs into ipro-NKs or iNKs. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.In some embodiments, said culture platform for obtaining NK cell precursors comprises deriving NK cells by contacting pro-NK cells with one or more of the artificial antigens introduced in the form of bead conjugates, plasma membrane particles and / or antigen presenting cells to stimulate NK proliferation, development and maturation. The methods disclosed herein utilize an optimized iCD34 culture platform that is free or essentially free of TGFβ receptor / ALK inhibitors.

[0253] In one embodiment of the method of generating pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from seeded iPSCs, the method comprises: (1) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the cells with a medium comprising a BMP activator, and optionally bFGF; (2) obtaining in the mesodermal cells the ability to differentiate into committed hemogenic endothelium by contacting the mesodermal cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (3) contacting the mesodermal cells with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF; and (4) contacting the mesodermal cells with a medium comprising a growth factor and promoter selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11. and (3) contacting the cells with a medium comprising one or more of a cytokine selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor, thereby differentiating and expanding the committed HE cells from the mesodermal cells capable of differentiation into committed hemogenic endothelium; and (4) contacting the HE cells with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproNK by contacting the HE cells with a medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, IL15, VEGF, and bFGF.In other embodiments, the method comprising differentiating the committed HE cells into pre-iproNK further comprises differentiating the pre-iproNK into pro-iNK or iNK by contacting the pre-iproNK cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising or essentially not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK cell precursors comprises NK cells during derivation by contacting the pro-NK cells with one or more of the artificial antigens for stimulating NK proliferation, development, and maturation, introduced in the form of bead conjugates, plasma membrane particles, and / or antigen presenting cells. In one embodiment, the above method further comprises seeding and expanding naive pluripotent cells by contacting the pluripotent cells with a medium comprising MEKi, GSKi, and ROCKi. In some embodiments, the method further comprises exposing the seeded iPSCs, mesoderm, and / or committed hemogenic endothelial mesoderm cells to a hypoxic tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. 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 sorting uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the sorting uses CD34 positive, CD93 negative. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor.In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin.In some embodiments, the medium in the above method is free or essentially free of a TGFβ receptor inhibitor.

[0254] In one embodiment of the method of generating pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from pluripotent stem cell-derived mesoderm, the method comprises: (1) differentiating the mesoderm by contacting the mesoderm with a medium comprising a BMP activator, a Wnt pathway activator, and bFGF to obtain mesoderm cells capable of differentiation into committed hemogenic endothelium; (2) contacting the mesoderm with one or more of the growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. (3) differentiating the committed HE cells into ipro-NK or iNK by contacting the cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproNK by contacting the HE cells with a medium containing one or more growth factors and cytokines selected from the group consisting of BMP activator, ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, IL15, VEGF, and bFGF. In another embodiment, the method comprising differentiating the committed HE cells into pre-iproNK further comprises differentiating the pre-iproNK into ipro-NK or iNK by contacting the pre-iproNK cells with a medium comprising one or more of growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and which is free or essentially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors.In some embodiments, the culture platform for obtaining NK cell precursors includes NK cells being derived by contacting pro-NK cells with one or more of artificial antigens for stimulating NK proliferation, development and maturation, introduced in the form of bead conjugates, plasma membrane particles and / or antigen presenting cells. In some embodiments, the method further comprises exposing mesoderm cells having the differentiation potential to mesoderm and / or committed hemogenic endothelium to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4 and / or CD93. 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 selection uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the selection uses CD34 positive, and CD93 negative. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the above method does not contain or is essentially free of a TGFβ receptor inhibitor.

[0255] In one embodiment of the method of generating pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from pluripotent stem cell-derived mesodermal cells capable of differentiation into committed hemogenic endothelium, the method comprises: (1) differentiating and expanding committed HE cells by contacting mesodermal cells capable of differentiation into committed hemogenic endothelium with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator; and (2) differentiating the committed HE cells into ipro-NK or iNK by contacting the HE cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproNK by contacting the HE cells with a medium comprising a BMP activator, a ROCK inhibitor, and one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating the committed HE cells into pre-iproNK further comprises differentiating the pre-iproNK into ipro-NK or iNK by contacting the pre-iproNK cells with a medium comprising one or more of a growth factor and cytokine selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising or essentially not comprising one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor.In some embodiments, the culture platform for obtaining NK cell precursors includes NK cells being derived by contacting pro-NK cells with one or more of artificial antigens for stimulating NK proliferation, development and maturation, which are introduced in the form of bead conjugates, plasma membrane particles and / or antigen presenting cells. In some embodiments, the method further comprises exposing the seeded pluripotent stem cells, mesoderm and / or mesoderm cells capable of differentiation into committed hemogenic endothelium to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4 and / or CD93. 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 selection uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In some embodiments, the selection uses CD34 positive, CD43 negative, and CD93 negative. In some embodiments, the selection uses CD34 positive and CD93 negative. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the above method does not contain or is essentially free of a TGFβ receptor inhibitor.

[0256] In one embodiment of a method for generating pluripotent stem cell-derived NK cell precursors (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from pluripotent stem cell-derived HE cells, the method comprises differentiating the committed HE cells into ipro-NK or iNK by contacting the committed HE cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the committed HE cells into pre-iproNK by contacting the HE cells with a medium comprising one or more of the growth factors and cytokines selected from the group consisting of BMP activators, ROCK inhibitors, and SCF, Flt3L, TPO, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating the committed HE cells into pre-iproNK further comprises differentiating the pre-iproNK into ipro-NK or iNK by contacting the pre-iproNK cells with a medium comprising one or more of growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising or essentially not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK cell precursors comprises NK cells during derivation by contacting the ipro-NK cells with one or more of artificial antigens for stimulating NK proliferation, development, and maturation, introduced in the form of bead conjugates, plasma membrane particles, and / or antigen presenting cells. In some embodiments, the method further comprises exposing the seeded pluripotent stem cells, mesoderm, and / or mesoderm cells capable of differentiation into committed hemogenic endothelium to a hypoxic partial pressure of about 2% to about 10%.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, CXCR4, and / or CD93. 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 other embodiments, the sorting uses CD34 positive, CD43 negative, and CD93 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, and CD93 negative. In some other embodiments, the sorting uses CD34 positive, CD93 negative. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the medium in the above methods is free or essentially free of a TGFβ receptor inhibitor.

