Methods and compositions for inducing hematopoietic cell differentiation

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 62 / 251,016 filed on 4 November 2015; International Application No. PCT / US16 / 14918 filed on 26 January 2016; and U.S. Provisional Patent Application No. 62 / 337,093 filed on 16 May 2015, the disclosures thereof incorporated herein by reference in their entirety.

[0002] The present invention relates as a whole to compositions and methods for producing all hematopoietic cells from pluripotent stem cells. Specifically, the present invention relates to an improved culture platform for producing all hematopoietic cells from pluripotent stem cells, including human induced pluripotent stem cells. [Background technology]

[0003] Human induced pluripotent stem cell (hiPSC) technology represents a highly promising and potentially limitless source of therapeutically promising hematopoietic cells for the treatment of numerous hematological and non-hematological malignancies, including cancer. To expand the potential of hiPSCs and genome-modified hiPSC technologies as allogeneic sources of hematopoietic cell therapies, it is essential to be able to efficiently and reproducibly generate not only hematopoietic stem cells and progenitor cells (HSCs) but also immune effector populations, including diverse subsets of T, B, NKT, and NK lymphoid cells and their progenitor cells.

[0004] In vitro induction of lymphocyte-forming hematopoiesis cells (HSCs) is complex due to the presence of at least two spatiotemporalally distinct waves of hematopoiesis (primary and secondary hematopoiesis) during embryonic development. Primary hematopoiesis begins in the extraembryonic yolk sac, giving rise to a transient and limited hematopoietic repertoire primarily consisting of embryonic erythroid cells and embryonic myeloid cells (though not HSCs). Only after the secondary hematopoietic wave occurs do early HSCs emerge from specialized endothelial progenitor cells within the ductus arteriosus structure called fate-determined hematopoietic endothelium (HE). Fate-determined HE then undergo endothelial hematopoietic transformation to produce HSCs, which subsequently migrate to the bone marrow, where they maintain a multilineage of hematopoiesis, including T, B, NKT, and NK lymphoid cells, throughout their life as a viable organism. Therefore, the development of HSCs and lymphoid effector cells from pluripotent stem cells depends on the accurate reproducibility of the intricate early embryonic hematopoietic developmental stages toward the secondary program through appropriately designed and effective methods and compositions.

[0005] Only a limited number of studies describe the induction of differentiation of hiPSCs into fate-determining hemoglobin (HE) cells in vitro. A major obstacle to the therapeutic use of hiPSCs is that these cells must first be co-cultured with mouse or human stromal cells in the presence of serum-containing media with unclear objectives for maintaining pluripotency and inducing differentiation. In addition, existing protocols employ strategies that involve culturing iPSCs to form embryoid bodies (EBs), which are heterogeneous cell aggregates containing various differentiated cells, including ectoderm, mesoderm, and endoderm cells. These procedures require the aggregation of pluripotent cells, for example, by spinning to form aggregates, by adhering and aggregating cells in wells, or by passively assembling and forming aggregates in suspension culture medium. The formed EBs are maintained in a differentiation induction culture system for a certain period, typically 7-10 days, to differentiate properly. After that, the EBs are transferred to adherent culture for further maturation or dissociated into single cells for cell type selection, with the aim of progressing to the next differentiation stage (Kennedy et al., Cell Reports 2012:1722-1735; Knorr, et al., Stem Cells Translational Medicine 2013 (2):274-283). For example, Kennedy et al. teach EB generation for iPSC differentiation by treating pluripotent cells with collagenase and trypsin to detach the cells and form small aggregates, which are then cultured to form EBs. While EB formation has been shown to promote the differentiation of pluripotent stem cells, the need for aggregate formation and subsequent EB formation is cumbersome, the cell number increases minimally during this process, and the cellular contents within the three-dimensional EB aggregates are exposed to factors in the culture medium inconsistently and unevenly, resulting in heterogeneous cell products with non-uniform differentiation stages. This poses a significant obstacle to the scalability and reproducibility of the manufacturing process, which needs to be efficient and streamlined.

