Methods and compositions for inducing differentiation of hematopoietic cells

Abstract

The present invention relates to methods and compositions for inducing differentiation of hematopoietic cells. The present invention provides culture platforms, cell culture media, and methods for differentiating pluripotent cells into hematopoietic cells. The present invention further provides pluripotent stem cell-derived hematopoietic cells generated using the culture platforms and methods disclosed herein that allow for feeder cell-free monolayer culture without EB formation. In particular, the pluripotent stem cell-derived hematopoietic cells of the present invention include, and are not limited to, iHSCs, permanent hemogenic endothelial cells, hematopoietic multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, and B cells.

Description

[0001] This application is a divisional application, based on divisional application number 202211397675.5. The original application was filed on July 26, 2016, with application number 201680073326.4, and is entitled "Method and Composition for Inducing Hematopoietic Cell Differentiation".

[0002] Related applications This application claims priority to U.S. Provisional Application No. 62 / 251,016, filed November 4, 2015; International Application No. PCT / US16 / 14918, filed January 26, 2016; and U.S. Provisional Application No. 62 / 337,093, filed May 16, 2015, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention generally relates to compositions and methods for producing cells of all hematopoietic lineages from pluripotent stem cells. More specifically, this invention relates to an improved culture platform for producing cells of all hematopoietic lineages from pluripotent stem cells (including human induced pluripotent stem cells). Background Technology

[0004] Human induced pluripotent stem cell (hiPSC) technology represents a promising and potentially limitless source of therapeutic hematopoietic cells for treating a variety of hematologic and non-hematologic malignancies, including cancer. To advance the prospects of hiPSCs and genome-engineered hiPSCs as allogeneic sources for hematopoietic cell therapy, it is essential to not only efficiently and reproducibly generate hematopoietic stem cells and progenitor cells (HSCs), but also to efficiently and reproducibly generate populations of immune effector cells, including diverse subsets of T, B, NKT, and NK lymphocytes and their progenitor cells.

[0005] In vitro-derived hematopoietic stem cells (HSCs) with the potential to generate lymphocytes are complicated by at least two transient and spatially distinct waves of hematopoiesis during embryonic development: primitive hematopoiesis and permanent hematopoiesis. Primitive hematopoiesis begins in the yolk sac outside the embryo and generates a transient and limited hematopoietic lineage, primarily consisting of primitive erythroid cells and bone marrow cells, but excluding HSCs. Neonatal HSCs emerge only during the subsequent permanent hematopoietic wave, from specialized endothelial progenitor cells within the arterial vessels (called permanent hematopoietic endothelial cells (HE)). Permanent HE then undergoes a transformation from endothelial cells to hematopoietic cells to produce HSCs, which eventually migrate to the bone marrow, where they maintain multi-lineage hematopoiesis, including T, B, NKT, and NK lymphocytes, throughout adult life. Therefore, the generation of HSCs and lymphoid effector cells from pluripotent stem cells depends on the ability to accurately reproduce the complex stages of early embryonic hematopoietic development according to a predetermined program through well-designed and validated methods and compositions.

[0006] Limited studies have described the directed differentiation of hiPSCs into permanent hematoxylin and eosinophils (HEs) in vitro. A major obstacle to using hiPSCs for therapeutic purposes has been the need to first co-culture these cells with mouse or human stromal cells in the presence of a medium containing an unknown serum to maintain pluripotency and induce differentiation. Additionally, existing protocols have employed strategies involving culturing iPSCs to form embryoid bodies (EBs), which are heterogeneous cell aggregates containing multiple differentiated cell types, including ectoderm, mesoderm, and endoderm cells. These procedures require the aggregation of pluripotent cells, for example, by centrifugation to form clots, allowing cells to settle and aggregate in wells, or permitting passive aggregation and clot formation in suspension cultures. The resulting EBs are maintained in a differentiation-inducing culture system for a certain period, typically seven to ten days, to allow for appropriate differentiation. The EBs are then transferred to an adhesive culture for further maturation or dissociated into single cells for cell type selection to proceed to subsequent differentiation steps. (Kennedy et al., *Cell Reports* 2012:1722-1735; Knorr et al., *Stem Cells Translational Medicine* 2013(2):274-283). For example, Kennedy et al. taught the generation of EBs for iPSC differentiation, in which pluripotent cells are treated with collagenase and trypsin to scrape off the cells to form small aggregates, which are then cultured to form EBs. EB formation has been shown to promote pluripotent stem cell differentiation; however, the formation of aggregates and subsequently EBs requires intensive labor, during which the cell number increases minimally, and the cellular contents in the three-dimensional EB aggregates are inconsistently and unevenly exposed to culture medium factors, resulting in heterogeneous cell products at a variable differentiation stage, and greatly hindering the scalability and reproducibility necessary for efficient and streamlined manufacturing processes.

[0007] Therefore, there is a need for methods and compositions that enable stem cells to differentiate into permanent hematopoiesis without relying on co-culture or serum-containing culture media and without requiring the formation of embryo-like aggregates as intermediates. Summary of the Invention

[0008] This invention generally relates to cell culture conditions, culture media, culture platforms, and methods for culturing stem cells and differentiating them into hematopoietic cells.

[0009] Specifically, the present invention provides a method and composition for generating hematopoietic cell lineages from permanent hematopoietic endothelial cells (HE) derived from pluripotent stem cells (including hiPSCs) without the formation of EB, under serum-free / feeder-free conditions and in a scalable monolayer culture platform. The range of cells that can be differentiated according to the method of the present invention includes pluripotent stem cells to progenitor cells focused on specific terminally differentiated and transdifferentiated cells, and various cell lineages that directly convert to hematopoietic fate without undergoing pluripotent intermediates. Similarly, the range of cells generated from stem cell differentiation includes pluripotent stem cells or progenitor cells to terminally differentiated stem cells, and all intermediate hematopoietic cell lineages.

[0010] This invention provides a method and composition for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in a monolayer culture, comprising contacting the pluripotent stem cells with a BMP pathway activator and optionally bFGF. Thus, pluripotent stem cell-derived mesoderm cells are obtained and expanded without the need for embryoid formation from the pluripotent stem cells. The mesoderm cells are then contacted with the BMP pathway activator, bFGF, and WNT pathway activator to obtain expanded mesoderm cells with permanent hematopoietic endothelial (HE) potential, without the need for embryoid formation from the pluripotent stem cells. By subsequent contact with bFGF and optionally a ROCK inhibitor and / or a WNT pathway activator, the mesoderm cells with permanent HE potential differentiate into permanent HE cells, which are also expanded during differentiation.

[0011] The method presented in this paper for obtaining hematopoietic lineage cells is superior to EB-mediated pluripotent stem cell differentiation because: EB formation produces moderate to minimal cell expansion; it does not allow monolayer culture, which is important for many applications that require uniform expansion and differentiation of said cells in a population; and it is laborious and inefficient.

[0012] This paper provides a monolayer differentiation platform that promotes differentiation into permanent hematopoietic endothelial cells, thereby generating hematopoietic stem cells and differentiated progeny, such as T, B, NKT, and NK cells. The demonstrated monolayer differentiation strategy achieves a combination of enhanced differentiation efficiency and large-scale expansion, enabling the delivery of a therapeutically relevant number of pluripotent stem cell-derived hematopoietic cells in various therapeutic applications. Furthermore, this invention discloses that monolayer culture using the methods provided herein generates functional hematopoietic lineage cells, achieving a full range of in vitro differentiation, in vitro regulation, and long-term in vivo hematopoietic self-renewal, remodeling, and transplantation. As used herein, iPSC-derived hematopoietic lineage cells include (but are not limited to) permanent hematopoietic endothelial cells, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0013] One aspect of the present invention provides a culture platform for obtaining pluripotent stem cell-derived hematopoietic lineage cells, comprising: (i) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, and optionally free of a TGFβ receptor / ALK inhibitor, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium is adapted for differentiation and expansion of mesodermal cells with perpetual hematopoietic endothelial cell potential; (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, and optionally free of a TGFβ receptor / ALK inhibitor, wherein the culture medium is adapted for obtaining mesodermal cells with perpetual hematopoietic endothelial cell potential; and (iii) a culture medium containing a BMP activator and optionally bFGF, wherein the culture medium is adapted for differentiation and expansion of pluripotent stem cells into mesodermal cells. In some embodiments, the pluripotent stem cells in the above culture platform are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the culture platform further comprises: (iv) a culture medium containing MEK inhibitors, GSK3 inhibitors and ROCK inhibitors but not TGFβ receptor / ALK inhibitors, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells.

[0014] In some embodiments of the culture platform described above, the culture platform further comprises additional culture media: (i) a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors, wherein the culture medium is suitable for the differentiation of pluripotent stem cell-derived pre-T cell progenitors into T cell progenitors or T cells; or (ii) a culture medium containing ROCK inhibitors, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, wherein the culture medium is suitable for the differentiation of pluripotent stem cell-derived perpetual hematopoietic endothelial cells into pre-T cell progenitors; and these additional culture media are suitable for generating pluripotent stem cell-derived T lineage cells.

[0015] In some embodiments of the culture platform described above, the culture platform may further include: (i) a culture medium containing one or more growth factors and cytokines, the cytokines being selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors and is suitable for the differentiation of pluripotent stem cell-derived pre-NK cell progenitors into NK cell progenitors or NK cells; or (ii) a culture medium containing ROCK inhibitors, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, wherein the culture medium is suitable for the differentiation of pluripotent stem cell-derived perpetual hematopoietic endothelial cells into pre-NK cell progenitors; and these additional culture media are suitable for generating pluripotent stem cell-derived NK lineage cells.

[0016] In one embodiment, the culture platform provided above further comprises: (i) a culture medium containing a BMP activator, one or more growth factors and cytokines, but without a ROCK inhibitor, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is suitable for the differentiation of pluripotent stem cell-derived pre-HSCs into hematopoietic pluripotent progenitor cells; and (ii) a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, wherein the culture medium is suitable for the differentiation of pluripotent stem cell-derived permatrophic hematopoietic endothelial cells into pre-HSCs; and these culture media are provided for the production of pluripotent stem cell-derived hematopoietic pluripotent progenitor cells.

[0017] Another aspect of the present invention provides compositions for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, said compositions comprising one or more of the following: (i) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines, optionally a Wnt pathway activator, and pluripotent stem cell-derived mesodermal cells with permanent hematopoietic endothelial cell potential, said cytokines being selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, said culture medium optionally excluding TGFβ receptor / ALK inhibitors, said culture medium being adapted to produce pluripotent stem cell-derived mesodermal cells with hematopoietic endothelial cell potential. (ii) a medium containing pluripotent stem cell-derived mesoderm cells with hematopoietic endothelial cell potential for differentiation into and expansion of permanent hematopoietic endothelial cells; and (iii) a medium containing BMP activator, bFGF and GSK3 inhibitor, but without TGFβ receptor / ALK inhibitor and containing pluripotent stem cell-derived mesoderm cells, wherein the medium is suitable for differentiation into and expansion of mesoderm cells with permanent hematopoietic endothelial cell potential from pluripotent stem cell-derived mesoderm cells; and (iii) a medium containing BMP activator and optionally present bFGF and iPSCs, wherein the medium is suitable for differentiation into and expansion of mesoderm cells from pluripotent stem cells.

[0018] In some embodiments of the composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and wherein one or more genetic imprints contained in the iPSC are retained in the hematopoietic lineage cells from which they differentiate.

[0019] In some embodiments of the composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, the composition comprises an additional culture medium, such as: (vi) a culture medium containing MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors but not TGFβ receptor / ALK inhibitors and containing pluripotent stem cells; wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, wherein one or more genetic imprints contained in the iPSC are retained in the hematopoietic lineage cells from which they differentiate.

[0020] In some embodiments of the above-described compositions for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, the compositions further comprise: (i) a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors, and containing pluripotent stem cell-derived pre-T cell progenitor cells, wherein the culture medium is suitable for the differentiation of pluripotent stem cell-derived pre-T cell progenitor cells into T cell progenitor cells or T cells; or (ii) a culture medium containing ROCK inhibitors, one or more growth factors and cytokines and optionally present BMP activators, and pluripotent stem cell-derived perennial hematopoietic endothelial cells, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, wherein the culture medium is suitable for the differentiation of perennial hematopoietic endothelial cells into pre-T cell progenitor cells. These additional culture media are suitable for generating pluripotent stem cell-derived T lineage cells.

[0021] In one embodiment of the above-described composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, the composition further comprises one or more culture media for generating pluripotent stem cell-derived hematopoietic pluripotent progenitor cells, wherein the culture media comprises: (i) a culture medium containing a BMP activator, one or more growth factors and cytokines, but without a ROCK inhibitor and containing pluripotent stem cell-derived pre-HSCs, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is suitable for pre-HSCs to differentiate into pluripotent hematopoietic progenitor cells; and / or (ii) a culture medium containing a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines, and pluripotent stem cell-derived permanent hematopoietic endothelial cells, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, wherein the culture medium is suitable for permanent hematopoietic endothelial cells to differentiate into pre-HSCs.

[0022] 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 containing a BMP activator and optionally bFGF, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cells into pluripotent stem cell-derived mesodermal cells; (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, and optionally excluding a TGFβ receptor / ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with permanent HE potential from mesodermal cells; and (iii) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, optionally a Wnt pathway activator, and optionally excluding a TGFβ receptor / ALK inhibitor, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cells into pluripotent stem cell-derived mesodermal cells; (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, and optionally excluding a TGFβ receptor / ALK inhibitor, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cells into ... (iv) A medium comprising a ROCK inhibitor, one or more growth factors and cytokines and optionally a BMP activator, and pluripotent stem cell-derived pluripotent endothelial cells, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, wherein the medium is suitable for differentiating pluripotent endothelial cells into pre-T cell progenitor cells; and (v) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, and containing pluripotent stem cell-derived pre-T cell progenitor cells, wherein the medium is suitable for differentiating pre-T cell progenitor cells into T cell progenitor cells or T cells.

[0023] In some embodiments of the culture platform described above for generating pluripotent stem cell-derived T lineage cells, the culture platform further comprises: (vi) a culture medium containing MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors, and optionally excluding TGFβ receptor / ALK inhibitors, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, wherein one or more genetic imprints contained in the iPSC are retained in the hematopoietic lineage cells differentiated from them.

[0024] 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 containing a BMP activator and optionally bFGF, wherein the culture medium is suitable for differentiating and expanding mesodermal cells from pluripotent stem cells; (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor and optionally excluding a TGFβ receptor / ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with permanent hematopoietic endothelial cell potential from mesodermal cells; and (iii) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, optionally a Wnt pathway activator and optionally excluding a TGFβ receptor / ALK inhibitor, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium... The medium is suitable for differentiating mesodermal cells with the potential to become permanent hematopoietic endothelial cells into permanent hematopoietic endothelial cells; (iv) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, wherein the culture medium is suitable for differentiating permanent hematopoietic endothelial cells into pre-NK cell progenitor cells; and (v) a culture medium containing one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor and is suitable for differentiating pre-NK cell progenitor cells into NK cell progenitor cells or NK cells.

[0025] In some embodiments of the culture platform described above for generating pluripotent stem cell-derived NK cells, the culture platform further comprises: (vi) a culture medium containing MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors but free of TGFβ receptor / ALK inhibitors, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, wherein one or more genetic imprints contained in the iPSC are retained in pluripotent stem cell-derived NK lineage cells differentiated from them.

[0026] Another aspect of the present invention provides a culture platform for generating pluripotent stem cell-derived permanent hematopoietic endothelial cells (iHE), the culture platform comprising: (i) a culture medium containing a BMP activator and optionally present bFGF, wherein the culture medium is adapted for differentiation and expansion of mesodermal cells from pluripotent stem cells; (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor and optionally free of a TGFβ receptor / ALK inhibitor, wherein the culture medium is adapted for obtaining mesodermal cells with permanent hematopoietic endothelial cell potential from pluripotent stem cell-derived mesodermal cells; and (iii) a culture medium containing a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is optionally free of a TGFβ receptor / ALK inhibitor, and wherein the culture medium is adapted for differentiation and expansion of permanent hematopoietic endothelial cells from mesodermal cells with permanent hematopoietic endothelial cell potential.

[0027] In some embodiments of a culture platform for generating pluripotent stem cell-derived permanent hematopoietic endothelial cells (iHE), the culture platform further comprises: (iv) a culture medium containing MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors but free of TGFβ receptor / ALK inhibitors, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells.

[0028] Another aspect of the present invention provides a culture platform for generating pluripotent stem cell-derived hematopoietic pluripotent progenitor cells, the culture platform comprising: (i) a culture medium containing a BMP activator and optionally bFGF, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cells into pluripotent stem cell-derived mesodermal cells; (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor and optionally excluding a TGFβ receptor / ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with permanent hematopoietic endothelial cell potential from pluripotent stem cell-derived mesodermal cells; and (iii) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium is optionally excluding a TGFβ receptor / ALK inhibitor. (iv) a culture medium containing a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L and IL11, wherein the culture medium is suitable for differentiating permanent hematopoietic endothelial cells into pre-HSCs; and (v) a culture medium containing a BMP activator, one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, wherein the culture medium is free of a ROCK inhibitor, wherein the culture medium is suitable for differentiating pre-HSCs into hematopoietic pluripotent progenitor cells. In some embodiments, the culture platform further comprises: (vi) a culture medium containing a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor but free of a TGFβ receptor / ALK inhibitor, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, pluripotent stem cells are iPSCs. In some embodiments, iPSCs are untreated iPSCs. In some embodiments, iPSCs contain one or more genetic imprints, wherein one or more genetic imprints contained in the iPSC are retained in hematopoietic cells derived from pluripotent stem cells from which they differentiate.

[0029] Another aspect of the present invention provides a method for guiding pluripotent stem cells to differentiate into permanent hematopoietic lineage cells, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate differentiation of pluripotent stem cells into and expansion of mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to initiate differentiation of mesodermal cells into and expansion of mesodermal cells with permanent hematopoietic lineage potential, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor; and (iii) contacting mesodermal cells with permanent hematopoietic lineage potential with the composition to initiate differentiation of pluripotent stem cells into and expansion of mesodermal cells with permanent hematopoietic lineage potential. Pluripotent stem cell-derived mesodermal cells with perpetual hematopoietic endothelial cell potential differentiate into and expand perpetual hematopoietic endothelial cells. The composition comprises a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor; and optionally, pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells with hematopoietic endothelial cells, and / or perpetual hematopoietic endothelial cells are subjected to a hypoxic pressure between about 2% and about 10%.

[0030] In some embodiments for guiding pluripotent stem cells to differentiate into hematopoietic lineage cells, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, wherein the composition is free of 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 untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and wherein one or more genetic imprints contained in the iPSC are retained in the pluripotent stem cell-derived hematopoietic cells from which they differentiate.

[0031] In some embodiments used to guide the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells does not involve the production of embryonic bodies and is carried out in a monolayer culture format.

[0032] In some embodiments of the above method, the obtained pluripotent stem cell-derived permanent hematopoietic endothelial cells are CD34+. In some embodiments, the obtained permanent hematopoietic endothelial cells are CD34+CD43-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CD43-CD93-. In some embodiments, the permanent hematopoietic endothelial cells are CD34+CD93-.

[0033] In some embodiments of the above method, the method further comprises (i) contacting pluripotent stem cell-derived permatrophic endothelial cells with a composition to initiate the differentiation of the permatrophic endothelial cells into pre-T cell progenitor cells, the composition comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally, (ii) contacting the pre-T cell progenitor cells with the composition to initiate the differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells, the composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but excluding one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor. In some embodiments of the method, the pluripotent stem cell-derived T cell progenitor cells are CD34+CD45+CD7+. In some embodiments of the method, the pluripotent stem cell-derived T cell progenitor cells are CD45+CD7+.

[0034] In further embodiments of the above-described method for guiding pluripotent stem cells to differentiate into hematopoietic lineage cells, the method further comprises: (i) contacting pluripotent stem cell-derived permatrophic hematopoietic endothelial cells with a composition to initiate the differentiation of the permatrophic hematopoietic endothelial cells into pre-NK cell progenitor cells, the composition comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally, (ii) contacting pluripotent stem cell-derived pre-NK cell progenitor cells with a composition comprising one or more growth factors and cytokines to initiate the differentiation of the pre-NK progenitor cells into NK cell progenitor cells or NK cells, the cytokines being selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor. 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 defined by NKp46+, CD57+, and CD16+.

[0035] Another aspect of the present invention provides a method for generating pluripotent stem cell-derived T-lineage cells, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate the differentiation of mesodermal cells into and expansion of pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but without a TGFβ receptor / ALK inhibitor, to initiate the differentiation of the mesodermal cells into and expansion of mesodermal cells with permanent HE potential; and (iii) contacting the mesodermal cells with permanent HE potential with a composition to initiate the differentiation of the mesodermal cells with permanent HE potential into and expansion of permanent hematopoietic endothelial cells, wherein the composition comprises a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the composition does not contain... (iv) Contacting perpetually hematopoietic endothelial cells with a composition to initiate differentiation of the perpetually hematopoietic endothelial cells into pre-T cell progenitor cells, the composition comprising a ROCK inhibitor, one or more growth factors and cytokines and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO and IL7; and (v) contacting the pre-T cell progenitor cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L and IL7 to initiate differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells, wherein the composition does not contain one or more of VEGF, bFGF, TPO, BMP activator and ROCK inhibitor; and optionally, subjecting the seeded pluripotent stem cells, mesodermal cells, mesodermal cells with perpetual HE potential and / or perpetually hematopoietic endothelial cells to a hypoxic pressure between about 2% and about 10%. In some embodiments, group II of the above method 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 pluripotent stem cells; and / or wherein the pluripotent stem cells are iPSCs. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments of the method, the differentiation of pluripotent stem cells into T cell lineages does not involve the production of embryonic bodies and is carried out in a monolayer culture format.

[0036] Another aspect of the present invention provides a method for generating pluripotent stem cell-derived NK lineage cells, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate differentiation of pluripotent stem cells into and expansion of mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally excluding a TGFβ receptor / ALK inhibitor, to initiate differentiation of mesodermal cells into and expansion of mesodermal cells with permanent HE potential; and (iii) contacting mesodermal cells with permanent HE potential with a composition comprising one or more growth factors and cytokines, a ROCK inhibitor, an optionally present Wnt pathway activator, and optionally excluding a TGFβ receptor / ALK inhibitor, to initiate differentiation of pluripotent stem cell-derived mesodermal cells with permanent HE potential into and expansion of pluripotent stem cell-derived permanent hematopoietic endothelial cells, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL1. 1; (iv) contacting pluripotent stem cell-derived perpetual hematopoietic endothelial cells with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines and optionally a BMP activator to initiate differentiation of pluripotent stem cell-derived perpetual hematopoietic endothelial cells into pre-NK cell progenitor cells, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and (v) contacting pluripotent stem cell-derived pre-NK cell progenitor cells with a composition to initiate differentiation of pluripotent stem cell-derived pre-NK cell progenitor cells into pluripotent stem cell-derived NK cell progenitor cells or NK cells, wherein the composition comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, but does not contain one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor; and optionally, subjecting the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, and / or perpetual hematopoietic endothelial cells to a hypoxia of about 2% to about 10%. In some embodiments, the method for generating pluripotent stem cell-derived NK lineage cells, in Group II, further comprises contacting iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor / ALK inhibitor, to seed and expand the iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the method for generating pluripotent stem cell-derived NK lineage cells does not involve generating embryonic bodies and is performed in a monolayer culture format.