[0257] In light of the above, one advantage of the culture platform contemplated herein is the absence of EB formation for differentiation of pluripotent stem cells, enhancing the viability and survival of single pluripotent cell culture, passage, and dissociation. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs are genomically modified. In some embodiments, the iPSCs are reprogrammed from immune cells of a particular donor or patient. Dissociation of cells into single cells, such as single cell suspensions, can be achieved by enzymatic or mechanical means. Any enzymatic agent known in the art that dissociates cells into single cells may be used in the methods of the present invention. In one embodiment, the dissociation agent is selected from trypsin / EDTA, TrypLE-Select, collagenase IV, and dispase. In dissociating cells according to the method contemplated herein, chelating agents such as EDTA, Accutase, or AccuMax may be used alone or in combination with enzymatic agents.To promote dissociation into single cells, the dissociation agent may be dissolved in calcium and magnesium-free PBS.To enhance cell survival during and after dissociation, in some embodiments, a survival-promoting substance is added, such as one or more growth factors, inhibitors of cell pathways involved in cell death and apoptosis, or conditioned medium.In one embodiment, the survival-promoting substance is a ROCK inhibitor, including but not limited to thiazovivin.

[0258] In some embodiments, the iPSCs for differentiation comprise a genetic imprint. In some embodiments, the genetic imprint of the pluripotent stem cell comprises (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions or substitutions in the genome of the pluripotent cell during or after reprogramming from a non-pluripotent cell to an iPSC; or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor-specific, disease-specific or therapeutic response-specific, and the pluripotent cells are reprogrammed from the source-specific immune cells, and the iPSCs retain the source therapeutic property, and the source therapeutic property is also included in hematopoietic cells derived from the iPSCs. In some embodiments, the genetically modified modalities include one or more of the following: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or derived cells. In some other embodiments, the genetically modified modalities include one or more of: (i) deletion or reduction in expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) introduction or increase in expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, TCR, or surface triggering receptors for bispecific or multispecific engagers. In some embodiments, the surface triggering receptor is universal, i.e., compatible with all effector cell types, and effector cells expressing this universal surface triggering receptor can bind to the same bispecific or multispecific engager regardless of their cell type. In some embodiments, the universal surface triggering receptor comprises an anti-epitope and a costimulatory domain, and the anti-epitope is specific for an epitope of the bispecific or multispecific engager.In some embodiments, the costimulatory domain of the universal surface triggering receptor comprises IL2 for enhanced canonical or non-cononical cell activation and effector cell function. In yet other embodiments, the hematopoietic cells comprise therapeutic properties of source-specific immune cells associated with one or more of the following: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) tolerance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0259] In some embodiments, the engager is cell type specific, i.e., the engager binds to and / or activates a particular immune cell type, hi certain embodiments, the engager is cell type independent, i.e., the engager binds to and / or activates multiple immune cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils.

[0260] In some embodiments, the iPSCs and derived hematopoietic cells are B2M null, HLA-E / G, PDL1, A 2A The cells may be one or more of R, CD47, LAG3 null, TIM3 null, TAP1 null, TAP2 null, tapasin null, NLRC5 null, PD1 null, RFKANK null, CITTA null, RFX5 null, and RFXAP null. These cells with modified HLA class I and / or modified HLA class II show improved in vivo persistence due to increased resistance to immune detection. Moreover, such cells can circumvent the HLA matching requirement in adoptive cell therapy, providing a universal, off-the-shelf source of therapeutic regimens.

[0261] In some embodiments, the iPSCs and derived hematopoietic cells thereof comprise one or more of HACD16, 41BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells have enhanced immune effector capabilities.

[0262] In some embodiments, the iPSCs and their derived hematopoietic cells contain triggering receptors on their surface for binding to bispecific or multispecific engagers, and such cells have enhanced tumor targeting specificity.

[0263] In some embodiments, the iPSCs and their derived hematopoietic cells are antigen-specific.

[0264] Cell culture and media collection techniques are outlined in: Hu et al., Curr. Opin. Biotechnol. 8:148, 1997; K. Kitano, Biotechnology 17:73, 1991; Curr. Opin. Biotechnol. 2:375, 1991; Birch et al., Bioprocess Technol. 19:251, 1990; “Teratocarcinomas and embryonic stem cells: A practical approach” (EJ Robertson, ed., IRL Press Ltd. 1987); “Guide to Techniques in Mouse Development” (PM Wasserman et al. eds., Academic Press 1993); “Embryonic Stem Cell Differentiation in vitro” (MV Wiles, Meth. Enzymol. 225:900, 1993); “Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy” (PD Rathjen et al., al., 1993). Differentiation of stem cells is reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 10:31, 1998.

[0265] In the present invention, strategies are provided for enriching cell populations with clear characterization at various stages of the method. In one embodiment, the method for enriching pluripotent stem cells from a cell population comprises dissociating the cells in the population and resuspending the cells to create a single cell suspension. The dissociated cells can be resuspended in any suitable solution or medium for cell maintenance or cell sorting. In certain embodiments, the pluripotent single cell suspension comprises a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and does not comprise a TFGβ inhibitor. In certain embodiments, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and / or the Rock inhibitor is thiazovivin.

[0266] In certain embodiments, the cell population has been positively selected for pluripotent cells by sorting and / or the population has been depleted of non-reprogrammed or non-pluripotent cells, resulting in a cell population enriched for pluripotent cells. In one embodiment, a single cell suspension is prepared and the single cells are then prepared for sorting, such as by staining for a pluripotency marker, for example with an appropriate antibody. Cells may be sorted by any suitable cell sorting method, such as by magnetic bead sorting or flow cytometry (FACS) sorting.

[0267] The cells may be selected based on one or more pluripotency markers or markers indicative of cell differentiation, such as, but not limited to, SSEA3 / 4 expression, TRA1-60 / 81 expression, TRA1-85 expression, TRA2-54 expression, GCTM-2 expression, TG343 expression, TG30 expression, CD9 expression, CD29 expression, CD133 / prominin expression, CD140a expression, CD56 expression, CD73 expression, CD105 expression, OCT4 expression, NANOG expression, SOX2 expression, KLF4 expression, SSEA1 expression (mouse), CD30 expression, SSEA5 expression, CD90 expression, and / or CD50 expression. In various embodiments, the cells are selected based on at least two, at least three, or at least four pluripotency or differentiation markers. In certain embodiments, the cells are selected based on expression of SSEA4, and in certain embodiments, based on expression of SSEA4 in combination with TRA1-81 and / or TRA1-60. In certain embodiments, cells are selected based on SSEA4 expression, TRA1-81 expression, or TRA1-60 expression, and / or CD30 expression. In one embodiment, cells are selected based on SSEA4, TRA1-81, and CD30. In another embodiment, cells are selected based on SSEA4, TRA1-60, and CD30. In certain embodiments, cell sorting using one or more surface differentiation markers includes, but is not limited to, CD13, CD26, CD34, CD45, CD31, CD46, and CD7, as well as pluripotency markers such as SSEA4, TRA1-81, and / or CD30.