[0006] In other words, there is a need for a method and composition that differentiates stem cells to secondary hematopoiesis without relying on co-culture or serum-containing media, and without requiring the formation of embryoid body aggregates as an intermediate. [Overview of the project]

[0007] The present invention relates, as a whole, to cell culture conditions, media, culture platform, and methods for proliferating and differentiating stem cells into hematopoietic cell prospective fate.

[0008] In particular, the present invention provides a method and composition for constructing hematopoietic cell lineages through fate-determined hematopoietic endothelium (HE) derived from pluripotent stem cells containing hiPSCs, without the need for EB formation, under serum-free / feeder-free conditions, and on an expandable, monolayer culture platform. The cells that can be differentiated according to the method of the present invention range from pluripotent stem cells to progenitor cells whose fate has been determined to specific well-differentiated cell types, and transdifferentiated cells, which are cells of various lineages that have directly transitioned to a hematopoietic fate without passing through pluripotent intermediates. Similarly, the cells resulting from the differentiation of stem cells also range from pluripotent stem cells or pluripotent progenitor cells to well-differentiated stem cells and all intervening hematopoietic cell lineages.

[0009] The present invention provides a composition and method for differentiating and proliferating hematopoietic cells from pluripotent stem cells in monolayer culture, comprising contacting pluripotent stem cells with a BMP pathway activator and optionally bFGF. Thus, mesodermal cells derived from pluripotent stem cells are acquired and proliferated without the formation of embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain proliferated mesodermal cells with the ability to differentiate into deterministic hematopoietic endothelial (HE) cells without the formation of embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF, and optionally a ROCK inhibitor and / or a WNT pathway activator, causes the mesodermal cells with the ability to differentiate into deterministic HE cells to differentiate into deterministic HE cells, which also proliferate during differentiation.

[0010] Because EB formation results in moderate to minimal cell proliferation, does not enable monolayer culture which is important for many applications requiring uniform proliferation and differentiation of cells within a population, and is laborious and inefficient, the methods provided herein for obtaining hematopoietic cells are superior to pluripotent stem cell differentiation via EB.

[0011] A monolayer differentiation platform is provided herein that promotes differentiation toward fate-determined hematopoietic endothelium, thereby inducing hematopoietic stem cells and resulting in differentiated offspring such as T cells, B cells, NKT cells, and NK cells. The demonstrated enhanced differentiation efficiency and mass proliferation combined monolayer differentiation strategy enable the delivery of therapeutically meaningful numbers of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. Furthermore, the invention discloses that monolayer culture using the method provided herein results in functional hematopoietic cells that enable in vitro differentiation, ex vivo regulation, and long-term in vivo hematopoietic self-renewal, reconstitution, and engraftment. As used herein, iPSC-derived hematopoietic cells include, but are not limited to, fate-determined hematopoietic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem cell progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

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

[0013] In some embodiments of the culture platform described above, the culture platform further comprises: (i) a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not containing one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors, and 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 medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, and suitable for the differentiation of pluripotent stem cell-derived fate-determined hematopoietic endothelium into pre-T cell progenitor cells; these additional media are suitable for the development of pluripotent stem cell-derived T cell lineage cells.

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

[0015] In one embodiment, the culture platform provided above comprises (i) a medium containing one or more growth factors and cytokines selected from the group consisting of a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but without a ROCK inhibitor, and suitable for the differentiation of pluripotent stem cell-derived pre-HSCs into hematopoietic pluripotent progenitor cells; and (ii) a 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, and suitable for the differentiation of pluripotent stem cell-derived fate-determined hematopoietic endothelium into pre-HSCs; these media enable the development of pluripotent stem cell-derived hematopoietic pluripotent progenitor cells.

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

[0017] In some embodiments of the composition for differentiation and proliferation of pluripotent stem cell-derived hematopoietic cells, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC. In some embodiments, the iPSC contains one or more genetic imprints, and the one or more genetic imprints contained in the iPSC are retained in hematopoietic cells derived therefrom.