[0037] Another aspect of the present invention provides a method for generating pluripotent stem cell-derived permanent hematopoietic endothelial cells, the method comprising: (i) contacting iPSCs with a composition comprising a BMP activator and optionally bFGF to initiate differentiation of pluripotent stem cells into and amplification of pluripotent stem cell-derived mesodermal cells; (ii) contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally without a TGFβ receptor / ALK inhibitor, to initiate differentiation of the pluripotent stem cell-derived mesodermal cells into and amplification of pluripotent stem cell-derived mesodermal cells with permanent HE potential; (iii) contacting the pluripotent stem cell-derived mesodermal cells with permanent HE potential into... Cell-derived mesodermal cells are contacted with a composition to initiate the differentiation of pluripotent stem cell-derived mesodermal cells with permanent hematopoietic stem cell (H&E) potential into and expand pluripotent stem cell-derived permanent hematopoietic endothelial cells. The composition comprises one or more growth factors and cytokines, a ROCK inhibitor, and optionally a Wnt pathway activator, and optionally does not contain a TGFβ receptor / ALK inhibitor. The cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11. Optionally, the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, and / or permanent hematopoietic endothelial cells are subjected to a hypoxic pressure between about 2% and about 10%. In some embodiments, the above method for generating pluripotent stem cell-derived permanent hematopoietic endothelial cells further comprises: contacting iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor / ALK inhibitor, to seed and expand the iPSCs; and / or wherein the iPSCs are untreated iPSCs. In some embodiments, iPSCs contain one or more genetic imprints, and wherein the one or more genetic imprints contained in the iPSCs are retained in permafrost hematopoietic endothelial cells derived from the pluripotent stem cells from which they differentiate. In some embodiments, the above-described method for differentiating iPSCs into permafrost hematopoietic endothelial cells does not involve the production of embryonic bodies and is performed in a monolayer culture format.

[0038] Another aspect of the present invention provides a method for generating pluripotent progenitor cells of a pluripotent stem cell lineage, comprising: (i) contacting iPSCs with a composition comprising a BMP activator and optionally bFGF to initiate differentiation of iPSCs into and expansion of pluripotent stem cell-derived mesodermal cells; (ii) contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but excluding a TGFβ receptor / ALK inhibitor, to initiate differentiation of the mesodermal cells into and expansion of mesodermal cells with permanent hematopoietic endometrial (HE) potential; and (iii) contacting the mesodermal cells with permanent HE potential with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, to initiate differentiation of the mesodermal cells with permanent HE potential into and expansion of permanent hematopoietic endothelial cells, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL1. 1. The composition thereof is free of TGFβ receptor / ALK inhibitors; (iv) contacting perpetual hematopoietic endothelial cells with a composition comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines to initiate differentiation of perpetual hematopoietic endothelial cells into pre-HSCs, wherein the cytokines are selected from the group consisting of: TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L and IL11; and (v) contacting pre-HSCs with a composition comprising a BMP activator, one or more growth factors and cytokines, but free of ROCK inhibitors, to initiate differentiation of the pre-HSCs into hematopoietic pluripotent progenitor cells, wherein the cytokines are selected from the group consisting of: TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11; and optionally, subjecting the seeded pluripotent stem cells, mesodermal cells and / or perpetual hematopoietic endothelial cells to a hypoxia of about 2% to about 10%. In some embodiments, the method described above for generating pluripotent stem cell-derived hematopoietic pluripotent progenitor cells further comprises contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor / ALK inhibitor, to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and wherein one or more genetic imprints contained in the iPSC are retained in the pluripotent stem cell-derived hematopoietic pluripotent progenitor cells differentiated from them. In some embodiments, differentiating pluripotent stem cells into hematopoietic pluripotent progenitor cells using the method described above does not involve the production of embryonic bodies and is performed in a monolayer culture format.

[0039] Another aspect of the present invention provides a composition comprising: one or more cell populations generated by a culture platform disclosed herein: (i) pluripotent stem cell source (iCD34) CD34+ permanent hematopoietic endothelial cells, wherein the iCD34 cells have the ability to differentiate into pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells and B cells, and wherein the iCD34 cells are CD34+CD43-; (ii) permanent hematopoietic endothelial cells (iHE), wherein the iHE cells are CD34+, and at least one of CD43-, CD93-, CXCR4-, CD73- and CXCR4-CD73-; (iii) pluripotent stem cell source permanent HSCs, wherein the iHSCs are CD34+CD45+; and (iv) hematopoietic pluripotent progenitor cells. (v) iMPP cells, wherein the iMPP cells are CD34+CD45+; (v) T cell progenitor cells, wherein the T cell progenitor cells are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells, wherein the T cells are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) NK cell progenitor cells, wherein the NK cell progenitor cells are CD45+CD56+CD7+; (viii) NK cells, wherein the NK cells are CD3-CD45+CD56+, and optionally further defined by NKp46+, CD57+ and CD16+; (ix) NKT cells, wherein the NKT cells are CD45+Vα24Jα18+CD3+; and (x) B cells, wherein the B cells are CD45+CD19+.

[0040] Another aspect of the invention provides one or more cell lines or clones produced using the methods disclosed herein: pluripotent stem cell sources (i) CD34+ permanent hematopoietic endothelial cells (iCD34), wherein the iCD34 cells have the ability to differentiate into pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, and NKT cells, and wherein the iCD34 cells are CD34+CD43-; (ii) permanent hematopoietic endothelial cells (iHE), wherein the iHE cell line or clone is CD34+, and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) permanent HSCs, wherein the iHSCs are CD34+CD45+; and (iv) hematopoietic pluripotent progenitor cells (iMPP). The iMPP cells are CD34+CD45+; (v) T cell progenitor cells, wherein the T cell progenitor cells are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells, wherein the T cells are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) NK cell progenitor cells, wherein the NK cell progenitor cells are CD45+CD56+CD7+; (viii) NK cells, wherein the NK cells are CD3-CD45+CD56+, and optionally further defined by NKp46+, CD57+ and CD16+; (ix) NKT cells, wherein the NKT cells are CD45+Vα24Jα18+CD3+; and (x) B cells, wherein the B cells are CD45+CD19+.

[0041] Another aspect of the present invention provides a method for promoting hematopoietic self-renewal, remodeling, or transplantation by using one or more of the following cell populations, cell lines, or clones generated by the disclosed method: (i) pluripotent stem cell sources: (i) CD34+ permanent hematopoietic endothelial cells (iCD34), wherein the iCD34 cells have the ability to differentiate into pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cell NK cells, and NKT cells, and wherein the iCD34 cells are CD34+CD43-; (ii) permanent hematopoietic endothelial cells (iHE), wherein the iHE cell line or clone is CD34+, and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) permanent HSCs, wherein the iHSCs are CD34+CD45+; (iv) hematopoietic stem cells (iHE). (v) Multipotent progenitor cells, wherein the iMPP cells are CD34+CD45+; (v) T cell progenitor cells, wherein the T cell progenitor cells are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells, wherein the T cells are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) NK cell progenitor cells, wherein the NK cell progenitor cells are CD45+CD56+CD7+; (viii) NK cells, wherein the NK cells are CD3-CD45+CD56+, and optionally further defined by NKp46+, CD57+ and CD16+; (ix) NKT cells, wherein the NKT cells are CD45+Vα24Jα18+CD3+; and (x) B cells, wherein the B cells are CD45+CD19+.

[0042] Another aspect of the present invention provides a method for generating hematopoietic lineage cells with enhanced therapeutic properties, the method comprising: obtaining iPSCs containing one or more genetic imprints; and guiding the iPSCs to differentiate into hematopoietic lineage cells. The directed differentiation step further comprises: (i) contacting pluripotent stem cells with a composition containing a BMP pathway activator and optionally present bFGF to obtain mesodermal cells; and (ii) contacting the mesodermal cells with a composition containing a BMP pathway activator, bFGF, and a Wnt pathway activator to obtain mesodermal cells with permanent hematopoietic endothelial (HE) potential, wherein the mesodermal cells with permanent hematopoietic endothelial (HE) potential are capable of providing hematopoietic lineage cells. Preferably, mesodermal cells and mesodermal cells with permanent hematopoietic stem cell (iPSC) potential are obtained in steps (i) and (ii) without using the step of forming embryonic bodies, and the resulting hematopoietic lineage cells comprise permanently hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells (MPPs), pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. Furthermore, the hematopoietic lineage cells retain the genetic imprints contained in the iPSCs for directed differentiation.

[0043] In some embodiments, the directed differentiation step in the above method further comprises: (i) contacting mesodermal cells with permanent HE potential with a composition comprising bFGF and a ROCK inhibitor to obtain permanent HE cells; (ii) contacting the permanent HE cells with a composition comprising a BMP activator and optionally a ROCK inhibitor and one or more growth factors and cytokines to obtain hematopoietic pluripotent progenitor cells (MPPs), wherein the cytokines are selected from the group consisting of: TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; and (iii) contacting the permanent HE cells with the composition to obtain pre-T cell progenitor cells. The composition comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more of the following: BMP activator, ROCK inhibitor, TPO, VEGF, and bFGF; or (iv) contacting the permanent HE cells with the composition to obtain pre-NK cell progenitors, NK cell progenitors, and / or NK cells, the 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 the following: BMP activator, ROCK inhibitor, VEGF, and bFGF.

[0044] To obtain iPSCs containing one or more genetic imprints, one method may be to introduce one or more genetic imprints into iPSCs by gene editing during or after reprogramming non-pluripotent cells into iPSCs, wherein the genetic imprints comprise one or more gene modification patterns, and the genetic imprints are introduced by genomic insertion, deletion, or substitution in the iPSC genome. Another method may be to (i) introduce one or more genetic imprints into iPSCs by obtaining source-specific immune cells with donor, disease, or treatment response specificity, wherein the immune cells exhibit retainable therapeutic properties; (ii) reprogram 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. Regarding the genetic modification patterns contained in the source cells, iPSCs, and / or iPSC-derived cells, they may include one or more of the following: safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of iPSCs or their derived cells.

[0045] In some embodiments, the gene modification pattern includes one or more of the following: (i) deletion or reduced expression of B2M, TAP1, TAP2, TAP-associated glycoprotein (Tapasin), NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2AThe expression of R, CAR, TCR, or surface triggering receptors targeting bispecific or multispecific conjugates is introduced or increased. In some embodiments, the above-described gene modification patterns are preserved in one or more of the following: pluripotent stem cell-derived permanent HE cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, and B cells. In some embodiments, the bispecific or multispecific conjugate is specific to one or more lymphocyte surface receptors and to one or more tumor-specific antigens on the surface of tumor cells. In certain embodiments, the surface triggering receptor is common to hematopoietic lineage cells (including T, NK, NKT, macrophages, and neutrophils). In some embodiments, the common surface triggering receptor comprises an anti-antigen determinant and a co-stimulatory domain, wherein the anti-antigen determinant is specific to the bispecific or multispecific conjugate. In some embodiments, the co-stimulatory domain comprises IL2. In some embodiments, lymphocyte surface receptors include one or more of surface-engineered patterns, CD3, CD16, CD64, and CD89; while tumor-specific antigens include one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In some embodiments, therapeutic properties of the derived specific immune cells include one or more of the following: (i) receptor expression targeting the antigen; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved target specificity while reducing extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0046] Another aspect of the invention provides hematopoietic lineage cells with enhanced therapeutic properties, said cells containing the same genetic imprints as those found in pluripotent stem cells derived from said pluripotent stem cells. In some embodiments, the genetic imprints of the pluripotent stem cells include (i) one or more genetic modification patterns obtained through genomic insertion, deletion, or substitution in the genome of the pluripotent cells during or after reprogramming non-pluripotent cells into iPSCs; or (ii) one or more source-specific immune cells that retain therapeutic properties, wherein said pluripotent cells are reprogrammed from source-specific immune cells. In some embodiments, the genetic modification patterns include one or more of the following: safety switch proteins, targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of iPSCs or their derived cells.

[0047] In some embodiments, the gene modification pattern includes one or more of the following: (i) deletion or reduced expression of B2M, TAP1, TAP2, TAP-associated glycoprotein (Tapasin), NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2A The expression of R, CAR, TCR, or surface triggering receptors targeting bispecific or multispecific conjugates is introduced or increased. In certain embodiments, the surface triggering receptor is universal to hematopoietic lineage cells, including T, NK, NKT, macrophages, and neutrophils. In some embodiments, the universal surface triggering receptor comprises an anti-antigen determinant and a co-stimulatory domain, wherein the anti-antigen determinant is specific to bispecific or multispecific conjugates. In some embodiments, the co-stimulatory domain comprises IL2. In still other embodiments, the hematopoietic lineage cells possess therapeutic properties of source-specific immune cells associated with one or more of the following: (i) receptor expression targeting an antigen; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved targeting specificity while reducing extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0048] In certain embodiments, the bispecific or multispecific conjugate is specific to a universal surface-triggered receptor and to one or more tumor-specific antigens on the surface of tumor cells. In some embodiments, the tumor-specific antigen comprises one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In certain embodiments, the therapeutic properties of the derived specific immune cells include one or more of the following: (i) receptor expression targeting the antigen; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved targeting specificity while reducing extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0049] A specific embodiment includes contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor before introducing one or more genetic imprints into iPSCs to seed and expand the cells.

[0050] In certain embodiments, the surface triggering receptor is universal to hematopoietic lineage cells, including T, NK, NKT, macrophages, and neutrophils. In some embodiments, the universal surface triggering receptor includes an anti-antigen determinant and a co-stimulatory domain, wherein the anti-antigen determinant is specific to bispecific or multispecific conjugates.

[0051] In some embodiments, the co-stimulatory domain includes IL2.

[0052] In certain embodiments, the bispecific or multispecific conjugate is specific to one or more tumor-specific antigens on the surface of tumor cells.

[0053] In some embodiments, hematopoietic lineage cells include permanent hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells (MPPs), pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, T cells include T regulatory cells (Tregs), central memory T cells (Tcms), stem cell memory cells (Tscms), and / or effector memory T cells (Tems). In certain embodiments, NK cells include adaptive NK cells.

[0054] In certain embodiments, hematopoietic lineage cells include one or more bispecific or multispecific conjugates targeting surface receptors. In some embodiments, the bispecific or multispecific conjugate a) is specific to the hematopoietic lineage cell type and wherein the conjugate is specific to surface receptors comprising CD3, CD16, CD64, or CD89; or b) is independent of the hematopoietic lineage cell type, wherein the hematopoietic lineage cells contain universal surface-triggered receptors and wherein the conjugate is specific to universal surface-triggered receptors.

[0055] A specific aspect relates to a method for treating a subject requiring cell therapy, comprising administering a therapeutically sufficient number of T-cell progenitor cells derived from induced pluripotent stem cells (iPSCs). In some embodiments, the subject is (i) a bone marrow or stem cell transplant candidate, or the subject has received chemotherapy or radiation therapy; (ii) has received bone marrow ablation or non-myeloablative chemotherapy or radiation therapy; (iii) suffers from a hyperplastic disorder or hematopoietic system cancer; (iv) suffers from a solid tumor; or (v) suffers from a viral infection or a disease associated with a viral infection; wherein the administered T-cell progenitor cells revitalize the thymus and reconstitute T cells in vivo. In some embodiments, the method of treating a subject requiring cell therapy further comprises administering a pharmaceutical composition comprising a bispecific or multispecific conjugate, wherein the bispecific or multispecific conjugate a) is specific to an effector cell type and wherein the conjugate is specific to a surface receptor comprising CD3, CD16, CD64, or CD89; or b) is independent of the effector cell type and wherein the conjugate is specific to a universal surface triggering receptor contained in T progenitor cells and T cells derived therefrom. In some embodiments, the genetic imprinting from source-specific immune cells comprises one or more of the following: (i) receptor expression targeting an antigen; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved target specificity while reducing extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0056] A pharmaceutical composition is also provided comprising a pharmaceutically acceptable medium and one or more hematopoietic lineage cells generated using the methods described herein with enhanced therapeutic properties. These hematopoietic lineage cells with enhanced therapeutic properties include permanent hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells (MPPs), pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. In some embodiments, the T cells comprise T regulatory cells (Tregs), central memory T cells (Tcms), stem cell memory cells (Tscms), and / or effector memory T cells (Tems). In some embodiments, the NK cells comprise adaptive NK cells. Further, therapeutic use of the above-described pharmaceutical compositions is provided, which is achieved by introducing the compositions into a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disease; a hematologic malignancy; a solid tumor; cancer; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0057] Another aspect of the present invention provides an antibody composition comprising one or more antibodies specific to at least one marker selected from the group consisting of: CCR10, CD164, CD95, CD144, CD166, lymphotoxin β 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, Notch 4. MSC, CD97, CD319, CD69, CD338, 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; wherein the above are used to identify permanently proliferating hematopoietic endothelial cells. In one embodiment, the antibody composition comprises an antibody specific for CD93; wherein cells that bind to the antibody specifically targeting CD93 have one or more of the phenotypes CD34+, CD43-, CD73-, CXCR4-, and CD73-CXCR4-.

[0058] Another aspect of the present invention provides a method for identifying perpetually producing hematopoietic endothelial cells differentiated from pluripotent stem cells, comprising (i) obtaining a differentiated cell population; (ii) introducing an antibody specifically targeting CD93 into the cell population; and (iii) identifying a cell population with a CD93 expression level of less than 1%. In some embodiments, the differentiated cell population comprises CD34+ or CD34+CD43- cells. In some embodiments, the method further comprises isolating CD34+ cells from the differentiated cell population; or isolating CD34+CD43- cells from the differentiated cell population.

[0059] Another method for generating pluripotent stem cell-derived permanent hematopoietic endothelial cells (HE cells) is provided, comprising: culturing pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell (HE) potential in a culture medium containing a Wnt pathway activator to obtain permanent HE cells. Obtaining permanent HE cells in the presence of a Wnt pathway activator increases the number and percentage of HE cells in the cell population compared to culturing in the absence of a Wnt pathway activator; improves HE differentiation efficacy; and / or improves HE cell content. In some embodiments, the culture 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 cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints as described above, which are retained in the pluripotent stem cell-derived permanent HE cells.

[0060] A particular aspect of the invention relates to antigen-specific iPSCs or derived hematopoietic lineage cells produced by the methods described herein. Certain aspects of the invention relate to compositions comprising antigen-specific iPSCs or derived hematopoietic lineage cells produced by the methods described herein. Some aspects of the invention relate to pharmaceutical compositions comprising antigen-specific iPSCs or derived hematopoietic lineage cells produced by the methods described herein and a pharmaceutically acceptable medium.

[0061] This document also provides a method for generating antigen-specific induced pluripotent stem cells (iPSCs) and derived hematopoietic lineage cells, comprising: (i) isolating primary antigen-specific T cells from a selected source having donor, disease, or treatment response specificity; (ii) reprogramming the primary antigen-specific T cells to obtain pluripotent stem cells; and (iii) guiding the pluripotent stem cells to differentiate into hematopoietic lineage cells. In some embodiments, the directed differentiation step comprises (i) contacting the pluripotent stem cells with a composition comprising a BMP pathway activator and optionally present 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 permanent hematopoietic endothelial (HE) potential without forming embryoid bodies. In some embodiments, isolated primary antigen-specific T cells are enriched as follows: (i) primary antigen-specific T cells are co-cultured with tumor cells, untransformed cells, dendritic cells, thymic epithelial cells, endothelial cells, or artificial antigen-presenting cells, plasma particles, or peptides expressing the antigen of interest; or primary antigen-specific T cells are sorted using a T cell receptor-specific binder specifically targeting the antigen of interest. In some embodiments, the primary antigen-specific T cells or enriched primary antigen-specific T cells may be regulated using transcription factors or small molecules to induce cell resuscitation.

[0062] A particular aspect of the invention relates to antigen-specific iPSCs or derived hematopoietic lineage cells produced by the methods described herein. Certain aspects of the invention relate to compositions comprising antigen-specific iPSCs or derived hematopoietic lineage cells produced by the methods described herein. Some aspects of the invention relate to pharmaceutical compositions comprising antigen-specific iPSCs or derived hematopoietic lineage cells produced by the methods described herein and a pharmaceutically acceptable medium.

[0063] In some embodiments, antigen-specific pluripotent stem cells obtained by the above methods can be further genetically engineered during or after reprogramming to include genetic imprints comprising one or more gene modification patterns through genomic insertion, deletion, or substitution. In some embodiments, the gene modification patterns comprise one or more of the following: safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; cell surface proteins that deliver secondary or tertiary antigen specificity; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of iPSCs or their derivatives. In some embodiments, the gene modification pattern includes one or more of the following: (i) deletion or reduced expression of B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2A The expression of R, CAR, TCR, or surface triggering receptors targeting bispecific or multispecific conjugates is introduced or increased. In some embodiments, the bispecific or multispecific conjugate is specific to a hematopoietic lineage cell type, and thus the conjugate is specific to a surface receptor selected from CD3, CD16, CD64, or CD89. In some embodiments, the surface triggering receptor is universal to hematopoietic lineage cells (including T, NK, NKT, macrophages, and neutrophils). In some embodiments, the bispecific or multispecific conjugate is independent of hematopoietic lineage cell type and is capable of conjugating to hematopoietic lineage cells containing a matching universal surface triggering receptor. In some embodiments, the universal surface triggering receptor includes an anti-antigen determinant and a co-stimulatory domain, wherein the anti-antigen determinant is specific to the antigen determinant contained in the bispecific or multispecific conjugate. In some embodiments, the co-stimulatory domain of the universal surface triggering receptor includes complete or partial IL2 for homologous or non-homologous cell activation and / or effector cell functional enhancement. In some embodiments, the bispecific or multispecific universal conjugate is specific to one or more tumor-specific antigens on the surface of tumor cells. In some embodiments, the tumor-specific antigen comprises one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In some embodiments, the bispecific or multispecific conjugate is specific to a universal surface-triggered receptor and to one or more tumor-specific antigens on the surface of tumor cells.

[0064] In some embodiments, antigen-specific iPSC-derived hematopoietic lineage cells include permanent hematopoietic endothelial cells (HE cells), hematopoietic stem cells and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells (MPPs), pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils.

[0065] Another aspect of the present invention provides a method for determining the clonality of induced pluripotent stem cells and their derived cells. The general method comprises reprogramming mature-derived T or B cells to obtain induced pluripotent stem cells (iPSCs); and detecting the presence of specific V(D)J recombinants in the iPSCs or hematopoietic lineage cells derived therefrom, said recombinants being identical to those contained in the mature T or B cells used to generate the iPSCs. In some embodiments, the method further comprises isolating iPSCs or hematopoietic lineage cells containing the same V(D)J recombinants as those in the mature-derived T or B cells. In some embodiments, the method comprises obtaining mature-derived T or B cells for reprogramming prior to reprogramming the source cells; and determining the V(D)J recombinants contained in immunoglobulins (Ig) or T-cell receptors (TCRs) specific to the mature-derived T or B cells.

[0066] Another aspect of the present invention provides a method for tracking adoptive cells in in vivo in a cell therapy, the method comprising: obtaining a blood, tissue, or tumor biopsy sample from a subject receiving adoptive cells for therapeutic use; isolating effector cells from the sample; and determining V(D)J recombinants in the effector cells; wherein the adoptive cells are derived from pluripotent stem cells reprogrammed from mature T or B cells; wherein the mature T or B cells contain specific V(D)J recombinants; and wherein the presence of V(D)J recombinants identical to those in the mature T or B cells indicates adoptive cell homing, retention, and / or expansion.

[0067] A pharmaceutical composition is also provided, comprising a pharmaceutically acceptable medium and one or more antigen-specific hematopoietic lineage cells produced using the methods described above, including permanent hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells (MPPs), pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells. In some embodiments, the T cells comprise T regulatory cells (Tregs), central memory T cells (Tcms), stem cell memory cells (Tscms), and / or effector memory T cells (Tems). In some embodiments, the NK cells comprise adaptive NK cells. Furthermore, a therapeutic use of the above pharmaceutical composition is provided, which is achieved by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disease; a hematologic malignancy; a solid tumor; cancer; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0068] In summary, the present invention provides a method and composition for achieving direct differentiation of pluripotent stem cells in a monolayer without generating embryonic bodies from pluripotent stem cells, thereby achieving the differentiation and expansion of mesodermal cells, permanent HE cells, and permanent HSCs, and thus enabling the acquisition of other hematopoietic lineage cells in a scalable and reliable manner with a very high level of efficiency. Attached Figure Description

[0069] Figure 1 A schematic diagram depicts a multi-stage culture method for the hematopoietic differentiation of induced pluripotent stem cells (iPSCs) into permanently hematopoietic endothelial cells (iHE) and multipotent progenitor cells (iMPP). It should be noted that by replacing the glass connective protein with Matrigel™, the culture can be transformed into a sufficiently defined state.