[0268] In some embodiments, the cell population undergoing reprogramming or the pluripotent cell population is depleted of differentiated cells.In one embodiment, the pluripotent cell population or the cell population induced to reprogramming may be depleted of cells with one or more cell surface markers of differentiated cells.Exemplary cell surface markers of differentiating cells include, but are not limited to, CD13, CD26, CD34, CD45, CD31, CD46, and CD7.In certain embodiments, CD13 is used as the surface marker of differentiating cells.

[0269] In other embodiments, the cell population is induced to differentiate into a desired lineage and depleted of pluripotent cells to obtain a population enriched for differentiating or differentiated cells. In some embodiments, the differentiated cell population comprises a cell population, such as ESCs or iPSCs, induced to differentiate into a specific lineage. In some embodiments, pluripotent cells can be depleted from the cell population using negative cell sorting methods described above ("panning"), such as sorting cells in said population by magnetic beads or FAC based on pluripotency markers. In some embodiments, a cell population comprising differentiated cells is sorted by FAC using pluripotency markers to obtain a fraction depleted of cells expressing the pluripotency marker. In other embodiments, the cell population is sorted by FAC based on differentiation markers, such as lineage specific markers including but not limited to CD13, CD26, CD34, CD45, CD31, CD46, and CD7 to obtain a fraction depleted of pluripotency markers. In some specific embodiments of the invention, CD13 is used as a surface marker for differentiating cells. D. Cell populations and cell lines generated from the methods and platforms provided herein

[0270] In some embodiments, the cells cultured after reprogramming are induced to differentiate for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 15 days, at least 18 days, at least 20 days, at least 22 days, at least 24 days, at least 26 days, at least 28 days, at least 30 days, at least 32 days, at least 35 days, at least 40 days, at least 42 days, or at least 45 days, or any number of days in between. In some embodiments, the cells cultured after reprogramming are induced for about 1-42 days, about 2-40 days, about 2-35 days, about 2-20 days, about 2-10 days, about 4-30 days, about 4-24 days, about 6-22 days, or about 8-12 days. In some embodiments, the cells are pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, enrichment provides a method for obtaining clonal pluripotent stem cell-derived differentiating cell colonies in a relatively short time, thereby improving the efficiency of generating pluripotent stem cell-derived differentiated cells at various stages. In one embodiment, enrichment provides a method for deriving CD34-expressing HE cells, CD34-expressing HSC cells, T cell progenitor cells or NK cell progenitor cells, and T cells or NK cells, thereby improving the efficiency of generating each of the cell populations. Enrichment may include sorting a cell population to identify and obtain cells expressing specific characteristic markers indicative of a differentiation stage / cell type. In some embodiments, the sorting uses CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34 positive. 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 selection uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.In some other embodiments, the sorting uses CD34 positive, CD43 negative, and CD93 negative. In some other embodiments, the sorting uses CD34 positive, and CD93 negative. Further enrichment methods include obtaining an enriched population of a desired cell type by depletion of cells expressing markers indicative of an undesired cell type.

[0271] Accordingly, one embodiment of the present invention provides a composition comprising one or more of the following cell populations, cell lines, or clonal cells: (i) pluripotent stem cell-derived CD34+ HE cells (iCD34) that have the potential to differentiate into multipotent progenitor cells and are CD34+CD43-; (ii) pluripotent stem cell-derived committed hemogenic endothelial (iHE) cells that are CD34+ and at least one of CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) CD34+CD45+, (v) pluripotent stem cell derived multipotent progenitor cells (iMPP); (v) pluripotent stem cell derived T cell progenitor cells (ipro-T) that are CD34+CD45+CD7+; (iv) pluripotent stem cell derived T cells (iT) that are CD45+CD4+CD3+ or CD45+CD8+CD3+; (vi) pluripotent stem cell derived NK cell progenitor cells (ipro-NK) that are CD45+CD56+CD7+CD3-; and (vii) pluripotent stem cell derived NK cells (iNK) that are CD45+CD56+NKp46+. In some embodiments, the compositions, cell populations, cell lines or clones described above are capable of cryopreservation. In some embodiments, the compositions, cell populations, cell lines or clones are capable of ambient storage conditions for more than 12 hours, more than 24 hours, more than 36 hours, more than 48 hours, but not more than 3 days, more than 4 days, more than 5 days, more than 6 days, or more than 1 week.

[0272] Another aspect of the invention provides a mixture comprising one or more of the following: (i) pluripotent stem cell-derived CD34+ HE cells (iCD34) and one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) pluripotent stem cell-derived committed hemogenic endothelium (iHE) and one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) pluripotent stem cell-derived committed HSC and one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2. (iv) pluripotent stem cell derived multipotent progenitor cells (iMPP), and iMPP-A; (v) pluripotent stem cell derived T cell progenitor cells (ipro-T), and one or more media selected from iTC-A2, and iTC-B2; (vi) pluripotent stem cell derived T cells (iTC), and iTC-B2; (vii) pluripotent stem cell derived NK cell progenitor cells (ipro-NK), and one or more media selected from iNK-A2, and iNK-B2; and / or (viii) pluripotent stem cell derived NK cells (iNK), and iNK-B2. a. iCD34-C comprises one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator; does not comprise a TGFβ receptor / ALK inhibitor; b. 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, Flt3L, and IL11; c. iTC-A2 comprises a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7; and optionally, a BMP activator; d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; said composition does not comprise one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; e. iNK-A2 comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator; f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15. E. Therapeutic Uses of iPSC-Derived Immune Cells

[0273] The present invention provides compositions comprising isolated populations or subpopulations of immune cells derived from iPSCs using the disclosed methods and compositions and suitable for cell-based adoptive therapy. In one embodiment, the isolated populations or subpopulations of immune cells comprise iPSC-derived HSC cells. In one embodiment, the isolated populations or subpopulations of immune cells comprise iPSC-derived T cells. In one embodiment, the isolated populations or subpopulations of immune cells comprise iPSC-derived NK cells. In some embodiments, the iPSC-derived immune cells are further conditioned ex vivo for improved therapeutic potential. In one embodiment, the isolated populations or subpopulations of iPSC-derived immune cells have an increased number or percentage of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated populations or subpopulations of iPSC-derived immune cells have an increased number or percentage of type I NKT cells. In another embodiment, the isolated populations or subpopulations of iPSC-derived immune cells have an increased number or percentage of acquired NK cells. In some embodiments, the isolated population or subpopulation of iPSC-derived HSC cells, T cells, or NK cells is allogeneic. In some other embodiments, the isolated population or subpopulation of iPSC-derived HSC cells, T cells, or NK cells is autologous.