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

[0019] In some embodiments of the above composition for the differentiation and proliferation of pluripotent stem cell-derived hematopoietic cells, the composition further comprises (i) a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors, and containing pluripotent stem cell-derived pre-T cell progenitor cells, and suitable for differentiation of the pluripotent stem cell-derived pre-T cell progenitor cells into T cell progenitor cells or T cells; or (ii) a medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator; and pluripotent stem cell-derived fate-determined hematopoietic endothelium, and suitable for differentiation of the fate-determined hematopoietic endothelium into pre-T cell progenitor cells. These additional media are suitable for the development of pluripotent stem cell-derived T cell lineage cells.

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

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

[0022] In some embodiments of the culture platform for producing pluripotent stem cell-derived T cells, the culture platform further comprises (vi) a culture medium that comprises a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and optionally does not contain a TGFβ receptor / ALK inhibitor, and is suitable for seeding and proliferation of pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in pluripotent stem cell-derived T cells differentiated therefrom.

[0023] Another aspect of the present invention provides a culture platform for producing pluripotent stem cell-derived NK cells, the culture platform comprising: (i) a medium comprising a BMP activator and optionally bFGF, and suitable for the differentiation and proliferation of mesodermal cells from pluripotent stem cells; (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally excluding a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells capable of differentiating from mesodermal cells into fate-determined hematopoietic endothelium; (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; optionally comprising a Wnt pathway activator; and optionally excluding a TGFβ receptor / ALK inhibitor A medium suitable for the differentiation of fate-determined hematopoietic endothelium from mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium, which does not contain (iv) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator, which is suitable for the differentiation of fate-determined hematopoietic endothelium into pre-NK cell progenitor cells; and (v) a medium suitable for the differentiation of pre-NK cell progenitor cells into NK cell progenitor cells or NK cells, which contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and does not contain one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor.

[0024] In some embodiments of the culture platform for producing pluripotent stem cell-derived NK cells, the culture platform further comprises (vi) a culture medium that contains a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but does not contain a TGFβ receptor / ALK inhibitor, and is suitable for seeding and proliferation of pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in pluripotent stem cell-derived NK cells differentiated therefrom.

[0025] A further aspect of the present invention provides a culture platform for producing pluripotent stem cell-derived fate-determined hematopoietic endothelium (iHE), the culture platform comprising: (i) a medium comprising a BMP activator and optionally bFGF, and suitable for the differentiation and proliferation of mesodermal cells from pluripotent stem cells; (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally excluding a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium from the pluripotent stem cell-derived mesodermal cells; and (iii) a medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, optionally excluding a TGFβ receptor / ALK inhibitor, and suitable for the differentiation and proliferation of fate-determined hematopoietic endothelium from mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium.

[0026] In some embodiments of the culture platform for producing pluripotent stem cell-derived fate-determined hematopoietic endothelium (iHE), the culture platform further comprises a medium containing (iv) a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, and suitable for seeding and proliferation of pluripotent stem cells.

[0027] A further aspect of the present invention provides a culture platform for producing pluripotent stem cell-derived hematopoietic pluripotent progenitor cells, the culture platform comprising: (i) a medium comprising a BMP activator and optionally bFGF, and suitable for the differentiation and proliferation of pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally excluding a TGFβ receptor / ALK inhibitor, and suitable for obtaining mesodermal cells from the pluripotent stem cell-derived mesodermal cells that have the ability to differentiate into fate-determined hematopoietic endothelium; (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator. (iv) A medium suitable for the differentiation and proliferation of fate-determined hematopoietic endothelium from mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium, which optionally does not contain a TGFβ receptor / ALK inhibitor; (iv) A medium containing one or more growth factors and cytokines selected from the group consisting of a BMP activator, a ROCK inhibitor, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, which is suitable for the differentiation of fate-determined hematopoietic endothelium into pre-HSCs; and (v) A medium containing one or more growth factors and cytokines selected from the group consisting of a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, which does not contain a ROCK inhibitor, which is suitable for the differentiation of pre-HSCs into hematopoietic pluripotent progenitor cells. In some embodiments, the culture platform further comprises: (vi) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and proliferation of pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in pluripotent stem cell-derived hematopoietic cells differentiated therefrom.