[0070] Figure 2 A schematic diagram depicts a multi-stage culture method for differentiating induced pluripotent stem cells into T cell progenitor cells (ipro-T) and fully differentiated T (iT) cells. It should be noted that by replacing the glass connective protein with Matrigel™, the culture can be transformed into a sufficiently defined culture.

[0071] Figure 3 A schematic diagram depicts a multi-stage culture method for differentiating induced pluripotent stem cells into NK cell progenitors (ipro-NK) and fully differentiated NK cells (iNK). It should be noted that by replacing the glass connective protein with Matrigel™, the culture can be transformed into a fully defined culture.

[0072] Figure 4A-C plots the flow-through cell measurement curves, depicting the emergence of iHE cells and the output of iCD34 and iHE cells (based on iPSC differentiation) over a 10-day timeframe. Calculations are based on snapshots of representative cultures, not optimized cultures.

[0073] Figure 5A -D describes modifications to the protocol, including inoculation density and growth factor titration, to improve HE output on day 10. A) Inoculation density on day 0 affects the HE population on day 10. B) BMP4 concentration from day 2 to day 6 affects the HE population on day 10. C) CHIR concentration on day 3.75 affects the HE population on day 10. D) Inoculation density on day 6 affects the HE population on day 10.

[0074] Figure 6A -B indicates that day 10 HE represents permanent hematopoiesis with pluripotency and dependent on the Notch signaling pathway. A) Morphological changes during the 7-day MPP analysis, and flow cytometry curves of newly emerging CD45 hematopoietic cells. B) Notch-dependent permanent CD45+ cells generated from iPSC-derived CD34+ cells during the iMPP analysis.

[0075] Figure 7A -B depicts the effect of differentiation under hypoxic conditions on the generation of iHE and iMPP hematopoietic progenitor cells. A) Monolayer differentiation under hypoxia increased the percentage of both iCD34-positive and iHE cells by day 10. B) On day 10, iCD34+HE cells generated under hypoxia conditions could further differentiate in iMPP analysis.

[0076] Figure 8A -D indicates the ability of day 10 iCD34 cells to be cryopreserved and maintain panhematopoietic and lymphoid potential. A) Day 10 cryopreserved iCD34 cells survive upon thawing and exhibit a phenotype similar to fresh iCD34+ cells. B) Viability of day 10 cryopreserved iCD34+ cells immediately after thawing. C) Day 10 cryopreserved iCD34+ cells survive during iMPP analysis and generate CD45+ hematopoietic cells. D) Day 10 cryopreserved iCD34+ cells survive and generate iT and iNK lymphocyte progenitor cells.

[0077] Figure 9A-B indicates that day 10 differentiation cultures can be transported overnight at ambient temperature without loss of HE potential. A) Day 7 cultures were maintained in an incubator (control) or treated for overnight transport, then reintroduced into the incubator for an additional two days of maintenance. The presence of iCD34 and iHE cells in the cultures (all on day 10) was then analyzed. In overnight transport cultures, T-flasks contained 30% culture medium and 70% substrate or 100% culture medium. B) Cell counts were calculated.

[0078] Figure 10A -C depicts early CD34+CD7+ T cell progenitors and mature CD4+ and CD8+ T cell subsets derived from hiPSCs using a CD45+CD56-gate selection strategy. A) Early T cell lineage markers indicate the presence of ipro-T cells as defined by CD34+ / CD7+. B) Mature T cell markers indicate the presence of mature T cells as defined by CD4+ or CD8+ cells. c) 5-day T cell differentiation, thus comparing the potential of CD34-positive cells from umbilical cord blood with iCD34-positive cells to generate ipro-T cells.

[0079] Figure 11A -C depicts early CD56+CD7+CD161+ NK cell progenitors and mature CD56+CD16+CD8+ NK cell subsets derived from hiPSCs using a CD45+ gate selection strategy. A) Early NK lineage markers indicate the presence of ipro-NK cells as defined by CD7 and CD56. B) Mature NK lineage markers indicate the presence of mature NK cells as defined by CD57, CD16, CD94, and CD56. c) NK cell differentiation at 5 days, thus comparing the potential of CD34-positive cells from umbilical cord blood with iCD34-positive cells to generate ipro-NK cells.

[0080] Figure 12A -C depicts a monolayer hiPSC hematopoietic differentiation platform that achieves a scalable expansion strategy not observed during EB formation. A. hiPSCs aggregate to form embryo-like bodies and differentiate for 14 days, followed by analysis of CD34 and CD43 expression. B. hiPSCs are seeded as a monolayer and differentiate for 8 days, followed by analysis of CD34, CD43, CXCR4, and CD73. C. CD34-positive cell counts and plotting against time for monolayer and EB-mediated hematopoietic differentiation.

[0081] Figure 13 A schematic diagram depicts a scalable expansion strategy for a monolayer hiPSC hematopoietic differentiation platform used to generate commercial iNK and iT cells. Calculations are based on snapshots of representative cultures, not optimized cultures.

[0082] Figure 14 This indicates that hiPSC-derived CD34-positive cells possess immunomodulatory properties by inhibiting CD3+ T cell survival.

[0083] Figure 15 We depicted mature CD4+ and CD8+ T cell subsets derived from hiPSCs on day 30 after HE isolation using a CD45+CD56-gate selection strategy.

[0084] Figure 16 This indicates that the feeder-based suspension culture supports the maturation of iCD34-derived NK cells.

[0085] Figure 17 This indicates that iCD34-derived iNK cells can respond to cytokine stimulation in order to secrete pro-inflammatory cytokines in a manner similar to that of peripheral blood NK cells.

[0086] Figure 18 This indicates that matrix-free differentiation of pro-NK cells derived from umbilical cord blood CD34-positive cells is faster than that of conventional matrix-based differentiation platforms using a CD45+ gate selection strategy.

[0087] Figure 19 Matrix-free differentiation of iPSC-derived iCD34+ cells into NK cells. DLL4 bound to the culture plate supports the differentiation of CD56+CD7+CD161+ NK cell progenitors, rather than CD11b+ bone marrow cells.

[0088] Figure 20 The study depicted the matrix-free differentiation of UCB CD34+ cells into T cells.

[0089] Figure 21 The study depicted the matrix-free differentiation of iPSC-derived iCD34+ cells into T cells.

[0090] Figure 22 The transplantation of hiPSC-derived iCD34+ cells was described.

[0091] Figure 23A -B indicates that iPSC-derived NK cells respond to cellular stimulation by secreting pro-inflammatory cytokines and possess cytotoxic functions similar to peripheral blood and cord blood NK cells. A. iPSC-derived (iNK) or peripheral blood-derived (pbNK) NK cells were unstimulated (US) or fed with cells at a 1:1 ratio for 4 hours, then collected and stained against CD45, CD56, and TNF-α and analyzed by flow cytometry. B. Effectors were assessed every 2 hours: cytotoxic iNK or cord blood-derived (CBNK) cells at target ratios of 1:1, 3:1, and 10:1 for 90 hours.

[0092] Figure 24The reprogramming of CAR-T cells into iPSCs (TiPSCs) was described based on PCR analysis of CAR (FTV106) integration into iPSCs derived from T cells transduced with FTV106, which retain the same genetic imprint as the source T cells.

[0093] Figure 25 This paper describes the screening of cell surface antibodies on hiPSC-derived CD34+ cells using a CD34+CD43-gate selection strategy, and the workflow for screening hiPSC-derived CD34+ antibodies. It depicts representative results from three independent classifications. Class I markers include cell surface proteins expressed at <1% on hiPSC-derived CD34+ cells. Class II markers include cell surface proteins expressed at 1%–99% on CD34+ cells. Class III markers include cell surface proteins expressed at >99% on CD34+ cells.

[0094] Figure 26 The analysis describes the anti-human antibodies against cell surface proteins expressed on hiPSC-derived CD34+ cells included in the analysis.

[0095] Figure 27 The expression of CD93 markers within the CD34+ population was depicted; and the expression of CXCR4 and CD73 in the CD34+CD93- and CD34+CD93+ portions of CD34+ cells were also described.

[0096] Figure 28A -B indicates the presence of permanent hematopoietic potential in the CD34+ population within the CD93- subset. A. After 10 days of differentiation into iNK cells, the absolute number of CD45+ hematopoietic cells and CD45+CD7+ lymphoprogenitor cells was assessed. B. After another 10 days of iNK differentiation, the presence of iNK cells in the culture was assessed based on the expression of CD56 and NKp30 markers.

[0097] Figure 29A -B indicates that regulation of WNT signaling improves iCD34 cell output. A. Cell cultures differentiated on day 6 in the presence of GSK3 inhibitor CHIR99021 (Wnt agonist) or IWP2 (WNT inhibitor). B. CHIR99021 increased the number and percentage of phenotype CD34+CD43-CD93-HE.

[0098] Figure 30A -B indicates that regulation of WNT signaling improves the output of iCD34 cells to panhematopoiesis and lymphocytes. A. CHIR99021 increases the production of CD45+ cells by regulated HE. B. CHIR99021 increases the production of NK progenitor cells by regulated HE.

[0099] Figure 31 This indicates that the absence of B2-microglobulin (B2M) in iPSCs leads to a lack of expression of HLA class I genes.

[0100] Figure 32A -D indicates that HLA class I knockout iPSCs differentiate into iCD34 HE and can further differentiate into pan-hematopoietic and lymphoid progenitor cells. A. B2M- / - iPSCs and wild-type iPSCs differentiate into HE after 10 days. B. B2M- / - iPSCs can differentiate into CD34+ HE at a similar frequency to wild-type controls. C. B2M- / - HE can generate CD45+ pan-hematopoietic progenitor cells at a similar efficiency to wild-type HE. D. B2M- / - iPSC-derived HE cells can generate iNK progenitor cells at a similar efficiency to wild-type HE.

[0101] Figure 33 This indicates that CD34+CD43-HE differentiated from B2M- / - iPSCs still exhibits HLA class I knockout phenotype.

[0102] Figure 34A -B indicates that HLA class I regulation on iPSCs enhances iPSC retention in immune-competent recipients. A. Transduced, HLA-modified iPSCs express HLA-E on their cell surface and maintain a pluripotent phenotype. B. In vivo teratoma fluorescein imaging 72 hours after B2M- / - HLAE iPSC injection shows enhanced retention compared to wild-type iPSCs.

[0103] Figure 35 This indicates that HLA-E expression was retained in D10 (CD34+CD43- HE cells) and D17 (CD45+ pan-hematopoietic progenitor cells) differentiated from B2M- / - HLA-E iPSCs.

[0104] Figure 36A -C indicates that HLA class I-regulated iPSCs can generate functional CD34+ HE. A. This indicates that B2M- / - HLA-E iPSCs can differentiate into CD34+ HE at a similar frequency to wild-type controls. B. This indicates that B2M- / - HLA-E iPSCs can differentiate into CD34+ HE in numbers comparable to wild-type controls. C. B2M- / - HLA-E HE can generate CD45+ pan-hematopoietic progenitor cells with a similar efficiency to wild-type HE.

[0105] Figure 37A-B indicates that iPSCs genetically engineered to express the high-affinity CD16 receptor and 41BBL co-stimulatory molecule maintained expression throughout differentiation into iCD34 cells. A. Undifferentiated cells on day 0; B. Differentiated cells on day 10.

[0106] Figure 38A -B indicates that iPSCs genetically engineered to express CD19 chimeric antigen receptor (CAR) and a truncated LNGFR cell surface marker as co-recognition markers of CAR maintained expression during differentiation into iCD34 cells. A. Undifferentiated cells on day 0; B. Differentiated cells on day 10.

[0107] Figure 39 We depicted cell type-specific expression of CAR driven by endogenous TCR promoters, and TCR expression and functional gene knockout caused by locus-specific insertion of CAR.

[0108] Figure 40A -B illustrates off-the-shelf targeting strategies for engaging cancer and other disease target cells with the entire effector lineage. A. A diagram illustrating hematopoietic effector cells derived from iPSCs, where corresponding lineage-specific triggering molecules can couple to conjugates that recognize specific target cells. B. A diagram illustrating the engineering of universal conjugates (specific and lineage-independent triggering molecules, including specific anti-antigen determinant conjugates) that are universally expressed on all derived hematopoietic cells.

[0109] Figure 41 This indicates that iPSCs genetically engineered to express the HACD16 receptor maintained expression until day 20 when they differentiated into iNK cells. Detailed Implementation

[0110] This invention generally relates to methods and compositions for differentiating stem cells into permanent hematopoietic cell fates. More specifically, the invention provides a multi-stage differentiation platform in which iPSCs or iPSC-derived cells can be induced to exhibit a permanent hematopoietic phenotype at different stages of development, ranging from permanent hematopoietic endothelial cells to fully differentiated hematopoietic cells, including T cells, B cells, NKT cells, and NK cells. That is, the invention provides methods and compositions for making cells more likely to exhibit a permanent hematopoietic fate (e.g., CD34+ permanent hematopoietic stem cells). Alternatively, the methods and compositions of the invention can be scalable to generate permanent hematopoietic endothelial cells (HE) from untreated iPSCs by avoiding the formation of EBs or aggregates.

[0111] A. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of this invention, the following terms are defined as follows. The article “a / an / the” is used herein to refer to one or more (i.e., at least one) grammatical object of the article. For example, “a component” means one component or more components.

[0112] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0113] The term “and / or” should be understood to mean one or both of the alternatives.

[0114] As used herein, the term "about" or "approximately" means that a quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" means a range of a quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length.

[0115] As used herein, the terms “substantially” or “basically” mean that the quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the terms “substantially identical” or “substantially identical” mean that the range of the quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length is approximately the same as that of a reference quantity, level, value, number, frequency, percentage, scale, size, amount, weight, or length.

[0116] As used herein, the terms “substantially free” and “substantially free” are used interchangeably and, when used to describe a composition (e.g., a cell population or culture medium), mean a composition that is free of the specified substance or its source, for example, 95%, 96%, 97%, 98%, or 99% free of the specified substance or its source, or undetectable, as measured by conventional methods. The terms “free” or “substantially free” of a component or substance in a composition also mean (1) that the composition does not contain any concentration of such component or substance, or (2) that the composition includes such component or substance that is functionally inert but at low concentrations. A similar meaning can be applied to the term “lacking”, which means that a particular substance or its source is absent in the composition.

[0117] As used herein, the term "obvious" refers to a range of quantities, levels, values, numbers, frequencies, percentages, scales, dimensions, quantities, weights, or lengths or events that are readily detectable by one or more standard methods. The term "inconspicuous" and its equivalents refer to a range of quantities, levels, values, numbers, frequencies, percentages, scales, dimensions, quantities, weights, or lengths or events that are not readily detectable or undetectable by standard methods. In one embodiment, an event is considered inconspicuous if the probability of its occurrence is less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, or less.

[0118] Throughout this specification, unless the context otherwise requires, the term "comprise / comprises / comprising" should be understood to imply that it includes the stated steps or components or a group of steps or components, but does not exclude any other steps or components or a group of steps or components. In certain embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0119] The phrase “composed of” means that it includes and is limited to anything that follows the phrase “composed of.” Therefore, the phrase “composed of” indicates that the listed components are necessary or required, and that no other components can exist.

[0120] "consistently composed of..." means including any of the components listed after the phrase, and is limited to other components that do not interfere with or affect the activity or effect specified for the listed components in this disclosure. Thus, the phrase "consistently composed of..." indicates that the listed components are required or necessary, but other components are optional and may or may not be present depending on whether they affect the activity or effect of the listed components.

[0121] Throughout this specification, references to "an embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, refer to specific features, structures, or characteristics described in connection with the embodiment, which are included in at least one embodiment of the invention. Therefore, the aforementioned phrases appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, specific features, structures, or characteristics can be combined in any suitable manner.

[0122] The term "in vitro" generally refers to activities occurring outside an organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside the organism, preferably with minimal alteration to natural conditions. In certain embodiments, an "in vitro" procedure involves obtaining living cells or tissues from an organism and culturing them in a laboratory apparatus, typically for several hours or up to about 24 hours under sterile conditions, but including up to 48 or 72 hours or longer, as applicable. In some embodiments, such tissues or cells may be collected and frozen, and later thawed for in vitro processing. Tissue culture experiments or procedures using living cells or tissues lasting longer than several days are typically considered "in vitro," but in some embodiments, this term may be used interchangeably with "in vitro."

[0123] The term "living body" generally refers to activities that take place inside an organism.

[0124] As used in this article, the term "archetypal stripe" refers to an early embryonic structure marked by the beginning of gastrulation or the formation of the three germ layers: mesoderm, endoderm, and ectoderm.

[0125] As used in this article, the term "mesoderm" refers to one of the three germ layers that appears during early embryonic development and produces various specialized cell types, including blood cells of the circulatory system, muscles, heart, dermis, bones, and other supporting and connective tissues.

[0126] As used herein, the terms "permanent hematopoietic endothelial cells" (HE) or "multipotent stem cell-derived permanent hematopoietic endothelial cells" (iHE) refer to a subset of endothelial cells that generate hematopoietic stem cells and progenitor cells during the transformation from endothelial cells to hematopoietic cells. Hematopoietic cell development in the embryo proceeds sequentially: from the lateral plate mesoderm to angiogenic cells to permanent hematopoietic endothelial cells and hematopoietic progenitor cells.

[0127] As used in this article, the term "hematopoietic stem cell" or "permanent hematopoietic stem cell" refers to CD34+ stem cells that are capable of producing mature bone marrow and lymphocyte types (including T cells, natural killer cells, and B cells).

[0128] As used herein, the terms "reprogramming," "dedifferentiation," "enhanced cell efficacy," or "enhanced developmental efficacy" refer to a method of improving cell efficacy or causing cells to dedifferentiate into a less differentiated state. For example, cells with increased cell efficacy have greater developmental plasticity (i.e., they can differentiate into more cell types) compared to the same cells in a non-reprogrammed state. In other words, reprogrammed cells are cells in a less differentiated state than the same cells in a non-reprogrammed state.

[0129] As used herein, the term "differentiation" is the process by which undifferentiated ("non-specialized") or weakly specialized cells acquire the characteristics of specialized cells (such as blood cells or muscle cells). Differentiated cells, or differentiation-inducing cells, are cells that are already at a more specialized ("specialized") position within a cell lineage. When applied to the differentiation process, the term "specialization" refers to a point in the differentiation pathway where a cell, under normal circumstances, would continue to differentiate into a specific cell type or a subpopulation of that cell type, and under normal circumstances, would not differentiate into a different cell type or revert to a weaker differentiated cell type.

[0130] As used herein, the term "differentiation marker gene" or "differentiation gene" refers to a gene whose expression indicates cell differentiation within cells (e.g., pluripotent cells). 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, CDH11, ZIC4, GA... L, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, M YC, 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.

[0131] As used in this article, the terms “differentiation marker gene profile” or “differentiation gene spectrum”, “differentiation gene expression profile”, “differentiation gene expression marker”, “differentiation gene expression map”, “differentiation gene map” or “differentiation gene marker” refer to the expression or expression level of multiple differentiation marker genes.

[0132] As used herein, the term "efficacy" refers to the sum of all developmental options available to the cell (i.e., developmental efficacy). Successive cell efficiencies include (but are not limited to) differentiation into totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells.

[0133] As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of a body or somatic organism (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuous developmental efficiency ranging from incomplete or partially pluripotent cells that cannot produce a complete organism (e.g., ectoderm stem cells or EpiSCs) to more primitive, multipotent cells that can produce a complete organism (e.g., embryonic stem cells).

[0134] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells derived from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed, to differentiate into tissues capable of differentiating into all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The resulting iPSCs do not refer to cells as they are found in nature. iPSCs as used herein include genome-engineered iPSCs, or iPSCs reprogrammed from immune cells of a preferred donor or patient, and therefore, iPSCs and / or derived lymphoid effector cells contain unique genetic imprints.

[0135] As used herein, the term “genetic imprinting” refers to genetic or epigenetic information that contributes to preferred therapeutic properties of a source cell or iPSC and is retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a “source cell” is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can further differentiate into specific cell types, including any hematopoietic lineage cell. Source cell-derived iPSCs and the differentiated cells derived from them are sometimes collectively referred to as “derived cells,” depending on the context. As used herein, genetic imprinting that confers preferred therapeutic properties is incorporated into iPSCs by reprogramming selected source cells that are specific to donor, disease, or treatment response, or by introducing genetic modification patterns into iPSCs through genome editing. In the aspects of source cells obtained from specially selected donors, disease, or treatment contexts, genetic imprints contributing to preferred therapeutic properties may include any background-specific genetic or epigenetic modifications exhibiting a retainable phenotype, i.e., preferred therapeutic property, which is transferred to derived cells of the selected source cells, regardless of whether the underlying molecular events are identified or not. Source cells specific to donor, disease, or treatment responses may contain genetic imprints that are retainable in iPSCs and derived hematopoietic lineage cells, including (but not limited to) pre-arranged single-specific TCRs, such as those from virus-specific T cells or constant-type natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, such as isotype for point mutations encoding high-affinity CD16 receptors in the selected donor; and predetermined HLA requirements, i.e., the selected HLA-matched donor cells exhibiting haplotypes as the population grows. As used herein, preferred therapeutic properties include derived cell transplantation, transport, homing, viability, self-renewal, survival, regulation and modulation of immune responses, improved survival and cytotoxicity. Preferred therapeutic properties may also involve receptor expression of the target antigen; HLA presentation or its absence; resistance to the tumor microenvironment; induction of bystander immune cells and immune regulation; improved target specificity while reducing extratumor effects; and resistance to therapies such as chemotherapy.

[0136] As used herein, the term "enhanced therapeutic properties" refers to cells whose therapeutic properties are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" will exhibit enhanced, improved, and / or strengthened therapeutic properties compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells can include (but are not limited to) cell transplantation, transport, homing, viability, self-renewal, survival, regulation and modulation of immune responses, survival rate, and cytotoxicity. Therapeutic properties of immune cells are also manifested through: receptor expression targeting antigens; HLA presentation or its absence; resistance to the tumor microenvironment; induction of bystander immune cells and immune modulation; improved target specificity while reducing extratumor effects; and resistance to therapies such as chemotherapy.

[0137] As used herein, the term "conjugate" refers to a molecule, such as a fusion polypeptide, that enables the formation of a connection between immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils) and tumor cells, and activates said immune cells. Examples of conjugates include (but are not limited to) bispecific T cell conjugates (BiTEs), bispecific killer cell conjugates (BiKEs), trispecific killer cell conjugates, or multispecific killer cell conjugates.

[0138] As used herein, the term "surface triggering receptor" refers to a receptor capable of triggering or initiating an immune response (e.g., a cytotoxic response). Surface triggering receptors can be engineered and expressed on effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, surface triggering receptors facilitate bispecific or multispecific antibody conjugation between effector cells and specific target cells (e.g., tumor cells), independent of the effector cell's native receptor and cell type. Using this approach, iPSCs containing a universal surface triggering receptor can be generated, and such iPSCs can then be differentiated into populations of various effector cell types expressing the universal surface triggering receptor. "Universal" means 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 couple or connect with conjugates that are recognizable by the surface triggering receptor and have the same antigenic determinant, regardless of the conjugate's tumor-binding specificity. In some embodiments, conjugates with the same tumor-targeting specificity are used for conjugation with the universal surface triggering receptor. In some embodiments, conjugates with different tumor-targeting specificities are used for conjugation with the universal surface triggering receptor. Therefore, it is possible to conjugate one or more effector cell types, thereby killing a specific type of tumor cell in some cases and two or more types of tumor cells in others. Surface trigger receptors typically contain a co-stimulatory domain for effector cell activation and an anti-antigen determinant specific to the antigenic determinant of the conjugate. Bispecific conjugates are specific to the anti-antigen determinant of the surface trigger receptor at one end and to the tumor antigen at the other end.