[0274] In some embodiments, iPSCs for differentiation contain a genetic imprint that conveys a desired therapeutic property in an effector cell, and said genetic imprint is retained and functional in differentiated hematopoietic cells derived from said iPSCs.

[0275] In some embodiments, the genetic imprint of the pluripotent stem cell comprises (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions or substitutions in the genome of the pluripotent cell during or after reprogramming from a non-pluripotent cell to an iPSC; or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor-specific, disease-specific, or therapeutic response-specific, and the pluripotent cell was reprogrammed from the source-specific immune cell, and the iPSC retains the source therapeutic property, and the source therapeutic property is also contained in hematopoietic cells derived from the iPSC.

[0276] In some embodiments, the genetically modified modalities include one or more of the following: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates; or proteins that promote the engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival of iPSCs or derived cells. In some other embodiments, the genetically modified modalities include one or more of the following: (i) deletion or reduction in expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) introduction or increased expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, TCR, or a surface triggering receptor for binding to a bispecific or multispecific engager.

[0277] In yet some further embodiments, the hematopoietic cells comprise therapeutic properties of source-specific immune cells associated with one or more of the following: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) tolerance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0278] In some embodiments, the iPSCs and derived hematopoietic cells include one or more of B2M-null, HLA-E / G-null, PDL1-null, A2AR-null, CD47-null, LAG3-null, TIM3-null, TAP1-null, TAP2-null, tapasin-null, NLRC5-null, PD1-null, RFKANK-null, CITTA-null, RFX5-null, and RFXAP-null. These cells with modified HLA class I and / or modified HLA class II show improved in vivo persistence due to increased resistance to immune detection. Moreover, such cells provide a universal, off-the-shelf source of therapeutic regimens, as they can circumvent HLA matching requirements in adoptive cell therapy.

[0279] In some embodiments, the iPSCs and derived hematopoietic cells comprise one or more of HACD16, 41BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells have enhanced immune effector capabilities.

[0280] In some embodiments, the iPSCs and their derived hematopoietic cells are antigen-specific.

[0281] By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases may be ameliorated. Examples of diseases include, but are not limited to, various autoimmune disorders including alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), certain types of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, certain types of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic erythroderma, lupus erythematosus, certain types of thyroiditis, certain types of uveitis, vitiligo, (Wegener's) granulomatosis with polyangiitis; acute leukemia, and hematopoietic malignancies, including but not limited to diffuse leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome; solid tumors, including but not limited to tumors of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus; and infectious diseases, including but not limited to HIV (human immunodeficiency virus)-associated disorders, RSV (respiratory syncytial virus)-associated disorders, EBV (Epstein-Barr virus)-associated disorders, CMV (cytomegalovirus)-associated disorders, adenovirus-associated disorders, and BK polyomavirus-associated disorders.

[0282] Certain embodiments of the present invention are directed to methods of treating a subject in need of treatment by administering to the subject a composition comprising any of the cells described herein. In certain embodiments, the terms "treat", "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of a complete or partial prevention of the disease, and / or therapeutic, in terms of a partial or complete cure of the disease and / or adverse effects caused by the disease. "Treatment", as used herein, encompasses any treatment of a disease in a mammal, including preventing the disease from arising in a subject susceptible to, but not yet diagnosed as suffering from, the disease; inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease. The therapeutic agent or composition may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, stabilizing or reducing undesirable clinical symptoms in the patient, is particularly important.

[0283] In certain embodiments, the subject has a disease, condition, and / or injury that can be treated, ameliorated, and / or improved by cell therapy. In some embodiments, it is contemplated that the subject in need of cell therapy is a subject with an injury, disease, or condition, whereby cell therapy (e.g., a therapy in which cellular material is administered to the subject) can treat, ameliorate, improve, and / or reduce the severity of at least one symptom associated with the injury, disease, or condition. Certain embodiments contemplate that the subject in need of cell therapy includes, but is not limited to, bone marrow or stem cell transplant candidates, subjects who have undergone chemotherapy or radiation therapy, subjects suffering from or at risk of suffering from a hyperproliferative disease or cancer (e.g., a hematopoietic hyperproliferative disease or cancer), subjects having or at risk of developing a tumor (e.g., a solid tumor), subjects suffering from or at risk of suffering from a viral infection or a disease associated with a viral infection.

[0284] Thus, the present invention further provides a pharmaceutical composition comprising a pluripotent cell-derived hematopoietic cell produced by the methods and compositions disclosed herein, and further comprising a pharma- ceutically acceptable medium. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived T cell produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived NK cell produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived CD34HE cell produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises a pluripotent cell-derived HSC cell produced by the methods and compositions disclosed herein.

[0285] In addition, the present invention provides therapeutic uses of the above pharmaceutical compositions by introducing the composition into a subject suitable for adoptive cell therapy having an autoimmune disorder; a hematopoietic tumor; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0286] The isolated pluripotent stem cell-derived hematopoietic cells may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% T cells, NK cells, NKT cells, CD34+HE cells, or HSCs. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic cells comprise about 95% to about 100% T cells, NK cells, NKT cells, CD34+HE cells, or HSCs. In some embodiments, the invention provides pharmaceutical compositions comprising purified T cells, NK cells, NKT cells, CD34+HE cells, or HSCs, such as compositions comprising an isolated population of about 95% T cells, NK cells, NKT cells, CD34+HE cells, or HSCs, for treating a subject in need of cell therapy.