[0028] Another aspect of the present invention provides a method for inducing differentiation of pluripotent stem cells into deterministic hematopoietic cells, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, optionally without a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells having deterministic HE differentiation potential from the mesodermal cells; (iii) the mesodermal cells having deterministic HE differentiation potential The method comprises contacting a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, and optionally not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of fate-determined hematopoietic endothelium from pluripotent stem cell-derived mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium; and optionally exposing pluripotent stem cells, pluripotent stem cell-derived mesoderm cells, mesoderm cells having hematopoietic endothelium, and / or fate-determined hematopoietic endothelium under a hypoxic partial pressure of approximately 2% to approximately 10%.

[0029] In some embodiments of the method for inducing differentiation of pluripotent stem cells into hematopoietic cells, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, to seed and proliferate the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in hematopoietic cells derived from the pluripotent stem cells differentiated therefrom.

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

[0031] In some embodiments of the above method, the fate-determined hematopoietic endothelial cells derived from pluripotent stem cells are CD34+. In some embodiments, the fate-determined hematopoietic endothelial cells are CD34+CD43-. In some embodiments, the fate-determined hematopoietic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the fate-determined hematopoietic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the fate-determined hematopoietic endothelial cells are CD34+CD43-CD93-. In some embodiments, the fate-determined hematopoietic endothelial cells are CD34+CD93-.

[0032] In some embodiments of the above method, the method further comprises (i) contacting pluripotent stem cell-derived fate-determined hematopoietic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator to induce differentiation of the fate-determined hematopoietic endothelium into pre-T cell progenitor cells; and optionally, (ii) 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, but not one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor to induce differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells. In some embodiments of the above method, the pluripotent stem cell-derived T cell progenitor cells are CD34+CD45+CD7+. In some embodiments of the above method, the pluripotent stem cell-derived T cell progenitor cells are CD45+CD7+.

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

[0034] Another aspect of the present invention provides a method for producing pluripotent stem cell-derived T cells, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells having the ability to differentiate into the fate-determined HE; (iii) contacting the mesodermal cells having the ability to differentiate into the fate-determined HE with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally comprising a Wnt pathway activator; and not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of mesodermal cells having the ability to differentiate into the fate-determined HE; (iv) Inducing differentiation and proliferation of hematopoietic endothelium; contacting the fate-determined hematopoietic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator to induce differentiation of the fate-determined hematopoietic endothelium into pre-T cell progenitor cells; 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, and not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor to induce differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells; optionally, the seeded pluripotent stem cells, mesoderm cells, mesoderm cells with fate-determined HE differentiation potential, and / or fate-determined hematopoietic endothelium may be exposed to a hypoxic partial pressure of approximately 2% to approximately 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 proliferate pluripotent stem cells; and / or the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs.In some embodiments, the iPSCs are naive iPSCs. In some embodiments of the method, the differentiation of the pluripotent stem cells into T cell lineages is performed without the formation of embryoid bodies and in a monolayer culture format.