[0139] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity of cell therapy or otherwise prevent adverse effects. In some cases, the expression of a safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have permanently incorporated the gene encoding the safety switch protein into their genome. This conditional regulation can be variable and may include control via small molecule-mediated post-translational activation and tissue-specific and / or timed transcriptional regulation. The safety switch can mediate the induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some cases, the safety switch protein is activated by exogenous molecules (e.g., prodrugs), which, upon activation, trigger apoptosis and / or cell death in the treated cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspasesin 9, thymidine kinase, cytosine deaminase, B-cell CD20, modified EGFR, and any combination thereof. In this strategy, prodrugs administered in adverse event conditions are activated by suicide gene products and kill transduced cells.

[0140] As used herein, the term "medicatically active protein or peptide" refers to a protein or peptide capable of exerting biological and / or pharmaceutical effects on an organism. Medicinally active proteins possess curative or palliative properties against a disease and can be administered to improve, alleviate, relieve, reverse, or reduce the severity of a disease. Medicinally active proteins also possess preventative properties and are used to prevent the onset of a disease or to reduce the severity of such a disease or pathological symptom when it manifests. Medicinally active proteins include intact proteins or peptides or pharmaceutically active fragments thereof. They also include pharmaceutically active analogs of said proteins or peptides or analogs of fragments of said proteins or peptides. The term "medicinally active protein" also refers to a variety of proteins or peptides that act in a cooperative or synergistic manner to provide therapeutic benefits. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0141] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases cellular signal transduction. Signal transduction refers to the transmission of molecular signals in chemically modified forms, which is achieved by recruiting protein complexes along pathways that ultimately trigger biochemical events in the cell. Signal transduction pathways are well-known in the field and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, TG point signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0142] As used herein, the term “targeting modality” refers to the genetic incorporation of molecules (e.g., peptides) into cells to promote antigen and / or antigenic determinant specificity, including (but not limited to) i) antigen specificity (when it involves a unique chimeric antigen receptor (CAR) or T-cell receptor (TCR); ii) conjugate specificity (when it involves a monoclonal antibody or a bispecific conjugate); iii) targeting transformed cells; iv) targeting cancer stem cells; and v) other targeting strategies in the absence of specific antigens or surface molecules.

[0143] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide that can exert biological and / or pharmaceutical effects on an organism. Examples of pharmaceutically active proteins or peptides include (but are not limited to) antibodies or fragments thereof, growth factors and / or cytokines.

[0144] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases cellular signal transduction. Signal transduction refers to the transmission of molecular signals in chemically modified forms, which is achieved by recruiting protein complexes along pathways that ultimately trigger biochemical events in the cell. Signal transduction pathways are well-known in the field and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, TG point signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0145] As used herein, the term “specific” can be used to refer to the ability of a molecule (e.g., a receptor or conjugate) to selectively bind to a target molecule, in contrast to nonspecific or nonselective binding.

[0146] As used herein, the term “adoptive cell therapy” refers to a cell-based immunotherapy, which, as used herein, refers to the infusion of autologous or allogeneic lymphocytes identified as genetically modified or unmodified T or B cells that have been expanded in vitro prior to the infusion.

[0147] As used herein, "therapeuticly adequate amount" includes, within its meaning, a non-toxic but sufficient and / or effective amount of the specific therapeutic and / or pharmaceutical composition mentioned herein to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health status, age, and stage and severity of the condition. In certain embodiments, a therapeutically adequate amount is sufficient and / or effective in reducing, decreasing, and / or improving at least one symptom associated with the disease or condition of the subject being treated.

[0148] As used herein, the term "embryonic stem cell" refers to naturally occurring pluripotent stem cells within the internal cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and generate all derived cells from the three primary germ layers during development: the ectoderm, endoderm, and mesoderm. They do not contribute to the outer membranes of the embryo or the placenta; that is, they are not totipotent.

[0149] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, pluripotent cells can also be called "partially differentiated cells." Pluripotent cells are well-known in the field, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotent" means that the cell can form many types of cells within a specified lineage, rather than cells from other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but they cannot form neurons. Therefore, the term "pluripotency" refers to a cell state whose developmental potential is less than that of totipotency and pluripotency.

[0150] Differentiation of pluripotent stem cells requires alterations to the culture system, such as changes to the stimulants in the culture medium or the physical state of the cells. Most traditional strategies utilize the formation of embryonic bodies (EBs) as a common and crucial intermediate step in initiating lineage-specific differentiation. EBs are three-dimensional clusters that have been shown to mimic embryonic development because they generate multiple lineages within their three-dimensional regions. Through the differentiation process, typically hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point they are typically further treated for several days to weeks to continue differentiating. EB formation is initiated by bringing pluripotent stem cells close together to form three-dimensional, multi-layered cell clusters, typically achieved through one of several methods, including allowing pluripotent cells to settle in droplets, allowing cells to settle in U-shaped bottom plates, or by mechanical agitation. Further differentiation cues are needed to promote EB development because aggregates maintained in pluripotent culture maintenance media do not form suitable EBs. Therefore, pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides inducing cues to the selected lineage. EB-based pluripotent stem cell culture typically induces the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) through moderate proliferation within EB cell clusters. While EBs have been shown to promote cell differentiation, they produce heterogeneous cells with variable differentiation states due to inconsistent exposure of cells in a three-dimensional structure to differentiation cues from the environment. Furthermore, the generation and maintenance of EBs are cumbersome. Additionally, cell differentiation via EBs is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0151] In contrast, "aggregate formation," distinct from "EB formation," can be used to expand pluripotent stem cell populations. For example, during aggregate-based pluripotent stem cell expansion, a culture medium that maintains proliferation and pluripotency is selected. Cell proliferation typically increases aggregate size, forming larger aggregates that can dissociate into smaller aggregates using conventional mechanical or enzymatic methods, thereby maintaining cell proliferation and increasing cell number within the culture. Unlike EB culture, cells cultured within aggregates maintain pluripotency markers. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0152] As used herein, “monolayer differentiation” is a term referring to a differentiation method that differs from differentiation through three-dimensional, multi-layered cell clusters, i.e., “EB formation.” Monolayer differentiation, along with other advantages disclosed herein, avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development, such as EB formation, differentiation toward a specific lineage is considered minimal compared to all three germ layer differentiations within EB.

[0153] Pluripotency can be determined in part by assessing the pluripotency characteristics of cells. Pluripotency characteristics include (but are not limited to): (i) pluripotent stem cell morphology; (ii) 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 composed of the three somatic cell lineages; and (vi) embryomorph formation composed of cells from the three somatic cell lineages.

[0154] Two types of pluripotency have been previously described: the “excited” or “metastable” pluripotent state is equivalent to ectodermal stem cells (EpiSCs) of late blastocysts, and the “initial” or “basal” pluripotent state is equivalent to the internal cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the initial or basal state further exhibits: (i) pre-activation or reactivation of the X chromosome in female cells; (ii) improved clonality and viability during single-cell culture; (iii) a general reduction in DNA methylation; (iv) reduced deposition of the H3K27me3 repressive chromatin marker on promoters of developmental regulatory genes; and (v) reduced expression of differentiation markers relative to excited-state pluripotent cells. Standard cell reprogramming methods (whereby exogenous pluripotent genes are introduced into somatic cells, expressed, and then silenced or removed from all pluripotent cells) typically exhibit the characteristics of an excited-state pluripotency. Under standard pluripotent cell culture conditions, such cells remain in an excited state unless exogenous transgene expression is maintained (where the characteristics of the basal state are observed).

[0155] As used in this article, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cells are characterized by a small, round shape, a high nucleus-to-cytoplasm ratio, a prominent nucleolus, and typical intercellular spacing.

[0156] As used herein, "feeder cells" or "feeder layers" are terms used to describe a 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, as the feeder cells provide growth factors and nutrients that support the second cell type. Feeder cells optionally originate 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 and immortalized mouse embryonic fibroblasts. When co-cultured with other cells, feeder cells can typically be inactivated by irradiation or treatment with an antimitotic agent (such as mitomycin) to prevent them from growing beyond their supporting cells. Feeder cells can include endothelial cells, stromal cells (such as epithelial cells or fibroblasts), and leukemia cells. Not limited to the foregoing, a particular type of feeder cell can be a human feeder layer, such as human skin fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEF). Generally, various feeder cells are capable of being used in part to maintain pluripotency, directly differentiate into a certain lineage, and promote maturation into specialized cell types, such as effector cells.

[0157] As used herein, a "feeder-free" (FF) environment refers to an environment, such as culture conditions, cell cultures, or culture media, that substantially does not contain a feeder layer or matrix cells, and / or has not been preconditioned by culturing feeder cells. A "preconditioned" medium refers to a culture medium collected after feeder cells have been cultured in it for a period of time (e.g., at least one day). Preconditioned media contain various mediators, including growth factors and cytokines secreted by feeder cells cultured in the medium.

[0158] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock or other domesticated animal.

[0159] "Multipotent factors" or "reprogramming factors" refer to agents that, alone or in combination with other agents, enhance cellular developmental efficacy. Multipotent factors include (but are not limited to) polynucleotides, peptides, and small molecules that can improve cellular developmental efficacy. Exemplary multipotent factors include, for example, transcription factors and small molecule reprogramming agents.

[0160] "Adhesion" refers to cells attaching to a container, such as cells attaching to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of a suitable culture medium. Certain cell types cannot be maintained or grown in a culture unless they adhere to the cell culture container. Certain cell types ("non-adhesive cells") maintain and / or proliferate in a culture without adhesion.

[0161] "Cultivation" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in a test tube environment. "Cell culture medium," "culture medium" (in all cases, the singular form "medium"), "supplement," and "culture medium supplement" refer to a nutrient composition for cultivating cell cultures.

[0162] "Cultivation" or "maintenance" refers to the maintenance, proliferation (growth), and / or differentiation of cells outside a tissue or body (e.g., in sterile plastic (or coated plastic) cell culture dishes or flasks). "Cultivation" or "maintenance" can utilize culture media as a source of nutrients, hormones, and / or other factors that contribute to cell proliferation and / or maintenance.

[0163] As used herein, a "dissociated" cell refers to a cell that has been substantially separated from other cells or surfaces (e.g., culture plate surfaces) or has been purified. For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in a test tube can dissociate from each other, for example, by enzymatic or mechanical dissociation into clusters, single cells, or a suspension of a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells dissociate from a culture plate or other surface. Thus, dissociation can involve interrupting the interaction between cells and the extracellular matrix (ECM) and substrates (e.g., culture surfaces), or interrupting the ECM between cells.

[0164] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and can refer to any T cell, such as cultured T cells, primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells derived from mammals. T cells can also differentiate from stem cells or progenitor cells. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any developmental stage, 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 monocytes (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, untreated T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc. Other types of helper T cells include, for example, Th3 (Treg), Th17, Th9, or Tfh cells. Other types of memory T cells include, for example, central memory T cells (Tcm cells), stem cell memory cells (Tscm cells), and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express T cell receptors (TCRs) or chimeric antigen receptors (CARs).

[0165] As used herein, the term "untreated T cells" or Tn refers to mature T cells, distinct from activated or memory T cells, which have not yet encountered their homologous antigens in their surroundings. Common characteristics of untreated T cells include surface expression of L-selectin (CD62L); lack of activation markers CD25, CD44, or CD69; and lack of memory CD45RO isoforms. They also express a functional IL-7 receptor composed of the subunit IL-7 receptor-α (CD127) and a common γ chain (CD132). In the untreated state, T cells are considered quiescent and undivided, and their homeostatic survival mechanisms require the common γ chain cytokines IL-7 and IL-15.

[0166] As used herein, the term “central memory T cells” or Tcm refers to a subpopulation or subset of T cells that, compared to effector memory T cells or Tcm, has lower expression of or pro-apoptotic signaling genes, such as Bid, Bnip3, and Bad, and higher expression of genes associated with transport to secondary lymphoid organs, including CD62L, CXCR3, and CCR7.

[0167] As used in this article, the terms “stem memory T cells” or “stem cell memory T cells” or Tscm refer to a subpopulation or subset of T cells that are capable of self-renewal and generating Tcm, Tem, and Teff (effective T cells), and that express CD27 and lymphocyte homing molecules such as CCR7 and CD62L, which are important characteristics that mediate long-term immunity.

[0168] As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As provided herein, NK cells can also differentiate from stem cells or progenitor cells. As used herein, the terms "adaptive NK cell" and "memory NK cell" are interchangeable and refer to a subset of NK cells phenotyped as CD3- and CD56+, expressing NKG2C and CD57 and optionally present CD16, but lacking expression of one or more of the following: PLZF, SYK, FceRɣ, and EAT-2. In some embodiments, the isolated CD56+ NK cell subset includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligand, NKp30, NKp40, NKp46, activating and repressive KIRs, NKG2A, and DNAM-1. CD56+ expression may be weak or strong.

[0169] As used herein, the term "NKT cell" or "natural killer T cell" refers to CD1d-specific T cells that express the T cell receptor (TCR). Unlike conventional T cells that 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 have been identified. The constant or type I NKT cell lineage expresses a very limited TCR: typical α Chain (V in humans) α 24-J α 18) With a limited range β Chain (V in humans) β 11) The combination. The second NKT cell population, called non-classical or non-constant type II NKT cells, shows a more heterogeneous TCR. αβ Utilization. Type I NKT cells are currently considered suitable for immunotherapy. Adaptive or constant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, αGalCer, CD161, and CD56. As provided in this article, NKT cells can also be differentiated from stem cells or progenitor cells.

[0170] As used herein, the terms “B lymphocyte” or “B cell” are used interchangeably and refer to a subset of lymphocytes defined by the expression of B cell receptors (BCR, Ig) CD19 or CD20, which contain both heavy and light chains of immunoglobulins, in the absence of T cell receptor (CD3). As provided herein, B cells can also be derived from stem cells or progenitor cells through directed differentiation. B cells encompass any subtype of B cells and can be at any developmental stage, including (but not limited to) proto-B cells, pre-B cells, untreated B cells, B-1 B cells, B-2 B cells, marginal zone B cells, follicular B cells, memory B cells, plasmablasts, plasma cells, and regulatory B cells.

[0171] B. Overview This invention generally relates to a multi-stage method for differentiating untreated pluripotent cells into non-pluripotent cells or partially differentiated cells (including mesodermal cells, permanent hematopoietic endothelial cells, permanent hematopoietic stem cells or progenitor cells, CD34+ cells, pluripotent progenitor cells (MPPs) (capable of differentiating into bone marrow, including neutrophil progenitor cells), T cell progenitor cells, NK cell progenitor cells) or fully differentiating them into final hematopoietic cells (e.g., T cells, B cells, NKT cells, or NK cells). The invention also relates to compositions used in the disclosed methods; and cell populations, cell lines, or clones produced using the disclosed methods.

[0172] Compared to methods used in the art, the present invention avoids the formation of EBs during iPSC differentiation. As provided, hematopoietic lineage cells derived from iPSCs are obtained by seeding cloned iPSCs in a TGFβ-free medium to maintain their pluripotency in a basal or untreated state, differentiating the cloned iPSCs in a monolayer form without EB formation, and applying appropriate combinations of small chemical molecules, growth factors, and cytokines using a stepwise strategy in the early and middle stages of differentiation. Therefore, the present invention enables the direct transfer of expanded cloned iPSCs to an adhesive culture in a monolayer form for immediate differentiation without the need for EB formation from the iPSCs.

[0173] This invention therefore provides a culture platform that enables stem cells to differentiate into permanent hematopoietic and functional hematopoietic lineage cells with high efficiency without using TGFβ receptor / ALK inhibitors, including SB431532. Furthermore, unlike previous studies, this invention also provides a culture platform using feeder-free, serum-free conditions that support direct differentiation of iPSC monolayer cultures without the need for iPSCs to form EB or aggregate intermediates.

[0174] C. Training Platform Existing methods for culturing pluripotent cells rely heavily on feeder cells or feeder-preconditioned media containing fetal bovine serum; however, such environments may not be suitable for producing cells for clinical and therapeutic use. For example, cells cultured in such foreign-contaminated environments are generally considered unsuitable for human cell transplantation because exposure to animal components may pose a serious risk of immune rejection and the transfer of unknown pathogens to treated patients, and may potentially reactivate animal retroviruses. Culture systems using animal-free media, such as the feeder-free environment covered in this article, facilitate the production of clinical-grade cell lines, specifically hESCs, hiPSCs, and pluripotent stem cell-derived HSC, T, B, NKT, or NK cell lines.

[0175] In certain embodiments, the feeder-free environment is substantially free of human feeder cells and is not pre-regulated with feeder cells (including, but not limited to, mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells). The feeder-free cell culture medium is suitable for culturing pluripotent cells, reprogrammed cells, single-cell culture of pluripotent cells, dissociation and passage, cell sorting of pluripotent cells, generation of basal-state pluripotent cells, maintenance of basal-state pluripotency, and induction of pluripotent cell differentiation. In certain embodiments, the feeder-free environment is used to induce pluripotency, improve reprogramming efficiency, increase or maintain cell efficacy, and / or induce differentiation. In some embodiments, the feeder-free environment is also substantially free of cytokines and growth factors, including bFGF.

[0176] In some aspects of the invention, one or more stages of the iPSC differentiation described above can be performed under feeder-free conditions. Such feeder-free conditions can take the form of monolayer culture and suspension culture, including (but not limited to) monolayer culture 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 perpetual hematopoietic endothelial cells is performed under monolayer feeder-free conditions. In yet another embodiment of the invention, differentiation of perpetual hematopoietic endothelial cells into hematopoietic stem cells is performed under monolayer feeder-free conditions. In one embodiment of the invention, differentiation of perpetual hematopoietic stem cells into pluripotent progenitor cells, T cell progenitor cells, or NK cell progenitor cells is performed under feeder-free suspension conditions, or under monolayer feeder-free conditions followed by feeder-free suspension conditions. In another embodiment of the invention, differentiation of T cell progenitor cells into fully differentiated T cells, or differentiation of NK cell progenitor cells into fully differentiated NK cells, is performed under feeder-free suspension conditions, or under monolayer feeder-free conditions followed by feeder-free suspension conditions.

[0177] Any suitable container or cell culture vessel can be used as a carrier for cell cultures in basal culture media and / or cell culture supplements. In some embodiments, however, coating the surface of the culture vessel with an adhesion-promoting matrix / substrate (e.g., collagen, fibronectin, RGD-containing peptides, gelatin, etc.) can promote cell attachment and, in certain embodiments, enhance the effects of the cell culture media and supplements disclosed herein. Substrates suitable for cell culture and passage are known in the art and include (but are not limited to) glass-linked proteins, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombin, mixtures of naturally occurring cell line-derived matrices (e.g., Matrigel™), and synthetic or artificial surfaces, such as polyamine monolayers and carboxyl-terminated monolayers. In some embodiments, providing feeder-free conditions involves culturing cells on a matrix-coated surface. In one embodiment, the culture platform covered herein comprises a matrix / substrate comprising Matrigel™ or glass-linked proteins. In some embodiments of the culture, Matrigel™ is used, and the culture is thus fully defined.

[0178] In some aspects of the invention, one or more stages of the iPSC differentiation described above can be performed under serum-free conditions. Examples of commercially available serum-free culture media suitable for cell attachment and / or induction include mTeSR™1, TeSR™2, or StemSpan™ from Stem Cell Technologies (Vancouver, Canada), primate ES / iPS cell culture medium from ReproCELL (Boston, MA), StemPro®-34 from Invitrogen (Carlsbad, CA), StemPro® hESC SFM from Invitrogen, and X-VIVO™ from Lonza (Basel, Switzerland).

[0179] In other embodiments, one or more culture media in the culture platform are feeder-free environments and optionally substantially free of cytokines and / or growth factors. In other embodiments, the cell culture medium contains supplements such as serum, extracts, growth factors, hormones, cytokines, etc. Generally, the culture platform comprises one or more stage-specific feeder-free serum-free culture media, each of which further comprises one or more of the following: nutrients / extracts, growth factors, hormones, cytokines, and culture medium additives. Suitable nutrients / extracts may include, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), a widely used basal medium for supporting the growth of many different mammalian cells; KOSR (knockout serum replacement); L-glutamic acid; NEAA (non-essential amino acids). Other culture medium additives may include (but are not limited to) MTG, ITS, βME, and antioxidants (e.g., ascorbic acid). In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, various interleukins (e.g., IL-1 to IL-18), various community-stimulating factors (e.g., granulocyte / macrophage community-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), and other cytokines that affect stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines are commercially available, for example, from R&D Systems (Minneapolis, Minn.), and can be natural or recombinant. In some other embodiments, the culture medium 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-associated tyrosine kinase 3 ligand (Flt3L); and one or more cytokines derived from leukemia inhibitory factor (LIF), namely IL3, IL6, IL7, IL11, and IL15.In some embodiments, the concentrations of growth factors / mitogens and cytokines are stage- and / or cell-type specific, determined empirically or in accordance with established cytokine technology guidelines.

[0180] Generally, techniques for differentiating induced pluripotent cells involve directly or indirectly modulating specific cellular pathways using methods based on polynucleotides, peptides, and / or small molecules. Cellular developmental efficacy can be modulated, for example, by contacting cells with one or more regulators. As used herein, “contact” can refer to culturing cells in the presence of one or more factors, such as small molecules, proteins, peptides, etc. In some embodiments, cells are contacted with one or more agents to induce cell differentiation. Such contact can occur, for example, by introducing one or more agents into cells during in vitro culture. Thus, contact can occur by introducing one or more agents into cells in a nutrient cell culture medium. The cells can be maintained in a culture medium containing one or more agents for a period of time sufficient for the cells to achieve the desired differentiation phenotype. In some other embodiments, “contact” occurs when one or more factors are introduced into cells via a vector. In some embodiments, one or more vectors are introduced via retroviruses, Sendai virus, adenoviruses, cell-free genomes, small loops, vector systems with expression cassettes, or mRNA.

[0181] In other embodiments, one or more stage-specific, feeder-free, serum-free culture media in the culture platforms disclosed herein further comprise one or more small molecules. In some embodiments, the culture platform comprises a cell culture medium containing GSK-3 inhibitors, MEK inhibitors, Rho kinase (ROCK) inhibitors, and does not contain or is free of small molecule inhibitors of the TGFβ / activin signaling pathway, including (but not limited to) TGFβ receptor or ALK5 inhibitors.

[0182] The culture platforms covered herein offer several advantages through the use of homogeneous populations of industrial-grade or clinical-grade pluripotent cells with reduced spontaneous differentiation and / or achieved basal state pluripotency. In one embodiment, homogeneous iPSCs are maintained in a composition comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor; and said composition is free of TGFβ receptor / ALK inhibitors. As used herein, the term “homogeneous” 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 if each cell expresses one or more of the same pluripotency markers, such as SSEA4 and TRA1-81, as intended herein. In one embodiment, a population is homogeneous if at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells are identical or substantially identical to the other cells in the population.

[0183] In various embodiments, the cell culture media in the culture platforms used herein for generating hematopoietic cell lineages to permanent hematopoietic endothelial cells do not contain or are substantially free of TGFβ / activin signaling pathway inhibitors, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In one embodiment, the culture platform comprises a seeding medium for maintaining untreated hiPSCs, the medium containing GSK-3 inhibitors, MEK inhibitors, and Rho kinase (ROCK) inhibitors. Not wishing to be bound by any particular theory, the inventors have found that while TGFβR / ALK5 inhibitors improve reprogramming efficiency, these inhibitors offset the long-term maintenance, quality, and homogeneity of pluripotent cell populations. That is, while inhibition of TGFβ pathway signaling improves cell reprogramming efficiency, the relief of this inhibition subsequently leads to the maintenance of pluripotent cell populations in in vitro culture systems, particularly in systems using feederless cells and single-cell enzymatic passages, where a homogeneous pluripotent population with reduced spontaneous differentiation and maintaining a “basal” or “untreated” pluripotent state is preferred. As used herein, the term “long-term” as measured by (but not limited to) passage number generally means at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more passages. As defined, “passage” refers to the act of allowing cells to divide again and seed them into multiple cell culture surfaces or containers when they have proliferated to the desired extent. Additionally, as disclosed herein, culturing metastable pluripotent cells in a medium containing GSK3 inhibitors and MEK inhibitors, and optionally a ROCK inhibitor, but without a TGFβR / ALK5 inhibitor, induces pluripotent cell conversion to achieve reduced spontaneous differentiation and / or basal state pluripotency.