[0287] In some embodiments, the pharmaceutical composition comprises an isolated population of pluripotent stem cell derived hematopoietic cells that comprises less than about 0.1%, less than about 0.5%, less than about 1%, less than about 2%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, or less than about 30% of iPSC-derived T cells, NK cells, NKT cells, CD34+HE cells, or HSCs. The isolated population of derived hematopoietic cells may in some embodiments comprise more than about 0.1%, more than about 0.5%, more than about 1%, more than about 2%, more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, or more than about 30% of T cells, NK cells, NKT cells, CD34+HE cells, or HSCs. In other embodiments, the isolated population of derived hematopoietic cells may comprise about 0.1% to about 1%, about 1% to about 3%, about 3% to about 5%, about 10% to about 15%, about 15% to about 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 60% to 70%, about 70% to 80%, about 80% to 90%, about 90% to 95%, or about 95% to about 100% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.

[0288] In certain embodiments, the derived hematopoietic cells may comprise about 0.1%, about 1%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% T cells, NK cells, NKT cells, CD34+ HE cells or HSCs.

[0289] As will be understood by those skilled in the art, both autologous and allogeneic immune cells can be used for cell therapy. Autologous cell therapy may have reduced infection, low probability of GvHD, and fast immune reconstitution. Allogeneic cell therapy may have immune-mediated graft-versus-malaria (GVM) effect and low recurrence rate. Based on the specific condition of the patient or subject who needs cell therapy, those skilled in the art can determine which specific type of therapy to administer.

[0290] In certain embodiments, the derived hematopoietic cells of the pharmaceutical composition of the invention are allogeneic to the subject. In certain embodiments, the derived hematopoietic cells of the pharmaceutical preparation of the invention are autologous to the subject. In an autologous transplant, the isolated population of derived hematopoietic cells is fully or partially HLA-matched to the patient. In another embodiment, the derived hematopoietic cells are not HLA-matched to the subject.

[0291] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is at least 0.1×10 5 Cells, at least 0.5 x 10 5 Cells, at least 1 x 10 5 Cells, at least 5 x 10 5 Cells, at least 10 x 10 5 Cells, at least 0.5 x 10 6 Cells, at least 0.75 x 10 6 Cells, at least 1 x 10 6 Cells, at least 1.25 x 10 6 Cells, at least 1.5 x 10 6 Cells, at least 1.75 x 10 6 Cells, at least 2 x 10 6 Cells, at least 2.5 x 10 6 Cells, at least 3 x 10 6 Cells, at least 4 x 10 6 Cells, at least 5 x 10 6 Cells, at least 10 x 10 6 Cells, at least 15 x 10 6 Cells, at least 20 x 10 6 Cells, at least 25 x 10 6 cells, or at least 30 × 10 6 It is a cell.

[0292] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 0.1×10 5 Cells ~ approx. 10×10 5 Cells; approx. 0.5×10 6 Cells ~ approx. 5 x 10 6 Cells; approx. 1 x 10 6 cells ~ approx. 3 x 106 Cells; approx. 1.5 x 10 6 Cells ~ approx. 2.5×10 6 cells; or approximately 2 x 10 6 Cells ~ approx. 2.5×10 6 It is a cell.

[0293] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 1×10 6 cells ~ approx. 3 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 5 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 10×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 20×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 30 x 10 6 cells, or approximately 20 x 10 6 Cells ~ approx. 30 x 10 6 It is a cell.

[0294] In some other embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 1×10 6 Cells ~ approx. 30 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 20×10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 10×10 6 cells, approximately 2.0 x 10 6 Cells ~ approx. 30 x 10 6 cells, approximately 2.0 x 10 6 Cells ~ approx. 20×10 6 cells, or approximately 2.0 x 10 6 Cells ~ approx. 10×10 6 It is a cell.

[0295] In yet another embodiment, the number of derived hematopoietic cells in the pharmaceutical composition is about 1×10 6 cells, approximately 2 x 10 6 cells, approximately 5 x 10 6 cells, approximately 7 x 10 6 cells, approximately 10 x 10 6 cells, approximately 15 x 10 6cells, approximately 17 x 10 6 cells, approximately 20 x 10 6 Cells approximately 25×10 6 cells, or approximately 30 x 10 6 It is a cell.

[0296] In one embodiment, the number of derived hematopoietic cells in the pharmaceutical composition is the number of immune cells in a partial or complete umbilical cord blood, i.e., at least 0.1×10 5 At least 0.5 x 10 cells / kg body weight 5 At least 1 x 10 cells / kg body weight 5 At least 5 x 10 cells / kg body weight 5 Cells / kg body weight, at least 10 x 10 5 At least 0.5 x 10 cells / kg body weight 6 At least 0.75 x 10 cells / kg body weight 6 At least 1 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 At least 1.5 x 10 cells / kg body weight 6 At least 1.75 x 10 cells / kg body weight 6 At least 2 x 10 cells / kg body weight 6 At least 2.5 x 10 cells / kg body weight 6 At least 3 x 10 cells / kg body weight 6 At least 4 x 10 cells / kg body weight 6 At least 5 x 10 cells / kg body weight 6 Cells / kg body weight, at least 10 x 10 6 At least 15 x 10 cells / kg body weight 6 At least 20 x 10 cells / kg body weight 6 At least 25 x 10 cells / kg body weight 6 cells / kg body weight, or at least 30 x 10 6 Cells / kg body weight.

[0297] The derived hematopoietic cells provided by the present invention can be administered to a subject without ex vivo or in vitro expansion prior to administration. In certain embodiments, an isolated population of derived hematopoietic cells is conditioned and treated ex vivo with one or more agents to obtain immune cells with improved therapeutic potential. The conditioned population of derived hematopoietic cells can be washed to remove the treatment agents, and the improved population is administered to a patient without further in vitro expansion of the population.

[0298] In other embodiments, the invention provides an isolated population of derived hematopoietic cells that are expanded prior to modulation of the isolated population or subpopulation of T lymphocytes with one or more agents. The isolated population of derived hematopoietic cells can be recombinantly engineered to express a TCR, CAR or other protein.

[0299] With respect to genetically modified derived hematopoietic cells expressing a recombinant TCR or CAR, either before or after the cells have been genetically modified, the cells may be modified using methods described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; Activation and proliferation can be achieved using the methods described in U.S. Patent Application Publication Nos. 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0300] In certain embodiments, the primary and costimulatory signals for the derived genetically modified hematopoietic cells can be provided by a variety of protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other in solution. In one embodiment, the agent providing the costimulatory signal can be bound to a cell surface and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that binds to the agents, e.g., artificial antigen presenting cells (aAPCs) disclosed in US Patent Publication Nos. 20040101519 and 20060034810, are contemplated for use in activating and expanding T lymphocytes of the present invention.

[0301] The compositions comprising the population of derived hematopoietic cells of the present invention can be sterile and suitable for administration to a human patient and can be in an administrable state (i.e., can be administered without further processing). In some embodiments, the therapeutic composition is in a state ready for infusion into a patient. An administrable cell-based composition refers to a composition that does not require any additional processing or manipulation prior to implantation or administration into a subject.

[0302] A sterile, therapeutically acceptable composition suitable for administration to a patient may contain one or more pharma- ceutically acceptable carriers (additives) and / or diluents (e.g., a pharma- ceutically acceptable medium, e.g., cell culture medium), or other pharma- ceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents are determined, in part, by the particular composition being administered and the particular method used to administer the therapeutic composition. Thus, there are a wide variety of suitable formulations of the therapeutic compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0303] In certain embodiments, the therapeutic cell composition comprising an isolated population of derived hematopoietic cells also comprises a pharma- ceutically acceptable cell culture medium. The therapeutic compositions comprising the derived hematopoietic cell populations disclosed herein can be administered enterally or parenterally, alone or together with other suitable compounds to achieve a desired therapeutic purpose.

[0304] The pharma- ceutically acceptable carrier and / or diluent must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the human subject being treated, which should further maintain or increase the stability of the therapeutic composition. The pharma-ceutically acceptable carrier may be liquid or solid, and is selected, taking into account the intended mode of administration, to provide the desired bulk, consistency, etc., when combined with the other components of the therapeutic composition of the present invention. For example, the pharma- ceutically acceptable carrier may be, but is not limited to, a binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.), an excipient (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.). Other pharma-ceutically acceptable carriers suitable for the compositions of the present invention include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0305] Such carrier solutions may also contain buffers, diluents and other suitable additives. A buffer refers to a solution or liquid that has a chemical makeup that neutralizes acids or bases without significantly changing the pH. Examples of buffers contemplated by the present invention include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), and saline (0.9% NaCl).

[0306] These pharma- ceutically acceptable carriers and / or diluents may be present in an amount sufficient to maintain a pH of the therapeutic composition of about 3 to about 10. That is, the buffering agent may be as much as about 5% of the total composition on a weight-by-weight basis. Electrolytes such as, but not limited to, sodium chloride and potassium chloride may also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH within one of the above pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH within the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0307] The sterile composition of the present invention can be a sterile solution or a sterile suspension in a non-toxic pharma- ceutically acceptable medium. A suspension can refer to a non-adherent state in which cells are not attached to a solid phase. For example, cells maintained in a suspension can be stirred and are not attached to a support such as a culture dish.

[0308] A suspension is a dispersion (mixture) of a finely divided chemical species combined with another chemical species, the former being so finely divided and mixed that it does not settle rapidly. A suspension can be prepared using a vehicle such as a liquid medium that contains a solution. In some embodiments, the therapeutic composition of the present invention is a suspension in which stem cells and / or progenitor cells are dispersed in an acceptable liquid medium or solution (e.g., saline or serum-free medium) and are not attached to a solid phase. In daily life, the most common suspension is a suspension of solids in liquid water. Acceptable diluents (e.g., vehicles and solvents) that can be used include water, Ringer's solution, isotonic sodium chloride (saline) solution, and serum-free cell culture medium. In some embodiments, hypertonic solutions are used to make suspensions. In addition, sterile, non-volatile oils are conventionally used as solvents or suspending media. In parenteral applications, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, and suspensions, emulsions, or implants. Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. In some embodiments, the infusion solution is isotonic with the target tissue. In some embodiments, the infusion solution is hypertonic with respect to the target tissue.

[0309] The pharma- ceutically acceptable carriers, diluents, and other components of the pharmaceutical compositions of the present invention ready for administration are obtained from US pharmaceutical grade reagents that allow the therapeutic compositions to be used in clinical therapy. Typically, these final reagents, including any media, solutions, or other pharma- ceutically acceptable carriers and / or diluents, are sterilized by methods conventional in the art, such as sterile filtration, and are tested for various undesirable contaminants, such as mycoplasma, endotoxin, or viral contamination, prior to use. The pharma- ceutical acceptable carriers, in one embodiment, are substantially free of natural proteins of human or animal origin and are suitable for preserving the cell population of the pharmaceutical composition, including hematopoietic stem and progenitor cells. The pharmaceutical compositions are intended to be administered to human patients, and therefore are substantially free of cell culture medium components, such as bovine serum albumin, horse serum, and fetal bovine serum.

[0310] The invention also provides, in part, the use of the pharma- ceutically acceptable cell culture medium, particularly the composition and / or medium, of the invention. Such compositions are suitable for administration to human subjects. In general, any medium that supports the maintenance, growth, and / or health of the derived hematopoietic cells of the invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharma-ceutically acceptable cell culture medium is serum-free and / or feeder-free.

[0311] The pharmaceutical composition may comprise a serum-free medium suitable for the storage of an isolated population of regulated derived hematopoietic cells. In various embodiments, the serum-free medium is animal-free and may optionally be protein-free. Optionally, the medium may contain biopharmaceutical acceptable recombinant proteins. Animal-free medium refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace the animal proteins originally contained in the animal-free medium, and these nutrients are obtained from synthetic, plant or microbial sources. Protein-free medium, in contrast, is defined as being substantially free of protein.

[0312] Those of skill in the art will appreciate that the above examples of media are illustrative and not limiting of media formulations suitable for use in the present invention, and that there are many suitable media known and available to those of skill in the art.

[0313] The pharmaceutical composition is substantially free of mycoplasma, endotoxin, and microbial contamination. In certain embodiments, the therapeutic composition contains less than about 10, about 5, about 4, about 3, about 2, about 1, about 0.1, or about 0.05 μg / ml of bovine serum albumin.