[0184] Achieving basic or untreated pluripotency of iPSCs is also crucial for obtaining hematopoietic lineage cells by differentiating iPSCs without forming EB intermediates. Furthermore, the efficiency of differentiating untreated iPSCs into permanent HE cells is significantly affected by using monolayer cultures without forming EB and its aggregates. In some embodiments, the culture platform comprises a medium containing a ROCK inhibitor and containing no or substantially no TGFβR / ALK5 inhibitors. In some other embodiments, the culture platform comprises a medium containing a GSK3 inhibitor but without TGFβR / ALK5 inhibitors, which facilitates the generation of permanent HE and / or permanent HSC cells using the culture platform provided herein.

[0185] 1. TGFβ receptor / ALK inhibitors TGFβ receptor (e.g., ALK5) inhibitors may include antibodies against TGFβ receptors (e.g., ALK5), dominant-negative variants of TGFβ receptors (e.g., ALK5), and antisense nucleic acids that inhibit the expression of TGFβ receptors (e.g., ALK5). Exemplary TGFβ receptor / ALK5 inhibitors include (but are not limited to) SB431542 (see, for example, 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-thiocarbamate (see, for example, Tojo et al., *Cancer Science* 96(11):791-800 (2005) and are available from, for example, Tocris. Bioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthidine; Wnt3a / BIO (see, for example, Dalton et al., WO2008 / 094597, which is incorporated herein by reference); GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide) (see, for example, Gellibert et al., Journal of Medicinal Chemistry 49(7):2210-2221 (2006)); SM16 (see, for example, Suzuki et al., Cancer Research 67(5):2351-2359) (2007)); IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, for example, Kim et al., Xenobiotica 38(3):325-339 (2008)); GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, for example, de Gouville et al., Drug News Perspective 19(2):85-90) (2006)); SB-505124 (2-(5-benzo[1,3]dioxacyclopenten-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, for example, DaCosta et al., Molecular Pharmacology 65(3):744-752 (2004)); and pyrimidine derivatives (see, for example, those listed in Stiefl et al. WO2008 / 006583, which are incorporated herein by reference).Furthermore, while it is not intended that "ALK5 inhibitor" encompass non-specific kinase inhibitors, it should be understood that "ALK5 inhibitor" includes inhibitors that inhibit ALK4 and / or ALK7 in addition to inhibiting ALK5, such as SB-431542 (see, for example, Inman et al., Journal of Molecular Pharmacology 62(1): 65-74 (2002)). Without intending to limit the scope of the invention, it is believed that ALK5 inhibitors affect the process of mesenchymal-to-epithelial cell transformation (MET). The TGFβ / activin pathway drives the epithelial-to-mesenchymal transformation (EMT). Therefore, inhibition of the TGFβ / activin pathway can promote the MET (i.e., reprogramming) process.

[0186] Given the data indicating the effect of ALK5 inhibition, it is believed that inhibition of the TGFβ / activin pathway will have a similar effect to ALK5 inhibition. Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with, or in place of, the ALK5 inhibitor as described in each paragraph herein. Exemplary TGFβ / activin pathway inhibitors include (but are not limited to): TGFβ receptor inhibitors, SMAD 2 / 3 phosphorylation inhibitors, SMAD 2 / 3 and SMAD 4 interaction inhibitors, and activators / agonists of SMAD 6 and SMAD 7. Furthermore, the following classification is for organizational purposes only, and those skilled in the art will recognize that compounds affect one or more points within the pathway, and therefore compounds can function in more than one defined category.

[0187] TGFβ receptor (TGFβR) inhibitors can include antibodies against the TGFβ receptor, dominant-negative variants of the TGFβ receptor, and siRNAs or antisense nucleic acids targeting the TGFβ receptor. Specific examples of TGFβ receptor inhibitors include (but are not limited to) SU5416; 2-(5-benzo[1,3]dioxane-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 ;LY580276;LY364947;LY2109761;SB-505124;SB-431542;SD-208;SM16;NPC-30345;Ki26894;SB-203580;SD-093;Glevec;3,5,7,2',4'-pentahydroxyflavone (morin);Activin-M108A;P144;Soluble TBR2-Fc;and antisense transfection of tumor cells targeting the TGFβ receptor. (See, for example, 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).)

[0188] SMAD 2 / 3 phosphorylation inhibitors may include antibodies against SMAD2 or SMAD3, dominant-negative variants 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 (morula). (See, for example, Wrzesinski, ibid.; Kaminska, ibid.; Shimanuki et al., Oncogene 26:3311-3320 (2007); and Kataoka et al., EP1992360, which are incorporated herein by reference).

[0189] Inhibitors of the SMAD2 / 3 and SMAD4 interaction can include antibodies against SMAD2, SMAD3, and / or smad4, dominant-negative variants of SMAD2, SMAD3, and / or smad4, and antisense nucleic acids targeting SMAD2, SMAD3, and / or smad4. Specific examples of inhibitors of the SMAD2 / 3 and SMAD4 interaction include (but are not limited to) Trx-SARA, Trx-xFoxH1b, and Trx-Lef1. (See, for example, Cui et al., Oncogene 24:3864-3874 (2005) and Zhao et al., Molecular Biology of the Cell, 17:3819-3831 (2006)).

[0190] Activators / promoters of SMAD 6 and SMAD 7 include (but are not limited to) antibodies against SMAD 6 or SMAD 7, dominant-negative variants of SMAD 6 or SMAD 7, and antisense nucleic acids targeting SMAD 6 or SMAD 7. Specific examples of inhibitors include (but are not limited to) Smad7-as PTO-oligonucleotides. (See, for example, US6534476 by Miyazono et al. and US2005119203 by Steinbrecher et al., both of which are incorporated herein by reference).

[0191] 2. WNT pathway promoters As used herein, the terms “Wnt signaling promoter,” “Wnt pathway activator,” “Wnt pathway activator,” or “Wnt pathway agonist” refer to agonists of the Wnt signaling pathway, including (but not limited to) one or more of the following agonists: Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, and Wnt16. Wnt pathway agonists further include (but are not limited to) one or more of the following polypeptides or fragments thereof: Dkk polypeptide, crescent polypeptide, cerberus polypeptide, axonin polypeptide, Frzb polypeptide, T-cytokine polypeptide, or dominant-negative heterozygous polypeptide.

[0192] Non-limiting examples of Wnt pathway agonists further include one or more of the following: nucleic acids containing a nucleotide sequence encoding a Wnt polypeptide, polypeptides containing an amino acid sequence of a Wnt polypeptide, nucleic acids containing a nucleotide sequence encoding an activated Wnt receptor, polypeptides containing an amino acid sequence of an activated Wnt receptor, small organic molecules that promote Wnt / β-oxocyanate signaling, small organic molecules that inhibit the expression or activity of Wnt antagonists, antisense oligonucleotides that inhibit the expression of Wnt antagonists, ribonucleases that inhibit the expression of Wnt antagonists, RNAi constructs that inhibit the expression of Wnt antagonists, siRNA or shRNA, antibodies that bind to and inhibit the activity of Wnt antagonists, nucleic acids containing a nucleotide sequence encoding a β-oxocyanate polypeptide, polypeptides containing an amino acid sequence of a β-oxocyanate polypeptide, nucleic acids containing a nucleotide sequence encoding a Lef-1 polypeptide, and polypeptides containing an amino acid sequence of a Lef-1 polypeptide.

[0193] Wnt pathway activators further include GSK3 inhibitors, such as nucleic acids containing nucleotide sequences encoding dominant-negative GSK-3, GSK3α, or GSK3β polypeptides; polypeptides containing amino acid sequences of dominant-negative GSK-3, GSK3α, or GSK3β polypeptides; small organic molecules that bind to and inhibit the expression or activity of GSK-3, GSK3α, or GSK3β; RNAi constructs, siRNAs, or shRNAs that bind to and inhibit the expression and / or activity of GSK-3, GSK3α, or GSK3β; antisense oligonucleotides that bind to and inhibit the expression of GSK-3, GSK3α, or GSK3β; antibodies that bind to and inhibit the expression and / or activity of GSK-3, GSK3β, or GSK3β; ribonucleases that bind to and inhibit the expression of GSK-3, GSK3α, or GSK3; and any GSK-3-independent agent that activates β-xanthine target genes and has an effect similar to GSK-3 inhibition.

[0194] 3. GSK3 inhibitors GSK3 inhibitors are specific exemplary Wnt pathway agonists suitable for use in the compositions covered herein, and may include (but are not limited to) polynucleotides, peptides, and small molecules. GSK3 inhibitors covered herein can reduce GSK3α / β expression and / or GSK3α / β activity. Illustrative examples of GSK3 inhibitors covered herein include (but are not limited to) anti-GSK3 antibodies, dominant-negative GSK3 variants, GSK3-targeting siRNAs, shRNAs, miRNAs, and antisense nucleic acids.

[0195] Other illustrative GSK3 inhibitors include (but are not limited to): Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, lithium, TDZD-8, BIO, BIO-acetone oxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazoline-4-yl)amine, pyridocarbazole-cyclopentadienylruthenium complex, TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidin-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, α-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrolo-2,5-dione, TWSl 19 pyrrolopyrimidine compounds, L803 H-KEAPPAPPQSpP-NH2 or its myristylated form, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-acetophenone, GF109203X, RO318220, TDZD-8, TIBPO and OTDZT.

[0196] In certain illustrative examples, the GSK3 inhibitor is CHIR99021, BIO, or camparone.

[0197] In a preferred embodiment, the GSK3 inhibitor is CHIR99021.

[0198] In another embodiment, the GSK3 inhibitor is BRD0705.

[0199] 4. ERK / MEK inhibitors ERK / MEK inhibitors applicable to the compositions covered herein include (but are not limited to) polynucleotides, peptides, and small molecules. The ERK / MEK inhibitors covered herein can reduce MEK or ERK expression and / or MEK or ERK activity. Illustrative examples of MEK / ERK inhibitors covered herein include (but are not limited to) anti-MEK or anti-ERK antibodies, dominant-negative MEK or ERK variants, and siRNAs, shRNAs, miRNAs, and antisense nucleic acids targeting MEK or ERK.

[0200] Other illustrative ERK / MEK inhibitors include (but are not limited to) PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSK1 120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEA1 19, AZD6244, FR180204, and PTK787.

[0201] Other illustrative MEK / ERK inhibitors include those compounds disclosed in international patent applications WO 99 / 01426, WO 02 / 06213, WO03 / 077914, WO 05 / 051301 and WO2007 / 044084.

[0202] Other illustrative examples of MEK / ERK inhibitors include the following compounds: 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide; 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide; 1-[6-(4-bromo-2- [Chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazol-5-yl]-2-hydroxy-acetone; 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazol-e-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide; 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzimidazol-5-carboxylic acid (2-hydroxy-ethoxy) 6-Amide; 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-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-e-5-carboxylic acid (2-hydroxy-ethoxy)-amide -Amide (hereinafter referred to as MEK inhibitor 1); 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (hereinafter referred to as MEK inhibitor 2); 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide, and their pharmaceutically acceptable salts.

[0203] In a preferred embodiment, the MEK / ERK inhibitor is PD98059.

[0204] 5. ROCK inhibitors Rho-associated kinase (ROCK) is a serine / threonine kinase that acts as a downstream effector of Rho kinase (it exists in three isoforms: RhoA, RhoB, and RhoC). ROCK inhibitors suitable for use in the compositions covered herein include (but are not limited to) polynucleotides, peptides, and small molecules. The ROCK inhibitors covered herein can reduce ROCK expression and / or ROCK activity. Illustrative examples of ROCK inhibitors covered herein include (but are not limited to) anti-ROCK antibodies, dominant-negative ROCK variants, ROCK-targeting siRNAs, shRNAs, miRNAs, and antisense nucleic acids.

[0205] The illustrative ROCK inhibitors covered herein include, but are not limited to, thiazovivin, Y27632, Fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxy-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, and the ROCK inhibitors disclosed in U.S. Patent No. 8,044,201 (which is incorporated herein by reference in its entirety).

[0206] In one embodiment, the ROCK inhibitor is thiazolinone, Y27632, or pyrintegrin.

[0207] In a preferred embodiment, the ROCK inhibitor is thiazolidinedione.

[0208] The amounts of small molecules in the compositions and cell culture media covered herein can be varied and optimized according to specific culture conditions, including the specific molecules and combinations used, the cell types cultured in the culture medium, and the specific application. In one embodiment, the concentration of the small molecule present in the composition is sufficient to induce pluripotency, improve reprogramming efficiency, enhance or maintain cell efficacy, or induce or maintain basal pluripotency.

[0209] Another aspect of the invention relates to Notch activators used in the invention. Notch encompasses all members of the Notch receptor family, including (but not limited to) Notch1. Notch activators include (but are not limited to) Notch receptor agonists. Notch agonists bind to Notch receptors and also initiate or mediate Notch receptor-related signaling events, such as causing cleavage of the intracellular domain of Notch and its 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 activators disclosed in EP 2606884, US 6689744, and US 5780300, the disclosures of which are incorporated herein by reference. In some embodiments, one or more Notch ligands may be introduced as soluble peptides or immobilized on a solid material. The solid material may include (but is not limited to) polystyrene plates or beads. Beads used to immobilize Notch ligands may be agarose beads, magnetic beads, and latex beads. In one embodiment, Notch ligand peptides bind / immobilize onto beads. In another embodiment, Notch ligand peptides bind / immobilize onto the surface of a polystyrene plate. In some embodiments, the immobilization of Notch ligands is non-covalent. In some embodiments, Notch ligand peptides are presented by cells.

[0210] Another aspect of the invention relates to BMP pathway activators, comprising those agents disclosed in the following disclosures: WO 2014011540, WO 2014062138, and WO 2005117994, the disclosures of which are incorporated herein by way of introduction. The BMP pathway activators used in this invention include (but are not limited to) BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In a non-limiting embodiment of the invention, the BMP pathway activator is BMP-4. BMPs are multifunctional cytokines and members of the transforming growth factor-β superfamily. Bone morphogenetic protein (BMP) receptors mediate BMP signaling by activating Smad. BMP ligands bind to BMP receptors BMPRI and BMPRII. Following phosphorylation of BMPRII, BMPRI is activated. Phosphorylation of BMPRI subsequently phosphorylates receptor-activated Smad proteins (R-Smads), which bind to the co-mediator -Smad (co-Smad) and enter the nucleus, where they regulate gene expression. In one embodiment, the BMP pathway activator is BMP4.

[0211] The present invention provides compositions for obtaining hematopoietic lineage cells from iPSCs through permanent HSCs differentiated from iPSCs or through permanent hematopoietic endothelial cells differentiated from iPSCs, and each of the methods does not involve the formation of EBs from iPSCs to achieve the desired cell differentiation.

[0212] 6. hiPSC Differentiation Platform I. iCD34 Platform One aspect of the present invention provides a culture platform for obtaining permanent hematopoietic endothelial cells using pluripotent stem cells. As used herein, permanent hematopoietic endothelial cells are a population of hematopoietic cells dedicated to permanent hematopoiesis with the ability to generate all hematopoietic cells, including (but not limited to) permanent HSCs, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, and / or B cells.

[0213] In one embodiment, the culture platform for obtaining permanent hematopoietic endothelial cells using pluripotent stem cells (including iPSCs) comprises a seeding medium containing MEKi, GSKi, and ROCKi. In some embodiments, the seeding medium is free of or substantially free of TGFβ receptor / ALK inhibitors. In one embodiment, the small molecule combination in the seeding medium of the present invention is shown in Table 1 as a fate maintenance medium (FMM). The components of the medium may be present in the medium in amounts within the concentration range shown in Table 1. In one embodiment, the iPSCs used to obtain permanent hematopoietic endothelial cells are cell lines generated using fate reprogramming medium (FRM) and further maintained in an FMM to establish and maintain a basal or untreated state of the iPSC cell line, which is suitable for stage-specific differentiation as disclosed herein. The basal or untreated iPSCs thus obtained are suitable for cryopreservation. In the present invention, the preserved iPSC cell lines or cloned iPSCs can be seeded in an FMM for daughter sequences to differentiate into permanent hematopoietic endothelial cells.

[0214] Table 1: Obtaining CD34+ permanent hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, and NK cell progenitor cells by inoculating untreated iPSC cultures: One aspect of the invention provides a culture medium for the differentiation and expansion of pluripotent stem cells (including iPSCs) into the mesoderm. In some embodiments, the iPSCs are untreated iPSCs. In one embodiment, the culture medium comprises a BMP activator, and optionally present bFGF, and a CD34 basal medium comprising a combination of small molecules as shown in Table 2. In some embodiments, the culture medium comprises extracellular matrix proteins. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines within the concentration ranges shown in Table 2. In some embodiments, the culture medium is fully defined, wherein glass connective protein is replaced with Matrigel™. In one embodiment, the culture medium described above for the differentiation and expansion of pluripotent stem cells into mesoderm further comprises between 0.2 and 50 ng of bFGF.

[0215] One aspect of the present invention provides a culture medium for obtaining mesodermal cells with permanent hematopoietic endothelial cell potential from pluripotent stem cells (including iPSCs). In some embodiments, the iPSCs are untreated iPSCs. In one embodiment, the culture 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 culture medium containing the GSK3 inhibitor is applied only according to mesodermal cell specifications to achieve permanent hematopoietic endothelial cell potential. In one embodiment, the culture medium containing the BMP activator, the GSK3 inhibitor, and bFGF further comprises a CD34 basal medium containing a combination of small molecules as shown in Table 3. In one embodiment, the above-described culture medium does not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the culture medium comprises extracellular matrix proteins. In other embodiments, the culture medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 3. In some embodiments, the culture medium is fully defined, wherein glass connective tissue is replaced with Matrigel™. One aspect of the present invention provides a culture medium for obtaining permanent hematopoietic endothelial cells from mesodermal cells. In some embodiments, the culture medium comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In one embodiment, the culture medium comprises VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor, and the CD34 basal medium comprises a small molecule combination as shown in Table 4. In one embodiment, the culture 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 culture medium for generating permanent HE from mesodermal cells comprises a ROCK inhibitor, a Wnt pathway activator, VEGF, bFGF, SCF, IL6, and IL11, and one or more of 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 culture medium containing VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor is free of one or more of Wnt pathway activators, TGFβ receptor / ALK inhibitors, IGF1, and EPO. In other embodiments, the culture media described herein contain small molecules, growth factors, and / or cytokines within the concentration ranges shown in Table 4. One aspect of the present invention provides a culture platform for obtaining pluripotent progenitor (MPP) cells from perpetual hematopoietic endothelial cells. The MPPs are capable of further differentiating into bone marrow, including neutrophil progenitor cells. In one embodiment, the culture platform comprises (i) a culture medium containing 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, wherein the culture medium is suitable for differentiating perpetual hematopoietic endothelial cells into pre-HSCs (Table 5). In another embodiment, the culture platform containing a culture medium for differentiating perpetual hematopoietic endothelial cells into pre-HSCs further comprises (ii) a culture medium containing a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is free of a ROCK inhibitor and suitable for differentiating the pre-HSCs into pluripotent progenitor cells. In some embodiments, the ROCK inhibitor is thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium described herein contains small molecules, growth factors, and / or cytokines within the concentration ranges shown in Table 5. II. iNK / iT Platform One aspect of the present invention provides a culture platform for generating T cell progenitors or T cells from perpetual hematopoietic endothelial cells. In one embodiment, the culture platform comprises (i) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; wherein the culture medium is suitable for differentiating perpetual hematopoietic endothelial cells into pre-iproT cells; and / or (ii) a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, and is suitable for differentiating the pre-iproT cells into T cell progenitors or T cells (Table 6). In some embodiments, the culture medium for differentiating perpetual hematopoietic endothelial cells into pre-iproT cells contains a ROCK inhibitor, SCF, Flt3L, TPO, and IL7; and is free of BMP activator. In some embodiments, the culture medium for differentiating pre-iproT cells into T cell progenitors or T cells contains SCF, Flt3L, and IL7. In some embodiments, the ROCK inhibitor is thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium herein contains small molecules, growth factors, and / or cytokines within the concentration ranges shown in Table 6. One aspect of the present invention provides a culture platform for generating NK cell progenitor cells or NK cells from perpetual hematopoietic endothelial cells. In one embodiment, the culture platform comprises (i) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, wherein the culture medium is suitable for differentiating perpetual hematopoietic endothelial cells into pre-iproNK cells (pre-iproNK); and / or (ii) a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, and is suitable for differentiating the pre-iproNK cells into NK cell progenitor cells or NK cells. In some embodiments, the culture medium for differentiating perpetual hematopoietic endothelial cells into pre-iproNK cells contains a ROCK inhibitor, SCF, Flt3L, TPO, IL3, IL7, and IL15; and is free of a BMP activator. In some embodiments, the culture medium for differentiating pre-iproNK cells into NK progenitor cells or NK cells contains SCF, Flt3L, and IL15. In some embodiments, the ROCK inhibitor is thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium herein contains small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 7. Another aspect of the present invention provides a culture platform for obtaining T cell progenitor cells or T cells, the culture platform comprising one or more of the following: (i) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L and IL7, and containing little or no VEGF, bFGF, BMP activators and ROCK inhibitors, the culture medium being suitable for differentiating pre-T cell progenitor cells into T cell progenitor cells or T cells; (ii) a culture medium comprising ROCK inhibitors, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO and IL7, the culture medium being suitable for differentiating perpetual hematopoietic endothelial cells into pre-T cell progenitor cells; (iii .... A culture medium containing inhibitors, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium is suitable for differentiating and expanding mesodermal cells into perpetual hematopoietic endothelial cells; (iv) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with perpetual hematopoietic endothelial cell potential; (v) a culture medium containing a BMP activator and optionally bFGF, wherein the culture medium is suitable for generating and expanding mesodermal cells from iPSCs; and (vi) a culture medium containing MEKi, GSKi, and ROCKi, and containing no or substantially no TGFβ receptor / ALK inhibitors, wherein the culture medium is suitable for seeding and expanding untreated iPSCs. In some embodiments, all of the above-described culture media contain no or substantially no 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 thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0216] In one embodiment, a culture platform for generating T cell progenitors or T cells comprises (i) a culture medium containing SCF, Flt3L, and IL7, and containing none or substantially none of VEGF, bFGF, BMP activators, and ROCK inhibitors, said culture medium being suitable for differentiating pre-T cell progenitors into T cell progenitors or T cells. In another embodiment, the culture platform for generating T cell progenitors or T cells comprising culture medium (i) further comprises (ii) a culture medium containing ROCK inhibitors, one or more growth factors and cytokines, and optionally a BMP activator, said cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, said culture medium being suitable for differentiating permafrost endothelial cells into pre-T cell progenitors. In another embodiment, the culture platform for generating T cell progenitors or T cells comprising culture media (i) and (ii) further comprises (iii) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the composition is suitable for differentiating and expanding mesodermal cells into perpetually hematopoietic endothelial cells. In yet another embodiment, the culture platform for generating T cell progenitors or T cells comprising culture media (i), (ii), and (iii) further comprises (iv) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with perpetually hematopoietic endothelial cell potential. In yet another embodiment, the culture platform for generating T cell progenitors or T cells comprising culture media (i), (ii), (iii), and (iv) further comprises (v) a culture medium containing a BMP activator and optionally bFGF, wherein the culture medium is suitable for generating and expanding mesodermal cells from iPSCs. In another embodiment, the culture platform for generating T cell progenitor cells or T cells comprising media (i), (ii), (iii), (iv), and (v) further comprises (vi) a culture medium containing MEKi, GSKi, and ROCKi, free of or substantially free of TGFβ receptor / ALK inhibitors, said culture medium being suitable for seeding and expanding untreated iPSCs. In some embodiments, all of the above-described culture media are free of or substantially 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 thiazolidinedione 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 to generate T cell progenitors or T cells. In some embodiments, Notch factors, including Jag1, Jag2, DLL-1, DLL-3, and DLL-4, may be introduced as soluble peptides, bead-bound peptides, surface-bound peptides, or cell-presented peptides.