[0314] With respect to mycoplasma contamination and microbial contamination, "substantially free of" as used herein means a negative reading in a commonly accepted test known to those skilled in the art. For example, mycoplasma contamination is determined by subculturing a sample of the therapeutic composition in broth medium with appropriate positive and negative controls and plating on agar plates at 37°C on days 1, 3, 7, and 14. The appearance of the sample is compared to that of the positive and negative controls under a microscope at 100x. Additionally, an inoculum of indicator cell culture is incubated for 3 and 5 days and examined for the presence or absence of mycoplasma by epifluorescence microscopy using a DNA-binding fluorescent dye at 600x. If the agar and / or broth medium procedures and the indicator cell culture procedures do not show any evidence of mycoplasma contamination, the sample is deemed satisfactory.

[0315] Organic solvent or suitable organic solvent generally refers to a liquid or gas containing carbon that dissolves solid, liquid, or gaseous solutes to produce a solution. A suitable organic solvent is an organic solvent that is suitable for ex vivo administration to mammalian cells or incubation with mammalian cells, and may also be suitable for in vivo administration to a subject, such as by having minimal toxicity or other inhibitory effects under ex vivo conditions (e.g., cell culture) or in vivo at the selected concentration, during the incubation or administration time. A suitable organic solvent should also be suitable for storage stability and handling of the agent described herein.

[0316] Examples of suitable organic solvents include, but are not limited to, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof. In certain embodiments, the composition or organic solvent is substantially free of methyl acetate, meaning that only trace amounts of methyl acetate should be present in the composition or solvent, preferably in undetectable amounts (e.g., as measured by high pressure liquid chromatography (HPLC), gas chromatography (GC), etc.).

[0317] An endotoxin-free container or composition means that the container or composition contains at most trace amounts of endotoxin (i.e., amounts that have no adverse physiological effects on a subject) or an undetectable amount of endotoxin. "Substantially endotoxin-free" means less endotoxin per dose of cells than the 5 EU / kg body weight / day total endotoxin allowed by the FDA for biologics, or 350 EU / total dose of cells for an average 70 kg person.

[0318] In one embodiment, the endotoxin-free container and / or composition is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% endotoxin-free. Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, although endotoxins can be present in gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), which are present in the outer membrane of various gram-negative bacteria and represent a central pathogenic property in the disease-causing ability of these bacteria. Small amounts of endotoxins can cause fever, lowering of blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects in humans. Thus, it is often desirable to remove most or all trace amounts of endotoxins from the formulation container, since even small amounts can cause adverse effects in humans. Endotoxins can be removed from the containers using methods known in the art, for example, the containers can be washed in a HEPA sterilizing washer with endotoxin-free water, depyrogenated at 250°C, and clean packaged in a HEPA sterilizing workstation located in a Class 100 / 10 cleanroom (e.g., a Class 100 cleanroom has no more than 100 particles larger than 0.5 microns in one cubic foot of air). EXAMPLES

[0319] The following examples are offered by way of illustration and not by way of limitation. Example 1: Generation and maintenance of hiPSCs

[0320] Somatic cells, including fibroblasts and blood cells, were induced to reprogram toward a pluripotent state using various combinations of factors, including but not limited to OCT4 / SOX2 / LargeT, OCT4 / SOX2, or OCT4 / SOX2 / NANOG / LargeT, in the presence of reprogramming medium containing inhibitors of the ROCK, MEK, GSK3 pathways, and TGFβ receptors (Valamehr et al. Sci Rep. 2012; 2: 213). 14 days after induction, the reprogramming population was switched to maintenance medium containing inhibitors of the ROCK, GSK3, and MEK pathways, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) (Valamehr et al. Stem Cell Reports 2014, 2(3): 366-381). Cells were maintained indefinitely in maintenance medium.

[0321] Approximately 3 weeks after induction, this reprogramming population was sorted into individual wells of a 96-well plate. Selected clones were characterized, and fully reprogrammed clones representative of naive hiPSCs were selected for differentiation studies (Valamehr et al. Stem Cell Reports 2014, 2(3): 366-381). To determine the % undifferentiated cells during maintenance and after differentiation, flow cytometry analysis was performed for surface co-expression of SSEA4, TRA181, and CD30.

[0322] Genomically modified iPSCs can also be generated during or after somatic cell reprogramming, for example, using the methods disclosed in US Application Serial Nos. 62 / 251,032 and 62 / 337,258, which are incorporated by reference in their entireties.

[0323] Furthermore, reprogrammed hiPSCs may be derived from selected somatic cells, including immune system cells such as T cells, of a preferred donor or patient because they have unique properties that contribute to the treatment of various diseases, including cancer, and / or are capable of differentiating into lymphoid effector cells with the same unique properties. Example 2: Identification of HE populations with hematopoietic differentiation and reconstitution and engraftment potential using the iCD34 culture platform

[0324] The iCD34 platform is a system optimized for hematopoietic cell differentiation. To initiate hematopoietic differentiation, hiPSCs were seeded in monolayers in maintenance medium on day 0 and allowed to attach and proliferate for approximately 24 hours. At this time point (day 1), the maintenance medium was removed and replaced with basal medium without maintenance factors. Around day 2, hematopoietic differentiation was initiated by switching the medium to iCD34-A (see Figure 1). As shown in Figure 1, this medium was supplemented with the growth factor bFGF on day 3 and later switch...

Claims

1. A hematopoietic cell having enhanced therapeutic properties comprising one or more genetic imprints contained in an induced pluripotent stem cell (iPSC), the hematopoietic cell being derived from the iPSC by in vitro differentiation induction, the one or more genetic imprints of the iPSC comprising: (a) one or more genetically modified modalities obtained through intragenomic insertions, deletions or substitutions in the genome of a pluripotent cell during or after reprogramming of a non-pluripotent cell into an iPSC; or (b) one or more retainable therapeutic properties of the source-specific immune cells that are donor-specific, disease-specific, or therapeutic response-specific, and the iPSCs are reprogrammed from the source-specific immune cells; Hematopoietic cells, including

2. The one or more genetically modified modalities may be: (i) introduction or increased expression of a chimeric antigen receptor (CAR), HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, TCR, or a surface triggering receptor for a bispecific or multispecific engager; or (ii) deletion or decreased expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP, The hematopoietic cell of claim 1 .

3. (a) the therapeutic properties of the source-specific immune cells include one or more of: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity; or (b) the hematopoietic cells of claim 1, comprising committed hemogenic endothelial cells, hematopoietic stem / progenitor cells (HSCs), hematopoietic multipotent progenitor cells (MPPs), pre-T cell precursors, pre-NK cell precursors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils.

4. (a) the T cells comprise regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem); or (b) The hematopoietic cell of claim 3, wherein the NK cell comprises an adaptive NK cell.

5. A composition comprising the hematopoietic cell according to any one of claims 1 to 4.

6. The composition of claim 5, further comprising a pharma- ceutically acceptable vehicle.

7. 1. A committed hemogenic endothelial (HE) cell or population thereof, comprising: (a) a committed hemogenic endothelial (HE) cell or population thereof capable of differentiating into a hematopoietic cell according to claim 1; (b) the committed HE cell comprises the one or more genetic imprints contained in the iPSC; (c) the committed HE cell is derived from the iPSC containing the one or more genetic imprints; (d) the committed HE cells comprise (i) hematopoietic multipotent progenitor cells (MPPs), pre-T cell progenitors, pre-NK cell progenitors, T cell progenitors, or NK cell progenitors; or (ii) effector cells, including T cells, NK cells, NKT cells, or B cells; and (e) the one or more genetic imprints are: (i) a chimeric antigen receptor (CAR), HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A ２Ａ (ii) introduction or increased expression of a surface triggering receptor for a R, TCR, or bispecific or multispecific engager; or (ii) deletion or decreased expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP, A fate-committed HE cell or population thereof comprising:

8. The committed HE cells are generated by inducing differentiation of iPSCs comprising the one or more genetic imprints into committed HE cells, the differentiation comprising: (i) contacting the iPSCs with a composition comprising a BMP pathway activator, and optionally bFGF, to obtain mesodermal cells; (ii) contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator to obtain mesodermal cells with committed hemogenic endothelial (HE) differentiation potential capable of providing hematopoietic lineage cells; and (iii) contacting a cell having committed HE differentiation potential with a composition comprising bFGF and a ROCK inhibitor to obtain committed HE cells; The fate-committed HE cells or populations thereof described in claim 7, wherein the mesodermal cells and mesodermal cells having the ability to differentiate into fate-committed HE cells are obtained without forming embryoid bodies in steps (i) and (ii).

9. (a) the WNT pathway activator is a GSK3 inhibitor, optionally wherein the GSK3 inhibitor is CHIR99021; or (b) the BMP pathway activator is BMP4, or a population thereof, of the fate-committed HE cell or population thereof of claim 8 .

10. 8. The committed HE cell or population thereof of claim 7, wherein the iPSCs comprising the one or more genetic imprints are obtained by (i) a genomic insertion of a sequence encoding a recombinant protein, or (ii) a genomic insertion or deletion that deletes or reduces expression of an endogenous gene.

11. 8. The fate-committed HE cell or population thereof of claim 7, wherein the fate-committed HE cell is cryopreserved, and the cryopreserved HE cell survives after thawing and retains the one or more genetic imprints.

12. 8. The fate-committed HE cell or population thereof of claim 7, wherein the one or more genetic imprints comprise one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival.

13. (i) the surface triggering receptor is ubiquitous on hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils; or (ii) the bi- or tri-specific engager is specific for a universal surface triggering receptor and specific for one or more tumor-specific antigens on the surface of tumor cells, or a population thereof, of the fate-committed HE cell of claim 12.

14. The fate-committed HE cell or population thereof of claim 7, wherein the effector cells comprising one or more genetic imprints comprise one or more of the following: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

15. 2. The committed hemogenic endothelial (HE) cell or population thereof capable of differentiating into a hematopoietic cell according to claim 1, wherein the committed HE cell comprises one or more genetic imprints contained in an iPSC from which the committed HE cell is derived, and the committed HE cell is generated by inducing differentiation of the iPSC comprising the one or more genetic imprints into the committed HE cell, the differentiation induction comprising: (i) contacting the iPSCs with a composition comprising a BMP pathway activator, and optionally bFGF, to obtain mesodermal cells; (ii) contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator to obtain mesodermal cells with committed hemogenic endothelial (HE) differentiation potential capable of providing hematopoietic lineage cells; and (iii) contacting a cell having committed HE differentiation potential with a composition comprising bFGF and a ROCK inhibitor to obtain committed HE cells; The mesodermal cells and mesodermal cells having the ability to differentiate into fate-committed HE cells are obtained without forming embryoid bodies in steps (i) and (ii), and are fate-committed HE cells or a population thereof.

16. 16. The fate-committed HE cell or population thereof of claim 15, wherein the fate-committed HE cell comprises one or more genetic imprints contained in the iPSC from which the fate-committed HE cell is derived, and the iPSC comprising the one or more genetic imprints is obtained by (i) a genomic insertion of a sequence encoding a recombinant protein, or (ii) a genomic insertion or deletion that deletes or reduces expression of an endogenous gene.

17. 16. The fate-committed HE cell or population thereof of claim 15, wherein the fate-committed HE cell is cryopreserved, and the cryopreserved HE cell survives after thawing and retains the one or more genetic imprints.

18. 16. The committed HE cell or population thereof of claim 15, wherein the HE cell or population thereof is capable of differentiating into a hematopoietic cell comprising the one or more genetic imprints, the hematopoietic cell comprising: (i) a hematopoietic multipotent progenitor cell (MPP), a pre-T cell progenitor cell, a pre-NK cell progenitor cell, a T cell progenitor cell or a NK cell progenitor cell; or (ii) an effector cell comprising a T cell, a NK cell, a NKT cell or a B cell.

19. The one or more genetic imprints are (a) (i) chimeric antigen receptor (CAR), HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A ２Ａ (ii) introduction or increased expression of a surface triggering receptor for a R, TCR, or bispecific or multispecific engager; or (ii) deletion or reduced expression of one or more of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP, or (b) one or more of the following proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates; or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, and / or survival.

20. (i) the surface triggering receptor is ubiquitous on hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils; or 20. The fate-committed HE cell or population thereof of claim 19, wherein (ii) the bi- or tri-specific engager is specific for a universal surface triggering receptor and specific for one or more tumor-specific antigens on the surface of tumor cells.

21. 20. The fate-committed HE cell or population thereof of claim 18, wherein the effector cells comprising said one or more genetic imprints comprise one or more enhanced therapeutic properties including one or more of: (i) expression of antigen targeting receptors; (ii) HLA presentation or lack thereof; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumoral effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

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