[0217] Another aspect of the present invention provides a culture platform for obtaining NK cell progenitor cells or NK cells, the culture platform comprising one or more of the following: (i) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors and is suitable for differentiating pre-NK cell progenitor cells into NK cell progenitor cells or NK cells; (ii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, wherein the culture medium is suitable for differentiating permafrost endothelial cells into pre-NK cell progenitor cells. (iii) A culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the composition is suitable for differentiation and expansion of mesodermal cells into perpetually hematopoietic endothelial cells; (iv) A culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with perpetually hematopoietic endothelial cell potential; (v) A culture medium containing a BMP activator and optionally bFGF, wherein the culture medium is suitable for generating and expanding mesodermal cells from iPSCs; (vi) A culture medium containing MEKi, GSKi, and ROCKi, and containing no or substantially no TGFβ receptor / ALK inhibitors, wherein the culture medium is suitable for seeding and expanding untreated iPSCs. In some embodiments, all of the above-described culture media contain no or substantially no 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 thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, NK maturation is performed using one or more artificial antigens that stimulate NK cell growth, development, and maturation, said artificial antigens being introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells.

[0218] In one embodiment, a culture platform for generating NK cell progenitors or NK cells comprises (i) a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors and is suitable for differentiating pre-NK cell progenitors into NK cell progenitors or NK cells. In some embodiments, NK maturation is performed using one or more artificial antigens that stimulate NK growth, development, and maturation, said artificial antigens being introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells. In another embodiment, the culture platform for generating NK cell progenitors or NK cells comprising culture medium (i) further comprises (ii) a culture medium containing ROCK inhibitors, one or more growth factors and cytokines, and optionally a BMP activator, said cytokines being selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, wherein said culture medium is suitable for differentiating permafrost endothelial cells into pre-NK cell progenitors. In another embodiment, the culture platform for generating NK cell progenitors or NK cells, comprising culture media (i) and (ii), further comprises (iii) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and the composition is suitable for differentiating and expanding mesodermal cells into perpetual hematopoietic endothelial cells. In yet another embodiment, the culture platform for generating NK cell progenitors or NK cells, comprising culture media (i), (ii), and (iii), further comprises (iv) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, suitable for obtaining mesodermal cells with perpetual hematopoietic endothelial cell potential. In yet another embodiment, the culture platform for generating NK cell progenitors or NK cells, comprising culture media (i), (ii), (iii), and (iv), further comprises (v) a culture medium containing a BMP activator and optionally bFGF, suitable for generating and expanding mesodermal cells from iPSCs. In another embodiment, the culture platform for generating NK cell progenitor cells or NK cells comprising media (i), (ii), (iii), (iv), and (v) further comprises (vi) a culture medium containing MEKi, GSKi, and ROCKi, and free of or substantially free of TGFβ receptor / ALK inhibitors, said culture medium being suitable for seeding and expanding untreated iPSCs. In some embodiments, all of the above-described culture media are free of or substantially 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 thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0219] One aspect of the present invention provides a culture platform for generating permanent hematopoietic endothelial cells, the culture platform comprising one or more of the following: (i) a culture medium for differentiation and expansion of mesodermal cells into permanent hematopoietic endothelial cells, the culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, the cytokines being selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; (ii) a culture medium for acquiring permanent hematopoietic potential in mesodermal cells, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a culture medium for differentiation and expansion of untreated iPSCs into mesodermal cells, the culture medium comprising a BMP activator and optionally bFGF; and (iv) an untreated iPSC seeding and expansion culture comprising MEKi, GSKi, and ROCKi, wherein the seeded culture is free of a TGFβ receptor / ALK inhibitor. In some embodiments, the permanent hematopoietic endothelial cells are CD34+. In some embodiments, all of the above-described culture media are free of or substantially 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 thiazolinone or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0220] In one embodiment, a culture platform for obtaining permanent hematopoietic endothelial cells comprises (i) a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the culture medium is suitable for the differentiation and expansion of mesodermal cells into permanent hematopoietic endothelial cells. In one embodiment, the culture platform containing culture medium (i) further comprises (ii) a culture medium containing a BMP activator, bFGF, and a GSK3 inhibitor, wherein the culture medium (ii) is suitable for imparting permanent hematopoietic potential to mesodermal cells. In another embodiment, the culture platform containing culture media (i) and (ii) further comprises (iii) a culture medium containing a BMP activator and optionally bFGF, wherein the culture medium (iii) is suitable for the differentiation and expansion of untreated iPSCs into mesodermal cells. In yet another embodiment, the culture platform comprising media (i), (ii), and (iii) further comprises (iv) a media comprising MEKi, GSKi, and ROCKi, and said media (v) is free of or substantially free of TGFβ receptor / ALK inhibitors, wherein said media (v) is suitable for inoculating and amplifying untreated iPSCs. In some embodiments, all of the above-described media are free of or substantially 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 thiazolidinedione or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0221] One aspect of the present invention provides a culture platform for generating CD34+ permanent hematopoietic endothelial cells, the culture platform comprising one or more of the following: (i) a culture medium for differentiating and expanding permanent hematopoietic endothelial cells from mesodermal cells, the culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, the cytokines being selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, wherein the permanent hematopoietic endothelial cells comprise CD34+ permanent hematopoietic endothelial cells; (ii) a culture medium for acquiring permanent hematopoietic potential in mesodermal cells, the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a culture medium for differentiating and expanding mesodermal cells from untreated iPSCs, the culture medium comprising a BMP activator and optionally bFGF; and (iv) an untreated iPSC seeding or expansion culture comprising MEKi, GSKi, and ROCKi, wherein the seeded culture contains no or substantially no TGFβ receptor / ALK inhibitor. In some embodiments, all of the above-described culture media are free of or substantially 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 thiazolidinedione or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0222] 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 culture medium for differentiating and expanding mesodermal cells from untreated iPSCs, the culture medium comprising a BMP activator and optionally present bFGF; and (ii) an untreated iPSC inoculation or expansion culture comprising MEKi, GSKi, and ROCKi, wherein the inoculated culture is free of or substantially free of a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above-described culture media are free of or substantially 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 thiazolidinedione or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, the culture platform for generating mesodermal cells may further comprise (iii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, wherein the culture medium is used to acquire permanent hematopoietic potential in the mesodermal cells. In some embodiments, the culture medium containing BMP activator, bFGF, and GSK3 inhibitor does not contain TGFβ receptor / ALK inhibitor.

[0223] C. Methods for obtaining permanent hematopoietic endothelial cells, pluripotent progenitor cells, T cells or NK cell progenitor cells, T cells and / or NK cells This invention provides a method for generating pluripotent stem cell-derived permanent hematopoietic cells using a multi-stage culture platform containing one or more culture media. The method is applicable to feeder-free conditions. Compared to methods known in the art, the method is also applicable to monolayer culture and therefore does not require EB formation or aggregate intermediates for pluripotent stem cell differentiation. The provided method generates and simultaneously expands pluripotent stem cell-derived permanent hematopoietic endothelial cells (iHE) CD34+ HE (iCD34), which can further differentiate into multipotent progenitor cells (iMPP), natural killer cell progenitor cells (ipro-NK), T cell progenitor cells (ipro-T), mature NK cells (iNK), and T cells (iT). Other aspects of the invention also provide a method for generating bone marrow cells differentiated from pluripotent stem cell-derived CD34+, HE, HSCs, and / or MPPs.

[0224] In one embodiment, the present invention provides a method for differentiating and expanding pluripotent cells into hematopoietic lineage cells via monolayer culture, comprising contacting the pluripotent cells with a BMP pathway activator and optionally bFGF, wherein pluripotent stem cell-derived mesoderm cells are obtained and expanded without the formation of embryonic bodies from the pluripotent stem cells; then, the pluripotent stem cell-derived mesoderm cells are contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial (HE) potential without the formation of embryonic bodies from the pluripotent stem cells. By subsequent contact with bFGF and optionally a ROCK inhibitor and / or a WNT pathway activator, the mesoderm cells with permanent HE potential differentiate into permanent HE cells, during which the permanent HE cells are also expanded.

[0225] Since EB formation does not induce cell proliferation, monolayer culture is impossible, laborious, and inefficient. Therefore, the method provided for obtaining hematopoietic lineage cells is superior to EB-mediated pluripotent stem cell differentiation. Furthermore, this invention discloses that monolayer culture using the method provided herein produces functional hematopoietic lineage cells, enabling long-term in vivo hematopoietic self-renewal, remodeling, and transplantation.

[0226] As detailed below, this invention provides a method for obtaining hematopoietic lineage cells from pluripotent cells by obtaining permanently generated hematopoietic endothelial cells. Specifically, this invention provides a method for guiding pluripotent cells to differentiate into hematopoietic lineage cells without forming EBs for differentiation.

[0227] I. iCD34 Platform 1. Derivation and amplification of permanent iHE One aspect of the present invention provides a method for generating permanent hematopoietic endothelial cells (iHE) using an optimized multi-stage approach. Generally, the method begins with a first stage in which pluripotent stem cells are seeded and expanded. The pluripotent stem cells are then differentiated into mesodermal cells, which are expanded during this stage. The expanded mesodermal population is then differentiated into a mesodermal population with permanent hematopoietic endothelial cell potential, and then the mesodermal population is differentiated into permanent hematopoietic endothelial cells and expanded. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. The present invention further provides a method for generating and expanding permanent hematopoietic endothelial cells (iHE), comprising: differentiating and expanding pluripotent stem cell-derived mesodermal cells, and obtaining mesodermal cells with permanent iHE potential, and then differentiating them into iHE. Alternatively, the present invention provides a method for generating and expanding permanent hematopoietic endothelial cells, comprising differentiating pluripotent stem cell-derived mesodermal cells with permanent iHE potential into permanent iHE. The method disclosed in this paper uses an optimized monolayer iCD34 culture platform without the formation of EB and contains little or no TGFβ receptor / ALK inhibitors.

[0228] In one embodiment of a method for generating perpetual hematopoietic endothelial cells (iHE) from pluripotent stem cells, the method comprises (1) differentiating and expanding the pluripotent stem cells into a mesodermal population by contacting the cells with a culture medium containing a BMP activator and optionally bFGF; (2) differentiating and expanding the mesodermal population by contacting the cells of the mesodermal population with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF to acquire perpetual HE potential in the mesodermal cells; and (3) differentiating and expanding the mesodermal cells with perpetual HE potential into perpetual HE cells by contacting the mesodermal cells with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iHE cells obtained by 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 utilizes CD34 positivity. In some embodiments, the sorting utilizes CD34 positivity and CD43 negativity. In some embodiments, the sorting utilizes CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, the sorting utilizes CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity. In some other embodiments, the sorting utilizes CD34 positivity, CD43 negativity, and CD93-. In some other embodiments, the sorting utilizes CD34 positivity and CD93 negativity. In some embodiments, the culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. 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, contacting the cells with a culture medium containing a GSK3 inhibitor is simply following mesodermal cell protocol to achieve permanent HE potential. In some embodiments, the method further comprises subjecting the seeded iPSCs and / or mesodermal cells to a low oxygen pressure between about 2% and about 10%. In some embodiments, the method further comprises seeding pluripotent stem cells by contacting the pluripotent cells with a culture medium containing MEKi, GSKi, and ROCKi, wherein the pluripotent stem cells expand.

[0229] In one embodiment of a method for generating perpetual hematopoietic endothelial cells (iHE) from seeded pluripotent stem cells, the method comprises (1) differentiating and expanding pluripotent stem cells into mesodermal cells by contacting them with a culture medium containing a BMP activator and optionally bFGF; (2) obtaining mesodermal cells with perpetual iHE potential by contacting them with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; and (3) differentiating and expanding the mesodermal cells with HE potential into perpetual HE cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iHE cells obtained by 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 above method further comprises sorting using CD34 positivity. In some embodiments, the sorting is using CD34 positivity and CD43 negativity. In some embodiments, the sorting is using CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, the sorting is using CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity. In some embodiments, the sorting is using CD34 positivity, CD43 negativity, and CD93 negativity. In some embodiments, the sorting is using CD34 positivity and CD93 negativity. In some embodiments, the culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the seeded iPSCs and / or mesodermal cells to a hypoxic pressure between about 2% and about 10%.

[0230] In one embodiment of a method for generating permanent hematopoietic endothelial cells (iHE) from pluripotent stem cell-derived mesoderm cells, the method comprises (1) obtaining mesoderm cells with permanent HE potential by contacting them with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; and (2) differentiating and expanding the mesoderm cells with permanent HE potential into permanent HE cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the iHE cells obtained by the above method express CD34. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. 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 is performed using CD34-positive and CD43-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting utilizes CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the sorting utilizes CD34-positive and CD93-negative cells. In some embodiments, the culture medium in the above methods is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the BMP activator in the methods is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above methods further include subjecting the mesodermal cells to a hypoxic pressure between about 2% and about 10%.

[0231] In one embodiment of a method for conferring permanent hematopoietic endothelial cell (iHE) potential to pluripotent stem cell-derived mesoderm cells, the method comprises contacting the mesoderm cells with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11, wherein the mesoderm cells are expanded. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the iHE cells obtained by 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 culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the mesoderm cells to a hypoxic pressure between about 2% and about 10%.

[0232] 2. Derivatization and expansion of pluripotent stem cell-derived mesodermal cells with permanent hematopoietic endothelial cell potential One aspect of the present invention provides a method for generating pluripotent stem cell-derived mesodermal cells using an optimized multi-stage approach. Generally, the method begins by seeding pluripotent stem cells. The seeded pluripotent stem cells are then allowed to develop into mesoderm, which further differentiates into mesodermal cells with permanent hematopoietic endothelial cell potential. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived mesodermal cells comprising: differentiating the seeded pluripotent stem cells into mesoderm, and then differentiating the mesoderm into mesodermal cells with permanent hematopoietic endothelial cell potential. The present invention further provides a method for generating pluripotent stem cell-derived mesodermal cells with permanent hematopoietic endothelial cell potential, and the method comprises differentiating the pluripotent stem cell-derived mesoderm into mesodermal cells with permanent hematopoietic endothelial cell potential. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that is free of or substantially free of TGFβ receptor / ALK inhibitors.

[0233] In one embodiment of a method for accrediting pluripotent stem cell-derived mesoderm cells to permanent hematopoietic endothelial cell potential, the method comprises (1) differentiating and expanding pluripotent stem cells into mesoderm cells by contacting them with a culture medium containing a BMP activator and optionally bFGF; and (2) accrediting the mesoderm cells to permanent hematopoietic endothelial cell potential by contacting them with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the pluripotent stem cells to a hypoxic pressure between about 2% and about 10%. In some embodiments, the above method further comprises seeding the cells by contacting them with a culture medium containing MEKi, GSKi, and ROCKi.

[0234] In one embodiment of a method for generating pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential from pluripotent stem cell-derived mesoderm, the method includes differentiating the mesoderm into mesoderm cells with permanent hematopoietic endothelial cell potential by contacting the mesoderm cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes subjecting the mesoderm cells to a low oxygen pressure between about 2% and about 10%.

[0235] 3. Derived from and expanded mesoderm by pluripotent stem cells One aspect of the present invention provides a method for generating pluripotent stem cell-derived mesodermal cells using an optimized multi-stage approach. Generally, the method begins with a first stage in which pluripotent stem cells are seeded, and then in a second stage, the seeded cells are differentiated into mesoderm. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. The method disclosed herein utilizes an optimized iCD34 culture platform that is free of or substantially free of TGFβ receptor / ALK inhibitors.

[0236] In one embodiment of a method for generating pluripotent stem cell-derived mesoderm from pluripotent cells, the method comprises differentiating and expanding seeded pluripotent stem cells into mesoderm cells by contacting the cells with a culture medium containing a BMP activator and optionally present bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the above method further comprises subjecting the seeded iPSCs to a hypoxic pressure between about 2% and about 10%. In some embodiments, the above method further comprises seeding and expanding iPSCs by contacting the pluripotent cells with a culture medium containing MEKi, GSKi, and ROCKi.

[0237] 4. Derivative hematopoietic pluripotent progenitor cells (iMPP) One aspect of the present invention provides a method for generating pluripotent stem cell-derived pluripotent progenitor cells (iMPPs) using an optimized multi-stage approach. Generally, the method begins by seeding pluripotent stem cells. The seeded cells are expanded and differentiated into mesodermal cells. The mesoderm is expanded and differentiated into mesodermal cells with permanent hematopoietic endothelial cell potential, and subsequently, the mesodermal cells are differentiated into permanent hematopoietic endothelial cells. Permanent hematopoietic stem cell (iHE) cells are expanded and differentiated into pre-HSCs, and then the pluripotent progenitor cells are capable of differentiating into bone marrow cells, including neutrophil progenitor cells. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived pluripotent progenitor cells (iMPPs) comprising differentiating the seeded pluripotent cells into mesoderm, differentiating the mesoderm into mesodermal cells with permanent hematopoietic endothelial cell potential, then differentiating the mesodermal cells into permanent iHE, and then differentiating the permanent iHE into iMPPs. This invention further provides a method for generating pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived mesoderm into mesoderm cells with permanent hematopoietic endothelial cell potential, then differentiating the mesoderm cells into permanent iHE cells, and then differentiating the permanent iHE cells into iMPPs. Alternatively, this invention provides a method for generating pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived mesoderm cells into permanent iHE cells, and then differentiating the permanent iHE cells into iMPPs. Additionally, this invention provides a method for generating pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived iHE cells into iMPPs. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. The methods disclosed herein use an optimized monolayer iCD34 culture platform without the formation of EB, said culture platform being free of or substantially free of TGFβ receptor / ALK inhibitors.

[0238] In one embodiment of a method for generating hematopoietic pluripotent progenitor cells (iMPPs) from pluripotent stem cells, the method comprises (1) differentiating and expanding pluripotent stem cells into mesodermal cells by contacting the pluripotent cells with a culture medium containing a BMP activator and optionally bFGF; (2) conferring permanent hematopoietic endothelial cell potential to the mesodermal cells by contacting the mesodermal cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; and (3) conferring permanent hematopoietic endothelial cell potential to the mesodermal cells with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally bFGF. The mesodermal cells are differentiated into and expanded into permanent HE cells by contacting a culture medium containing a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (4) the permanent HE cells are differentiated into iMPPs by contacting the HE cells with a culture medium containing a BMP activator, one or more growth factors and cytokines, and optionally a ROCK inhibitor, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In some embodiments, the above methods further include seeding and expanding the cells by contacting pluripotent stem cells with a culture medium containing MEKi, GSKi, and ROCKi. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-HSCs by contacting them with a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating permanent HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting them with a culture medium containing a BMP activator and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the culture medium in the method described above is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the above method further comprises subjecting the seeded pluripotent stem cells, mesoderm, and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia of approximately 2% to approximately 10%. In some embodiments, the iHE cells obtained by the above method express CD34.In some embodiments, the above method further comprises sorting iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting is performed using CD34-positive and CD43-negative cells. In some embodiments, the sorting is performed using CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting is performed using CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the sorting is performed using CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the sorting is performed using CD34-positive and CD93-negative cells. 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 ROCK inhibitor is Y27632 or thiazolinone.

[0239] In one embodiment of a method for generating pluripotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesoderm, the method comprises (1) conferring permanent hematopoietic endothelial cell potential upon mesoderm cells by contacting them with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; (2) differentiating and expanding permanent hematopoietic endothelial cell potential in mesoderm cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (3) differentiating the permanent hematopoietic endothelial cells into iMPPs by contacting them with a culture medium containing a BMP activator, one or more growth factors and cytokines, and optionally a ROCK inhibitor, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In some embodiments, pluripotent stem cells are iPSCs. In some embodiments, iPSCs are untreated iPSCs. In some embodiments, the method described above further comprises differentiating the permanent HE cells into pre-HSCs by contacting them with a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating permanent HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting them with a culture medium containing a BMP activator and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the culture medium in the method described above is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the above method further comprises subjecting mesodermal cells and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a low oxygen pressure of about 2% to about 10%.

[0240] In one embodiment of a method for generating pluripotent stem cell-derived pluripotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential, the method comprises (1) differentiating and expanding the pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential into permanent HE cells by contacting the cells with a culture medium containing one or more growth factors and cytokines, a ROCK inhibitor, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (2) differentiating the permanent HE cells into iMPPs by contacting the permanent HE cells with a culture medium containing a BMP activator and one or more growth factors and cytokines and optionally a ROCK inhibitor, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-HSCs by contacting them with a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating permanent HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting them with a culture medium containing a BMP activator and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the culture medium in the method comprises free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the method further comprises subjecting mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxic pressure between about 2% and about 10%.

[0241] In one embodiment of a method for generating pluripotent progenitor cells (iMPPs) from permanent HE cells derived from pluripotent stem cells, the method includes differentiating the permanent HE cells into iMPPs by contacting them with a culture medium containing a BMP activator, one or more growth factors and cytokines, and optionally a ROCK inhibitor, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the method further includes differentiating the permanent HE cells into pre-HSCs by contacting them with a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating permanent hematopoietic stem cells (HE cells) into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting them with a culture medium containing a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and the culture medium being free of or substantially free of ROCK inhibitors. In some embodiments, the culture medium in the above method is free of or substantially free of TGFβ receptor inhibitors. In some embodiments, the above method further comprises subjecting mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia of between about 2% and about 10%.

[0242] 5. Obtain pluripotent stem cell-derived T cell progenitor cells (ipro-T) or T cell-iCD34 platform and iT platform One aspect of the present invention provides a method for generating pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells using an optimized multi-stage approach. Generally, the method begins by differentiating and expanding pluripotent stem cells into mesodermal cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. The mesodermal cells are then differentiated into mesodermal cells with permanent hematopoietic endothelial cell potential. Subsequently, the mesodermal cells with permanent hematopoietic endothelial cell potential are differentiated into permanent hematopoietic endothelial cells, while the permanent hematopoietic endothelial cells are expanded in a culture medium. The permanent hematopoietic endothelial cells are then differentiated into pre-iproT cells, and then into T cell progenitor cells (proto-T), which are capable of continuously differentiating into T cells in the same culture medium. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived T cell progenitor cells (ipro-T) or T cells, comprising differentiating seeded pluripotent stem cells into mesoderm, differentiating the mesoderm into mesodermal cells with permanent hematopoietic endothelial cell potential, then differentiating the mesodermal cells with permanent hematopoietic endothelial cell potential into iHE, and then differentiating the iHE into T cell progenitor cells or T cells. The present invention further provides a method for generating pluripotent stem cell-derived T cell progenitor cells (ipro-T) or T cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesodermal cells with permanent hematopoietic endothelial cell potential, then differentiating the mesodermal cells into iHE, and then differentiating the iHE into ipro-T or T cells. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived T cell progenitor cells or T cells, comprising differentiating pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential into iHE, and then differentiating the iHE into ipro-T or T cells. Additionally, this invention provides a method for generating pluripotent stem cell-derived T cell progenitor cells (ipro-T) or T cells, comprising differentiating pluripotent stem cell-derived iHE into ipro-T or T cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. The method disclosed herein utilizes an optimized iCD34 culture platform that is free of or substantially 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.

[0243] In one embodiment of a method for generating pluripotent stem cell-derived T cell progenitors (ipro-T) or 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 culture medium containing a BMP activator and optionally bFGF; (2) differentiating the mesoderm into mesoderm cells with permanent hematopoietic endothelial cell potential by contacting the mesoderm cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; and (3) differentiating the mesoderm cells with permanent hematopoietic endothelial cell potential with a culture medium containing a ROCK inhibitor, one or more growth factors, and... The mesodermal cells are differentiated into permanent HE cells by contacting a culture medium containing cytokines and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (4) the permanent HE cells are differentiated into ipro-T or iT cells by contacting a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more factors, wherein the optionally one or more factors are selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above methods further include seeding and expanding the cells by contacting pluripotent stem cells with a culture medium containing MEKi, GSKi, and ROCKi. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-iproT cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating permanent HE cells into pre-iproT cells further comprises differentiating the pre-iproT cells into ipro-T cells or iT cells by contacting them with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the method comprises generating pluripotent stem cell-derived pro-T cells using 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, bead-bound peptides, surface-bound peptides, or cell-presented peptides.In some embodiments, the method further comprises subjecting seeded pluripotent stem cells, mesoderm, and / or mesodermal cells with perpetual hematopoietic endothelial cell potential to a hypoxia of approximately 2% to approximately 10%. In some embodiments, iHE cells obtained by 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 in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazolidinedione. In some embodiments, the culture medium in the method contains no or substantially no TGFβ receptor inhibitors.

[0244] In one embodiment of a method for generating pluripotent stem cell-derived T cell progenitor cells (ipro-T) from pluripotent stem cell-derived mesoderm, the method comprises (1) differentiating the mesoderm into mesoderm cells with permanent hematopoietic endothelial cell potential by contacting the mesoderm cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; (2) differentiating the mesoderm cells with permanent hematopoietic endothelial cell potential into permanent HE cells by contacting the mesoderm cells with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (3) differentiating the permanent HE cells into ipro-T or iT cells by contacting the permanent HE cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more factors, wherein the optionally present one or more factors are selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, pluripotent stem cells are iPSCs. In some embodiments, iPSCs are untreated iPSCs. In some embodiments, the method described above further comprises differentiating the permanent HE cells into pre-iproT cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating permanent HE cells into pre-iproT cells further comprises differentiating the pre-iproT cells into ipro-T cells or iT cells by contacting them with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the method described above comprises generating pluripotent stem cell-derived pro-T cells using 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, bead-bound peptides, surface-bound peptides, or cell-presented peptides. In some embodiments, the method further comprises subjecting mesodermal and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia of about 2% to about 10%. In some embodiments, iHE cells obtained by the method express CD34.In some embodiments, the above method further comprises sorting iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. 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 ROCK inhibitor is Y27632 or thiazolinone. In some embodiments, the culture medium in the above method contains no or substantially no TGFβ receptor inhibitors.

[0245] In one embodiment of a method for generating pluripotent stem cell-derived T cell progenitors (ipro-T) or T cells (iT) from pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential, the method comprises (1) differentiating the mesoderm cells with permanent hematopoietic endothelial cell potential into permanent hematopoietic stem cell (HE) cells by contacting the mesoderm cells with permanent hematopoietic endothelial cell potential with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (2) differentiating the permanent HE cells into ipro-T or iT cells by contacting the permanent HE cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7 and optionally one or more factors, wherein the optionally one or more factors are 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 untreated iPSCs. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-iproT cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating permanent HE cells into pre-iproT cells further comprises differentiating the pre-iproT cells into ipro-T cells or iT cells by contacting them with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the method comprises generating pluripotent stem cell-derived pro-T cells using 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, bead-bound peptides, surface-bound peptides, or cell-presented peptides. In some embodiments, the method further comprises subjecting mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia pressure between about 2% and about 10%. In some embodiments, iHE cells obtained by 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 in the method is BMP4.In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazolinone. In some embodiments, the culture medium used in the above methods contains no or substantially no TGFβ receptor inhibitors.

[0246] In one embodiment of a method for generating pluripotent stem cell-derived T cell progenitors (ipro-T) or T cells (iT) from pluripotent stem cell-derived HE cells, the method comprises differentiating the permanent HE cells into ipro-T or T cells by contacting them with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more other factors. The optionally present factors are 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 untreated iPSCs. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-iproT cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a BMP activator, the cytokines being selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating permanent HE cells into pre-iproT cells further comprises differentiating the pre-iproT cells into ipro-T cells or iT cells by contacting the pre-iproT cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the method comprises generating pluripotent stem cell-derived pro-T cells using 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, bead-bound peptides, surface-bound peptides, or cell-presented peptides. In some embodiments, the method further comprises subjecting the pluripotent stem cell-derived HE cells to a hypoxic pressure between about 2% and about 10%. In some embodiments, the iHE cells obtained by the method express CD34. In some embodiments, the above method further comprises sorting iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. 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 ROCK inhibitor is Y27632 or thiazolinone. In some embodiments, the culture medium in the above method contains no or substantially no TGFβ receptor inhibitors.

[0247] 6. Obtain pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or NK cells – iCD34 platform and iNK platform One aspect of the present invention provides a method for generating pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or NK cells (iNK) using an optimized multi-stage approach. Generally, in some embodiments, the method begins by seeding pluripotent stem cells. The pluripotent stem cells are then developed into mesodermal cells, which are expanded and subsequently differentiated into mesodermal cells with permanent hematopoietic endothelial cell potential. The mesodermal cells with permanent hematopoietic endothelial cell potential are then differentiated into and expanded into permanent hematopoietic endothelial cells. The HE cells are capable of differentiating into pre-iproNK, and then into NK cell progenitor cells (proto-NK), which are capable of continuously differentiating into NK cells in the same culture medium. Alternatively, the present invention provides a method for generating pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or NK cells, comprising differentiating the seeded pluripotent stem cells into mesoderm, differentiating the mesoderm into mesodermal cells with permanent hematopoietic endothelial cell potential, then differentiating the mesodermal cells with permanent hematopoietic endothelial cell potential into iHE, and then differentiating the iHE into NK cell progenitor cells. This invention further provides a method for generating pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or NK cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesoderm cells with permanent hematopoietic endothelial cell potential, then differentiating the mesoderm cells with permanent hematopoietic endothelial cell potential into iHE, and then differentiating the iHE into ipro-NK or iNK. Alternatively, this invention provides a method for generating pluripotent stem cell-derived NK cell progenitor cells or iNK, comprising differentiating pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential into iHE, and then differentiating the iHE into ipro-NK or iNK. Additionally, this invention provides a method for generating pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or NK cells, comprising differentiating pluripotent stem cell-derived iHE into ipro-NK or iNK. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises obtaining NK cells by contacting progenitor NK cells with one or more artificial antigens that stimulate NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells. The method disclosed herein utilizes an optimized iCD34 culture platform that is free of or substantially free of TGFβ receptor / ALK inhibitors.

[0248] In one embodiment of a method for generating pluripotent stem cell-derived NK cell progenitors (ipro-NK) or NK cells (iNK) from seeded iPSCs, the method comprises (1) differentiating and expanding the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a culture medium containing a BMP activator and optionally bFGF; (2) conferring permanent hematopoietic endothelial cell potential upon the mesodermal cells by contacting the mesodermal cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; and (3) conferring permanent hematopoietic endothelial cell potential upon contacting the mesodermal cells with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally bFGF. The mesodermal cells are differentiated into and expanded into permanent HE cells by contacting a culture medium containing a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (4) the permanent HE cells are differentiated into ipro-NK or iNK cells by contacting a culture medium containing one or more growth factors and cytokines and optionally one or more factors, wherein the cytokines are selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and the optionally one or more factors are selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, pluripotent stem cells are iPSCs. In some embodiments, iPSCs are untreated iPSCs. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-iproNK cells by contacting them with a culture medium containing 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 permanent HE cells into pre-iproNK cells further comprises differentiating the pre-iproNK cells into proto-iNK cells or iNK cells by contacting them with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises obtaining NK cells by contacting proto-NK cells with one or more artificial antigens that stimulate NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells. In one embodiment, the above method further comprises inoculating and expanding the pluripotent cells by contacting untreated pluripotent cells with a culture medium containing MEKi, GSKi, and ROCKi.In some embodiments, the method further comprises subjecting seeded iPSCs, mesoderm, and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia of approximately 2% to approximately 10%. In some embodiments, iHE cells obtained by 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 utilizes CD34-positive and CD43-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting utilizes CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the sorting utilizes CD34-positive and CD93-negative cells. 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 ROCK inhibitor is Y27632 or thiazolinone. In some embodiments, the culture medium used in the above methods contains no or substantially no TGFβ receptor inhibitors.

[0249] In one embodiment of a method for generating pluripotent stem cell-derived NK cell progenitors (ipro-NK) or NK cells (iNK) from pluripotent stem cell-derived mesoderm, the method comprises (1) differentiating the mesoderm by contacting it with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF to obtain mesoderm cells with permanent hematopoietic endothelial cell potential; and (2) differentiating the mesoderm cells with permanent hematopoietic endothelial cell potential by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, to obtain mesoderm cells with permanent hematopoietic endothelial cell potential. (2) Obtaining permanent HE cells, wherein the cytokines are selected from the group consisting of: VEGF, bFGF, SCF, IL6, and IL11; and (3) differentiating the permanent HE cells by contacting them with a culture medium containing one or more growth factors and cytokines, and optionally one or more factors, to obtain ipro-NK or iNK, wherein the cytokines are selected from the group consisting of: SCF, Flt3L, IL3, IL7, and IL15, and the optionally one or more factors are selected from the group consisting of: VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, pluripotent stem cells are iPSCs. In some embodiments, iPSCs are untreated iPSCs. In some embodiments, the above method further comprises differentiating the permanent HE cells into pre-iproNK by contacting them with a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of: SCF, Flt3L, TPO, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating permanent HE cells into pre-iproNK cells further comprises differentiating the pre-iproNK cells into ipro-NK or iNK cells by contacting the pre-iproNK cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises obtaining NK cells by contacting progenitor NK cells with one or more artificial antigens that stimulate NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells. In some embodiments, the method further comprises subjecting mesoderm and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxic pressure between about 2% and about 10%. In some embodiments, the iHE cells obtained by the 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 utilizes CD34-positive and CD43-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting utilizes CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the sorting utilizes CD34-positive and CD93-negative cells. 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 ROCK inhibitor is Y27632 or thiazolinone. In some embodiments, the culture medium in the above method contains no or substantially no TGFβ receptor inhibitors.

[0250] In one embodiment of a method for generating pluripotent stem cell-derived NK cell progenitors (ipro-NK) or NK cells (iNK) from pluripotent stem cell-derived mesoderm cells with permanent hematopoietic endothelial cell potential, the method comprises (1) differentiating the mesoderm cells with permanent hematopoietic endothelial cell potential into permanent HE cells by contacting them with a culture medium containing a ROCK inhibitor, one or more growth factors and cytokines, and optionally a Wnt pathway activator, wherein the cytokines are selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (2) differentiating the permanent HE cells into ipro-NK or iNK by contacting them with a culture medium containing one or more growth factors and cytokines, and optionally one or more factors, wherein the cytokines are selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and the optionally one or more factors are 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 untreated iPSCs. In some embodiments, the method further comprises differentiating the permanent HE cells into pre-iproNK cells by contacting them with a culture medium containing 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 permanent HE cells into pre-iproNK cells further comprises differentiating the pre-iproNK cells into ipro-NK cells or iNK cells by contacting them with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises obtaining NK cells by contacting progenitor NK cells with one or more artificial antigens that stimulate NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells. In some embodiments, the method further comprises subjecting the seeded pluripotent stem cells, mesoderm, and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia of approximately 2% to approximately 10%. In some embodiments, the iHE cells obtained by 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 utilizes CD34-positive and CD43-negative methods. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD73-negative methods. In some other embodiments, the sorting utilizes CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative methods. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD93-negative methods. In some embodiments, the sorting utilizes CD34-positive and CD93-negative methods. 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 ROCK inhibitor is Y27632 or thiazolidinedione. In some embodiments, the culture medium in the above methods contains no or substantially no TGFβ receptor inhibitors.

[0251] In one embodiment of a method for generating pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or NK cells (iNK) from pluripotent stem cell-derived HE cells, the method includes differentiating the permanent HE cells into ipro-NK or iNK by contacting them with a culture medium containing one or more growth factors and cytokines, optionally one or more of the factors present, wherein the cytokines are selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and the optionally one or more factors are 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 untreated iPSCs. In some embodiments, the method further includes differentiating the permanent HE cells into pre-iproNK by contacting them with a culture medium containing a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines, wherein the cytokines are selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating permanent HE cells into pre-iproNK cells further comprises differentiating the pre-iproNK cells into ipro-NK cells or iNK cells by contacting the pre-iproNK cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises obtaining NK cells by contacting ipro-NK cells with one or more artificial antigens that stimulate NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of bead-binding, plasma membrane granules, and / or antigen-presenting cells. In some embodiments, the above method further comprises subjecting seeded pluripotent stem cells, mesoderm, and / or mesodermal cells with permanent hematopoietic endothelial cell potential to a hypoxia of about 2% to about 10%. In some embodiments, the iHE cells obtained by the above method express CD34. In some embodiments, the above method further includes sorting iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting utilizes CD34-positive and CD43-negative cells. In some embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting utilizes CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some other embodiments, the sorting utilizes CD34-positive, CD43-negative, and CD93-negative cells.In some other embodiments, the sorting utilizes CD34-positive and CD93-negative samples. 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 ROCK inhibitor is Y27632 or thiazolidinedione. In some embodiments, the culture medium in the above methods contains no or substantially no TGFβ receptor inhibitors.

[0252] Based on the foregoing, one advantage of the culture platform covered herein is the enhanced viability and survival of cultured, passaged, and dissociated single pluripotent cells for pluripotent stem cell differentiation without the formation of EBs. In some embodiments, pluripotent stem cells are iPSCs. In some embodiments, iPSCs are untreated iPSCs. In some embodiments, iPSCs are genomically engineered. In some embodiments, iPSCs derived from immune cells of a specific donor or patient are reprogrammed. Dissociation of cells into single cells, such as single-cell suspensions, can be achieved by enzymatic or mechanical means. Any enzymatic reagent known in the art for dissociating cells into single cells can be used in the methods of the present invention. In one embodiment, the dissociation reagent is selected from trypsin / EDTA, TrypLE-Select, collagenase IV, and dispersase. According to the methods covered herein, chelating agents such as EDTA, AccuMax, or AccuMax can also be used alone or in combination with enzymatic reagents for cell dissociation. The dissociation reagent can be dissolved in calcium- and magnesium-free PBS to promote dissociation into single cells. To enhance cell survival during and after dissociation, in some embodiments, survival-promoting substances are added, such as one or more growth factors, inhibitors of cellular pathways involving cell death and apoptosis, or conditioned media. In one embodiment, the survival-promoting substance is a ROCK inhibitor, including (but not limited to) thiazolinone.

[0253] In some embodiments, the iPSCs used for differentiation contain genetic imprints. In some embodiments, the genetic imprints in pluripotent stem cells include (i) one or more genetic modification patterns acquired during or after the reprogramming of non-pluripotent cells into iPSCs through genomic insertion, deletion, or substitution in the pluripotent cell genome; or (ii) one or more source-specific immune cells that retain therapeutic properties, and wherein the pluripotent cells are reprogrammed from source-specific immune cells, and wherein the iPSCs retain source therapeutic properties, which are also present in the iPSC-derived hematopoietic lineage cells. In some embodiments, the genetic modification patterns include one or more of the following: safety switch proteins, targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of iPSCs or their derived cells. In some other embodiments, the gene modification pattern comprises one or more of the following: (i) deletion or reduced expression of B2M, TAP1, TAP2, TAP-associated glycoprotein, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) introduction or increase of expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, TCR, or a surface triggering receptor for a bispecific or multispecific conjugate. In some embodiments, the surface triggering receptor is universal, i.e., compatible with any effector cell type, and effector cells expressing the universal surface triggering receptor are capable of coupling with the same bispecific or multispecific conjugate, regardless of their cell type. In some embodiments, the universal surface triggering receptor comprises an anti-antigen determinant and a co-stimulatory domain, wherein the anti-antigen determinant is specific to the antigen determinant in the bispecific or multispecific conjugate. In some embodiments, the co-stimulatory domain of a universal surface-triggered receptor includes IL2 for typical or atypical cell activation and effector cell function enhancement. In still other embodiments, hematopoietic lineage cells include therapeutic properties of source-specific immune cells associated with one or more of the following: (i) receptor expression targeting an antigen; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved target specificity while reducing extratumor effects; (v) resistance to therapies such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0254] In some embodiments, the conjugate is cell type specific, i.e., the conjugate binds to and / or activates a specific immune cell type. In certain embodiments, the conjugate is cell type independent, i.e., the conjugate binds to and / or activates multiple immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils.

[0255] In some embodiments, iPSCs and their derived hematopoietic cells comprise one or more of the following: B2M knockout, HLA-E / G, PDL1, A 2A R, CD47, LAG3 knockout, TIM3 knockout, TAP1 knockout, TAP2 knockout, TAP-associated glycoprotein knockout, NLRC5 knockout, PD1 knockout, RFKANK knockout, CITTA knockout, RFX5 knockout, and RFXAP knockout. These cells, possessing modified HLA class I and / or II, exhibit enhanced resistance to immune detection and thus improved in vivo retention. Furthermore, these cells avoid the need for HLA matching in adoptive cell therapy, thereby providing a universal, off-the-shelf source of therapeutic options.

[0256] In some embodiments, iPSCs and their derived hematopoietic cells comprise one or more of HACD16, 41BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells possess enhanced immune effector capabilities.

[0257] In some embodiments, iPSCs and their derived hematopoietic cells contain surface trigger receptors for coupling with bispecific or multispecific conjugates. Such cells exhibit improved tumor-targeting specificity.

[0258] In some embodiments, iPSCs and their derived hematopoietic cells are specific to antigens.

[0259] Overview of cell culture and culture medium collection techniques 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., ed., Academic Press 1993); "Embryonic Stem Cell Differentiation in vitro" (MV Wiles, *Meth. Enzymatic Methods*). "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy" (PD Rathjen et al., 1993). Stem cell differentiation is reviewed in Robertson, *Meth. Cell Biol.* 75:173, 1997; and Pedersen, *Reprod. Fertil. Dev.* 10:31, 1998.

[0260] In this invention, strategies for enriching cell populations with specific characteristics are provided at different stages of the method. In one embodiment, a method for enriching pluripotent stem cells from a cell population includes preparing a single-cell suspension by dissociating and resuspending the cells in the population. The dissociated cells can be resuspended in any suitable solution or culture medium for cell maintenance or cell sorting. In a particular embodiment, the single pluripotent cell suspension contains a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor but lacks a TFGβ inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and / or the Rock inhibitor is thiazolidinedione.

[0261] In one particular embodiment, cell populations are sorted to actively select pluripotent cells and / or depleted populations of non-reprogrammed or non-pluripotent cells, thereby obtaining a pluripotent cell population. In one embodiment, a single-cell suspension is prepared, and then single cells are prepared for sorting, for example by staining against markers of pluripotency using, for example, an appropriate antibody. Cells can be sorted using any suitable cell sorting method, such as magnetic beads or flow cytometry (FACS).

[0262] Cells can be sorted based on one or more markers of pluripotency or markers indicating cell differentiation, including (but not limited to) the expression of SSEA3 / 4, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / convexin, CD140a, CD56, CD73, CD105, OCT4, NANOG, SOX2, KLF4, SSEA1 (mouse), CD30, SSEA5, CD90, and / or CD50. In various embodiments, cells are sorted based on at least two, at least three, or at least four markers of pluripotency or differentiation. In some embodiments, cells are sorted based on SSEA4 expression, and in certain specific embodiments, cells are sorted based on the expression of SSEA4 in combination with TRA1-81 and / or TRA1-60. In some embodiments, cells are sorted based on the expression of SSEA4, TRA1-81, or TRA1-60 and / or CD30. In one embodiment, cells are sorted based on SSEA4, TRA1-81, and CD30. In another embodiment, cells are sorted based on SSEA4, TRA1-60, and CD30. In some embodiments, cell sorting using one or more surface markers of differentiation includes (but is not limited to) CD13, CD26, CD34, CD45, CD31, CD46, and CD7, as well as pluripotent markers such as SSEA4, TRA1-81, and / or CD30.

[0263] In some embodiments, differentiated cells in a reprogrammed cell population or pluripotent cell population are depleted. In one embodiment, cells in a pluripotent cell population or cell population induced to be reprogrammed, or cells possessing one or more cell surface markers of differentiated cells, are depleted. Illustrative examples of cell surface markers of differentiated cells include (but are not limited to) CD13, CD26, CD34, CD45, CD31, CD46, and CD7. In a particular embodiment, CD13 is used as a surface marker of differentiated cells.

[0264] In other embodiments, cell populations are induced to differentiate into desired lineages and depleted of pluripotent cells to obtain a rich population of positively differentiated or differentiated cells. In some embodiments, the differentiated cell population comprises cell populations that have been induced to differentiate into a specific lineage, such as ESCs or iPSCs. In some embodiments, pluripotent cells in the cell population can be depleted using the aforementioned negative cell sorting techniques (“panning”), such as sorting cells in the population based on magnetic beads or pluripotency marker-based FACs. In some embodiments, the cell population containing differentiated cells is sorted using pluripotency marker-based FACs, and a depleted portion of cells expressing the pluripotency marker is obtained. In other embodiments, the cell population is sorted using differentiation marker-based FACs to obtain a depleted portion of the pluripotency marker, such as lineage-specific markers, including (but not limited to) CD13, CD26, CD34, CD45, CD31, CD46, and CD7. In some specific embodiments of the invention, CD13 is used as a surface marker for differentiated cells.

[0265] D. Cell populations and cell lines generated using the methods and platform presented in this paper In some embodiments, the cells cultured after reprogramming are induced to differentiate over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 22, 24, 26, 28, 30, 32, 35, 40, 42, or 45 days, or any number of days in between. In some embodiments, the cells cultured after reprogramming are induced for about 1 to 42 days, 2 to 40 days, 2 to 35 days, 2 to 20 days, 2 to 10 days, 4 to 30 days, about 4 to 24 days, about 6 to 22 days, or about 8 to about 12 days. In some embodiments, the cells are pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are untreated iPSCs. In one embodiment, enrichment provides a method for obtaining a clonal pluripotent stem cell-derived differentiated cell population in a relatively short time, thereby improving the efficiency of generating pluripotent stem cell-derived differentiated cells at different stages. In one embodiment, enrichment provides a method for obtaining CD34-expressing HE cells, CD34-expressing HSC cells, T or NK cell progenitor cells, and T or NK cells, thereby improving the efficiency of generating each of the cell populations. Enrichment may include sorting the cell population to identify and obtain cells expressing specific characteristic markers indicating differentiation stage / cell type. In some embodiments, the sorting utilizes CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting utilizes CD34 positivity. In some embodiments, the sorting utilizes CD34 positivity and CD43 negativity. In some embodiments, the sorting utilizes CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, the sorting utilizes CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity. In some other embodiments, the sorting utilizes CD34 positivity, CD43 negativity, and CD93 negativity. In some other embodiments, the sorting utilizes CD34 positivity and CD93 negativity. Another enrichment method involves depleting cells expressing markers representing undesirable cell types to obtain enriched clusters of the desired cell types.

[0266] Therefore, one aspect of the present invention provides a composition comprising one or more cell populations, cell lines, or clones of the following: (i) pluripotent stem cell-derived CD34+ HE cells (iCD34), wherein the iCD34 cells have the ability to differentiate into pluripotent progenitor cells, and wherein the iCD34 cells are CD34+CD43-; (ii) pluripotent stem cell-derived perpetual hematopoietic endothelial cells (iHE), wherein the iHE cell line or clone is CD34+, and at least one of CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) pluripotent stem cell-derived pluripotent progenitor cells (iMPP), wherein the iMPP cells are CD34+CD45+; (v) pluripotent stem cells The composition comprises: (iv) pluripotent stem cell-derived T progenitor cells (ipro-T), wherein the T progenitor cells are CD34+CD45+CD7+; (vi) pluripotent stem cell-derived T cells (iT), wherein the T cells are CD45+CD4+CD3+ or CD45+CD8+CD3+; (vi) pluripotent stem cell-derived NK progenitor cells (ipro-NK), wherein the NK progenitor cells are CD45+CD56+CD7+CD3-; and (vii) pluripotent stem cell-derived NK cells (iNK), wherein the NK cells are CD45+CD56+NKp46+. In some embodiments, the above compositions, cell populations, cell lines, or clones are suitable for cryopreservation. In some embodiments, the compositions, cell populations, cell lines, or clones are suitable for ambient storage conditions for more than 12 hours, 24 hours, 36 hours, or 48 hours, but no longer than 3 days, 4 days, 5 days, 6 days, or one week.

[0267] Another aspect of the present invention provides a mixture comprising one or more of the following: (i) pluripotent stem cell sources, namely CD34+ HE cells (iCD34), and one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) permanent hematopoietic endothelial cells (iHE), and one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) permanent HSCs, and one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2. (iv) Culture medium for iMPP and iMPP-A; (v) Culture medium for iTC-A2 and iTC-B2; (vi) Culture medium for iTC and iTC-B2; (vii) Culture medium for iNK-A2 and iNK-B2; (vii) Culture medium for iNK-A2 and iNK-B2; and / or (viii) Culture medium for iNK-B2 and iNK-B2; wherein a. iCD34-C contains a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, IL11, IGF and EPO, and optionally present Wnt pathway activators; and does not contain TGFβ receptor / ALK inhibitors; b. iMPP-A contains 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 contains 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 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L and IL7; wherein the composition does not contain 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, and optionally a BMP activator, wherein the cytokines are selected from the group consisting of: SCF, Flt3L, TPO, IL3, IL7, and IL15, and f. iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15.

[0268] E. Therapeutic uses of iPSC-derived immune cells This invention provides a composition comprising an isolated population or subset of immune cells derived from iPSCs using the methods and compositions disclosed herein, wherein the immune cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subset of immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subset of immune cells comprises iPSC-derived T cells. In one embodiment, the isolated population or subset of immune cells comprises iPSC-derived HSC cells. In some embodiments, the iPSC-derived immune cells are further modulated in vitro to improve therapeutic potential. In one embodiment, the isolated population or subset of immune cells derived from iPSCs comprises an increased number or ratio of untreated T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subset of immune cells derived from iPSCs comprises an increased number or ratio of type I NKT cells. In another embodiment, the isolated population or subset of immune cells derived from iPSCs comprises an increased number or ratio of adaptive NK cells. In some embodiments, the isolated HSC cells, T cells, or NK cell populations or subsets derived from iPSCs are allogeneic. In some other embodiments, the isolated HSC cells, T cells, or NK cell populations or subsets derived from iPSCs are autogenous.

[0269] In some embodiments, the iPSCs used for differentiation contain a genetic imprint of the desired therapeutic properties of the effector cells, which is retained and function in the differentiated hematopoietic cells derived from the iPSCs.

[0270] In some embodiments, the genetic imprinting in pluripotent stem cells includes (i) one or more genetic modification patterns obtained during or after reprogramming non-pluripotent cells into iPSCs by genomic insertion, deletion, or substitution in the pluripotent cell genome; or (ii) one or more source-specific immune cells that retain therapeutic properties and are specific to donor, disease, or treatment response, wherein the pluripotent cells are reprogrammed from source-specific immune cells, wherein the iPSCs retain source therapeutic properties, and the source therapeutic properties are also contained in the iPSC-derived hematopoietic lineage cells.

[0271] In some embodiments, the gene modification pattern comprises one or more of the following: safety switch proteins, targeting patterns, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of iPSCs or their derived cells. In some other embodiments, the gene modification pattern comprises one or more of the following: (i) deletion or reduced expression of B2M, TAP1, TAP2, TAP-associated glycoproteins, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) introduction or increase of expression of HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, TCR, or surface trigger receptors coupled to bispecific or multispecific conjugates.

[0272] In some other embodiments, the hematopoietic lineage cells include therapeutic properties of source-specific immune cells associated with one or more of the following: (i) receptor expression targeting antigens; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved target specificity while reducing extratumor effects; (v) resistance to therapies such as chemotherapy; and (vi) improved homing, survival, and cytotoxicity.

[0273] In some embodiments, iPSCs and their derived hematopoietic cells comprise one or more of the following: B2M knockout, HLA-E / G, PDL1, A 2A R, CD47, LAG3 knockout, TIM3 knockout, TAP1 knockout, TAP2 knockout, TAP-associated glycoprotein knockout, NLRC5 knockout, PD1 knockout, RFKANK knockout, CITTA knockout, RFX5 knockout, and RFXAP knockout. These cells, possessing modified HLA class I and / or II, exhibit enhanced resistance to immune detection and thus improved in vivo retention. Furthermore, these cells avoid the need for HLA matching in adoptive cell therapy, thereby providing a universal, off-the-shelf source of therapeutic options.

[0274] In some embodiments, iPSCs and their derived hematopoietic cells comprise one or more of HACD16, 41BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells possess enhanced immune effector capabilities.

[0275] In some embodiments, iPSCs and their derived hematopoietic cells are specific to antigens.

[0276] Many diseases can be improved by introducing the immune cells of this invention into subjects suitable for adoptive cell therapy. Examples of diseases include a variety of autoimmune disorders, including (but not limited to) alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Hugh Grant's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatous polyangiitis (Wegener's disease); hematologic malignancies, including This includes (but is not limited to) acute and chronic 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 infections, including (but not limited to) HIV (human immunodeficiency virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cell cytomegalovirus), adenovirus-related conditions, and BK polyomavirus-related conditions.

[0277] Specific embodiments of the invention relate to a method of treating a subject by administering to a subject in need a composition comprising any of the cells described herein. In specific embodiments, the terms “treatment” and the like are generally used herein to refer to achieving a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing the disease, and / or therapeutic in terms of partially or completely curing it, for the sake of disease and / or adverse effects attributable to said disease. As used herein, “treatment” encompasses any treatment of a mammalian disease and includes: preventing the development of said disease in a subject who may be susceptible to it but has not yet been diagnosed with it; inhibiting said disease, i.e., halting its development; or alleviating said disease, i.e., causing said disease to regress. The therapeutic agent or composition may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is of particular interest, wherein the treatment stabilizes or reduces adverse clinical symptoms in the patient.

[0278] In certain embodiments, the subject suffers from a disease, symptom, and / or lesion that can be treated, alleviated, and / or improved by cell therapy. Some embodiments anticipate subjects requiring cell therapy to be those suffering from an lesion, disease, or symptom, wherein the cell therapy (e.g., a therapy that delivers cellular material to the subject) is capable of treating, alleviating, improving, and / or reducing the severity of at least one symptom associated with said lesion, disease, or symptom. Some embodiments anticipate subjects requiring cell therapy to include (but are not limited to) bone marrow or stem cell transplant candidates, subjects who have received chemotherapy or radiation therapy, subjects who have or are at risk of developing a hyperplastic condition or cancer (e.g., hyperplastic syndrome or hematopoietic cancer), subjects who have a tumor or are at risk of developing a tumor (e.g., a solid tumor), and subjects who have a viral infection or a disease associated with a viral infection or are at risk of developing a viral infection or a disease associated with a viral infection.

[0279] Accordingly, the present invention further provides a pharmaceutical composition comprising pluripotent hematopoietic lineage cells prepared by the methods and compositions disclosed herein, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable medium. In one embodiment, the pharmaceutical composition comprises pluripotent T cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent NK cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent CD34 HE cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent HSCs prepared by the methods and compositions disclosed herein.

[0280] Additionally, the above-mentioned pharmaceutical composition is provided for therapeutic use, which is achieved by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disease; a hematologic malignancy; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0281] The isolated pluripotent stem cell-derived hematopoietic lineage cells may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, CD34+ HE cells, or HSCs. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic lineage cells have about 95% to about 100% T cells, NK cells, NKT cells, CD34+ HE cells, or HSCs. In some embodiments, the present invention provides pharmaceutical compositions having purified T cells, NK cells, NKT cells, CD34+ HE cells, or HSCs, such as compositions having an isolated population of about 95% T cells, NK cells, NKT cells, CD34+ HE cells, or HSCs, for treating subjects requiring cell therapy.

[0282] In some embodiments, the pharmaceutical composition comprises an isolated pluripotent stem cell-derived hematopoietic lineage cell population having less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% iPSC-derived T cells, NK cells, NKT cells, CD34+ HE cells, or HSCs. In some embodiments, the isolated derived hematopoietic lineage cell population may have more than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% T cells, NK cells, NKT cells, CD34+ HE cells, or HSCs. In other embodiments, the isolated derived hematopoietic lineage cell population may have about 0.1% to about 1%, about 1% to about 3%, about 3% to about 5%, about 10% to about 15%, about 15% to 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.

[0283] In a particular embodiment, the derived hematopoietic lineage cells may have 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.

[0284] As those skilled in the art will understand, both autologous and allogeneic immune cells can be used in cell therapy. Autologous cell therapy can reduce infection, lower the probability of GvHD, and accelerate immune remodeling. Allogeneic cell therapy can have immune-mediated graft-versus-malignant disease (GVM) effects and a lower relapse rate. Based on the specific conditions of the patient or subject requiring cell therapy, those skilled in the art can determine which specific type of therapy to administer.

[0285] In certain embodiments, the derived hematopoietic lineage cells in the pharmaceutical composition of the present invention are allogeneic relative to the subject. In certain embodiments, the derived hematopoietic lineage cells in the pharmaceutical formulation of the present invention are autologous relative to the subject. For autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA matched relative to the patient. In another embodiment, the derived hematopoietic lineage cells are not HLA matched relative to the subject.

[0286] In some embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is at least 0.1 × 10⁻⁶. 5 1 cell, at least 0.5 × 10 5 10 cells, at least 1×10 5 1 cell, at least 5 × 10 5 10 × 10 cells, at least 10 × 10 5 1 cell, at least 0.5 × 10 6 1 cell, at least 0.75 × 10 6 10 cells, at least 1×10 6 1.25 × 10⁶ cells 6 1.5 × 10⁶ cells 6 1.75 × 10⁶ cells 6 1 cell, at least 2 × 10 6 1 cell, at least 2.5 × 10 6 1 cell, at least 3 × 10 6 1 cell, at least 4 × 10 6 1 cell, at least 5 × 10 6 10 × 10 cells, at least 10 × 10 6 15 × 10 cells, at least 15 × 10 6 1 cell, at least 20 × 10 6 1 cell, at least 25 × 10 6 One cell or at least 30 × 10 6 Each cell.

[0287] In some embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is about 0.1 × 10⁻⁶. 5 From one cell to approximately 10 × 10 5 10 cells; approximately 0.5 × 106 From one cell to approximately 5 × 10 6 1 × 10⁻⁶ cells; approximately 1 × 10⁻⁶ 6 From one cell to approximately 3 × 10 6 1.5 × 10⁻⁶ cells; approximately 1.5 × 10⁻⁶ 6 From one cell to approximately 2.5 × 10⁻⁶ 6 10 cells; or approximately 2 × 10 6 From one cell to approximately 2.5 × 10⁻⁶ 6 Each cell.

[0288] In some embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is about 1 × 10⁻⁶. 6 From one cell to approximately 3 × 10 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 5 × 10 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 10 × 10 6 10 × 10⁶ cells; approximately 10 × 10⁶ 6 From one cell to approximately 20 × 10 6 10 × 10⁶ cells; approximately 10 × 10⁶ 6 From one cell to approximately 30 × 10 6 10 cells; or approximately 20 × 10 6 From one cell to approximately 30 × 10 6 Each cell.

[0289] In some other embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is from about 1 × 10⁻⁶ cells to about 30 × 10⁻⁶ cells. 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 20 × 10 6 1.0 × 10⁶ cells; approximately 1.0 × 10⁶ cells 6 From one cell to approximately 10 × 10 6 10 cells; approximately 2.0 × 10 6 From one cell to approximately 30 × 10 6 10 cells; approximately 2.0 × 10 6 From one cell to approximately 20 × 10 6 10 cells; or approximately 2.0 × 10⁻⁶ cells. 6 From one cell to approximately 10 × 10 6 Each cell.

[0290] In other embodiments, the number of derived hematopoietic lineage cells in the pharmaceutical composition is approximately 1 × 10⁻⁶. 6 1 cell, approximately 2 × 10 6 1 cell, approximately 5 × 10 6 1 cell, approximately 7 × 10 6 One cell, approximately 10 × 10 6 One cell, approximately 15 × 106 17 × 10⁶ cells 6 One cell, approximately 20 × 10 6 One cell, approximately 25 × 10 6 One cell or approximately 30 × 10 6 Each cell.

[0291] In one embodiment, the number of derived hematopoietic lineage cells in the pharmaceutical composition is the number of immune cells in a portion or a single umbilical cord blood sample, or at least 0.1 × 10⁻⁶ cells. 5 Cells / kg body weight, at least 0.5 × 10⁻⁶ 5 Cells / kg body weight, at least 1×10 5 Cells / kg body weight, at least 5×10 5 Cells / kg body weight, at least 10×10 5 Cells / kg body weight, at least 0.5 × 10⁻⁶ 6 Cells / kg body weight, at least 0.75 × 10⁻⁶ 6 Cells / kg body weight, at least 1×10 6 Cells / kg body weight, at least 1.25 × 10⁻⁶ 6 Cells / kg body weight, at least 1.5 × 10 6 Cells / kg body weight, at least 1.75 × 10⁻⁶ 6 Cells / kg body weight, at least 2×10 6 Cells / kg body weight, at least 2.5 × 10⁻⁶ 6 Cells / kg body weight, at least 3×10 6 Cells / kg body weight, at least 4×10 6 Cells / kg body weight, at least 5×10 6 Cells / kg body weight, at least 10×10 6 Cells / kg body weight, at least 15 × 10 6 Cells / kg body weight, at least 20 × 10 6 Cells / kg body weight, at least 25 × 10⁻⁶ 6 Cells / kg body weight or at least 30 × 10⁻⁶ 6 Cells per kg of body weight.

[0292] The derived hematopoietic lineage cells provided by this invention can be administered to subjects without prior in vitro or in vitro expansion. In a particular embodiment, the isolated derived hematopoietic lineage cell population is an immune cell that has been modulated and treated in vitro with one or more reagents to obtain improved therapeutic potential. The modulated derived hematopoietic lineage cell population can be washed to remove the treatment agents, and the improved population can be administered to the patient without further in vitro expansion of the population.

[0293] In other embodiments, the present invention provides an isolated, derived hematopoietic lineage cell population, which, after expansion, is modulated with one or more reagents into an isolated T lymphocyte population or subset. The isolated, derived hematopoietic lineage cell population can be recombined to express TCR, CAR, or other proteins.

[0294] For genetically engineered hematopoietic lineage cells expressing recombinant TCRs or CARs, whether before or after gene modification, the cells can be activated and expanded using methods described in the following documents: for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 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.

[0295] In some embodiments, different approaches can be used to provide primary and co-stimulatory signals to derived hematopoietic lineage cells. For example, the reagent providing each signal can be present in solution or conjugated to a surface. When conjugated to a surface, the reagent can be conjugated to the same surface (i.e., “cis” formation) or to individual surfaces (i.e., “trans” formation). Alternatively, one reagent can be conjugated to a surface and another reagent can be present in solution. In one embodiment, the reagent providing the co-stimulatory signal can bind to the cell surface and the reagent providing the primary activation signal can be present in solution or conjugated to a surface. In some embodiments, both reagents can be present in solution. In another embodiment, the reagent can be in a soluble form and then cross-linked to a surface, such as cells expressing Fc receptors or antibodies or other binding agents to which the reagent will bind, as disclosed in U.S. Patent Application Publications Nos. 20040101519 and 20060034810 concerning artificial antigen-presenting cells (aAPCs), which are intended to be used in this invention for activating and expanding T lymphocytes.

[0296] Compositions comprising the hematopoietic lineage cell populations of the present invention can be sterile and suitable for and ready for administration (i.e., can be administered to human patients without any further processing). In some embodiments, the therapeutic composition is ready for infusion into a patient. Ready-to-administer cell-based compositions mean that the composition requires no further processing or manipulation prior to transplantation or administration to the recipient.

[0297] Suitable therapeutically acceptable sterile compositions for administration to patients may include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable media, such as cell culture media), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition being administered and the specific method of administration. Therefore, the therapeutic compositions of the present invention have a variety of suitable formulations (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0298] In certain embodiments, therapeutic cell compositions having isolated derived hematopoietic lineage cell populations also have pharmaceutically acceptable cell culture media. Therapeutic compositions comprising derived hematopoietic lineage cell populations as disclosed herein can be administered alone via enteral or parenteral administration, or in combination with other suitable compounds, to achieve desired therapeutic goals.

[0299] Pharmaceutically acceptable carriers and / or diluents must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the human subjects being treated. Additionally, the stability of the therapeutic composition should be maintained or enhanced. Pharmaceutically acceptable carriers can be liquid or solid and are selected according to a predetermined planned administration method to provide the desired bulk density, consistency, etc., when combined with other components in the therapeutic composition of the present invention. For example, pharmaceutically acceptable carriers can be (but are not limited to) binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, dicalcium phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium glycolate starch, etc.), or wetting agents (e.g., sodium lauryl sulfate, etc.). Other pharmaceutically acceptable carriers suitable for the compositions of the present invention include (but are not limited to) water, salt solutions, ethanol, polyethylene glycol, gelatin, amyl starch, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0300] Such carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer is a solution or liquid whose chemical composition neutralizes an acid or base without causing a significant change in pH. Examples of buffers contemplated in this invention include (but are not limited to) Durbectos-buffered saline (PBS), Ringer's solution, 5% dextrose aqueous solution (D5W), and standard / physiological saline (0.9% NaCl).

[0301] These pharmaceutically acceptable carriers and / or diluents may be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer may comprise up to about 5% (by weight) of the total composition. The therapeutic composition may also include electrolytes, such as (but not limited to) sodium chloride and potassium chloride. 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 solution with a pH in one of the said pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0302] The sterile composition of the present invention can be a sterile solution or suspension present in a pharmaceutically acceptable, non-toxic medium. Suspension can refer to a non-adhesive condition, wherein cells do not adhere to a solid carrier. For example, cells maintained in a suspension can be agitated to prevent them from adhering to a carrier, such as a culture dish.

[0303] A suspension is a dispersion (mixture) in which finely divided substances combine with another substance, wherein the constituents are so finely divided and mixed that they cannot settle rapidly. Suspensions can be prepared using a mediator (e.g., a liquid medium, including 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, such as physiological saline or serum-free culture medium, and do not adhere to a solid carrier. In everyday life, the most common suspensions are those that are solid in liquid water. Acceptable diluents (e.g., mediators and solvents) that can be used are water, Ringer's solution, isotonic sodium chloride (physiological saline) solution, and serum-free cell culture medium. In some embodiments, a hypertonic solution is used to prepare the suspension. Additionally, sterile, non-volatile oils are conventionally used as solvents or suspension media. In parenteral administration, mediators are particularly suitable as solutions (preferably oils or aqueous solutions) as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or polydextrose. In some embodiments, the infusion solution is isotropic relative to the patient's tissue. In some embodiments, the infusion solution is hypertonic relative to the patient's tissue.

[0304] Pharmaceutically acceptable carriers, diluents, and other components constituting the ready-to-use pharmaceutical compositions of the present invention are derived from U.S. pharmaceutical-grade reagents permitted for use in clinical therapy. Typically, these finished reagents, including any media, solutions, or other pharmaceutically acceptable carriers and / or diluents, are sterilized according to conventional methods in the art, such as filtration sterilization, and tested for a variety of undesirable contaminants (e.g., mycoplasma, endotoxins, or viral contamination) prior to use. In one embodiment, the pharmaceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for storing cell populations, including hematopoietic stem cells and progenitor cells, in the pharmaceutical composition. The pharmaceutical composition is intended for administration to human patients and is therefore substantially free of cell culture components, such as bovine serum albumin, horse serum, and fetal bovine serum.

[0305] This invention also provides in part the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the invention. Such compositions are suitable for administration to human subjects. Generally, any culture medium supporting the maintenance, growth, and / or health of the derived hematopoietic lineage cells of the invention is suitable as a pharmaceutical cell culture medium. In certain embodiments, a pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium.

[0306] The pharmaceutical composition may have a serum-free culture medium suitable for storing regulated and isolated derived hematopoietic lineage cell populations. In various embodiments, the serum-free culture medium is non-animal and may optionally be protein-free. Optionally, the culture medium may contain biomedically acceptable recombinant proteins. A non-animal culture medium is one in which the components are derived from non-animal sources. The recombinant protein replaces the protozoan protein in the non-animal culture medium, and the nutrients are der...

Claims

1. A method for generating hematopoietic lineage cells with enhanced therapeutic properties, comprising: a) Obtain induced pluripotent stem cells (iPSCs) containing one or more genetic imprints; b) Guiding the iPSCs to differentiate into hematopoietic lineage cells, wherein guiding differentiation includes: (i) Contacting iPSCs with a composition containing a BMP pathway activator and optionally present bFGF to obtain mesodermal cells; and (ii) Contact the mesodermal cells with a composition comprising BMP pathway activator, bFGF and WNT pathway activator to obtain mesodermal cells with permanent hematopoietic endothelial (HE) potential, wherein the mesodermal cells with permanent hematopoietic endothelial (HE) potential are capable of providing hematopoietic lineage cells; Mesodermal cells and mesodermal cells with permanent HE potential are obtained in steps (i) and (ii) without the step of forming embryonic bodies; The hematopoietic lineage cells mentioned above include permanent hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells (MPPs), pre-T cell progenitor cells, pre-NK cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, or B cells; and The hematopoietic lineage cells therein retain the genetic imprints contained in the iPSCs.

2. The method of claim 1, wherein obtaining iPSCs comprising one or more genetic imprints further comprises: a) Introducing one or more genetic imprints into an iPSC during or after genome editing of a non-pluripotent cell into an iPSC, wherein the genetic imprints comprise one or more patterns of gene modification introduced by genome insertion, deletion, or substitution in the genome of the iPSC; or b) Introducing one or more genetic imprints into the iPSC as follows i. Obtaining source-specific immune cells that are specific to a donor, disease, or treatment response, wherein the immune cells exhibit therapeutic properties that can be retained; and ii. Reprogram the source-specific immune cells into iPSCs; and optional iii. Introducing additional genetic imprints into the iPSCs of step (ii) by gene editing during or after the reprogramming of the source-specific immune cells into iPSCs.

3. The method according to claim 2, wherein the gene modification mode comprises one or more of the following: safety switch proteins, targeting modes, receptors, signal transduction molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, survival, immune response regulation and / or survival of the iPSCs or their derivative cells.

4. The method according to claim 2, wherein the gene modification pattern comprises one or more of the following: (i) deletion or reduced expression of B2M, TAP1, TAP2, TAP-associated glycoprotein (Tapasin), NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2A The expression of R, CAR, TCR, or surface-triggered receptors targeting bispecific or multispecific conjugates is introduced or increased.

5. The method of claim 4, wherein the surface triggering receptor is common to the hematopoietic lineage cells comprising T, NK, NKT, macrophages and neutrophils.

6. The method of claim 5, wherein the universal surface-triggered receptor comprises an antiantigen determinant and a co-stimulatory domain, wherein the antiantigen determinant is specific to the bispecific or multispecific conjugate.

7. The method of claim 6, wherein the co-stimulatory domain comprises IL2.

8. The method of claim 4, wherein the bispecific or multispecific conjugate is specific to the universal surface-triggered receptor and to one or more tumor-specific antigens on the surface of tumor cells.

9. The method of claim 8, wherein the tumor-specific antigen comprises one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA.

10. The method of claim 1, wherein the therapeutic properties of the source-specific immune cells comprise one or more of the following: (i) receptor expression targeting the antigen; (ii) HLA presentation or its absence; (iii) resistance to the tumor microenvironment; (iv) induction of bystander immune cells and immune modulation; (iv) improved target specificity while reducing extratumor effects; (v) resistance to therapies such as chemotherapy; and (vi) improved homing, retention, and cytotoxicity.

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