[0035] A further aspect of the present invention provides a method for producing NK cell lineage cells derived from pluripotent stem cells, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of mesodermal cells from the pluripotent stem cells; and (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, to produce mesodermal cells with fate-determining HE differentiation ability from the mesodermal cells. (iii) To induce cell differentiation and proliferation; (iii) to contact mesodermal cells having fate-determined HE differentiation potential with a composition comprising one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; a ROCK inhibitor; optionally a Wnt pathway activator; and optionally not a TGFβ receptor / ALK inhibitor, thereby differentiating and proliferating pluripotent stem cell-derived fate-determined hematopoietic endothelium from the pluripotent stem cell-derived mesodermal cells having fate-determined HE differentiation potential. (iv) Inducing the differentiation of pluripotent stem cell-derived fate-determined hematopoietic endothelium into preNK cell progenitor cells by contacting the pluripotent stem cell-derived preNK cell progenitor cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator; and (v) Differentiating pluripotent stem cell-derived preNK cell progenitor cells with SCF, Flt3L, IL3, IL7, and I The method comprises contacting the pluripotent stem cell-derived pre-NK cell progenitor cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of L15, but not comprising one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors, to induce differentiation of the pluripotent stem cell-derived pre-NK cell progenitor cells into pluripotent stem cell-derived NK cell progenitor cells or NK cells; and optionally exposing seeded pluripotent stem cells, pluripotent stem cell-derived mesoderm cells, and / or fate-determined hematopoietic endothelium under a hypoxic partial pressure of about 2% to about 10%.In some embodiments, the method for producing group II pluripotent stem cell-derived NK cells further comprises contacting the iPSCs with a composition containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, and then seeding and growing the iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method for producing pluripotent stem cell-derived NK cells is embryoid-free and in a monolayer culture format.

[0036] Another aspect of the present invention provides a method for producing fate-determined hematopoietic endothelium derived from pluripotent stem cells, the method comprising: (i) contacting iPSCs with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) contacting pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of pluripotent stem cell-derived mesodermal cells having fate-determined HE differentiation potential from pluripotent stem cell-derived mesodermal cells; and (iii) The method comprises contacting pluripotent stem cell-derived mesoderm cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; a ROCK inhibitor; and optionally a Wnt pathway activator, but optionally free of a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of pluripotent stem cell-derived hematopoietic endothelium from the pluripotent stem cell-derived mesoderm cells having the ability to differentiate into fate-determined HE; and optionally exposing seeded pluripotent stem cells, pluripotent stem cell-derived mesoderm cells, and / or fate-determined hematopoietic endothelium under a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the above method for producing pluripotent stem cell-derived fate-determined hematopoietic endothelial cells further comprises contacting iPSCs with a composition containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, and then seeding and growing the iPSCs; and / or the iPSCs are naive iPSCs. In some embodiments, the iPSCs contain one or more genetic imprints, and the one or more genetic imprints contained in the iPSCs are retained in pluripotent stem cell-derived fate-determined hematopoietic endothelial cells differentiated therefrom. In some embodiments, the above method for differentiating iPSCs into fate-determined hematopoietic endothelial cells does not produce embryoid bodies and is in a monolayer culture format.

[0037] Another aspect of the present invention provides a method for producing pluripotent hematopoietic progenitor cells derived from pluripotent stem cells, the method comprising: (i) contacting iPSCs with a composition comprising a BMP activator and optionally bFGF to induce differentiation and proliferation of pluripotent stem cell-derived mesodermal cells from the iPSCs; (ii) contacting the pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of the fate-determined HE-differentiation-capable mesodermal cells from the mesodermal cells; (iii) contacting the fate-determined HE-differentiation-capable mesodermal cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, but not comprising a TGFβ receptor / ALK inhibitor, to induce differentiation and proliferation of the fate-determined HE-differentiation-capable mesodermal cells; (iv) Inducing differentiation and proliferation of fate-determined hematopoietic endothelium from mesodermal cells with E differentiation potential; (iv) Inducing differentiation of fate-determined hematopoietic endothelium into pre-HSCs by contacting the fate-determined hematopoietic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of BMP activators, ROCK inhibitors, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; and (v) Inducing differentiation of pre-HSCs into BMP-activated hematopoietic endothelium The method comprises contacting the pre-HSCs with a composition comprising a primordial agent, one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but without a ROCK inhibitor, to induce differentiation of the pre-HSCs into hematopoietic pluripotent progenitor cells; and optionally exposing the seeded pluripotent stem cells, mesoderm cells, and / or fate-determined hematopoietic endothelium under a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the above method for producing pluripotent stem cell-derived hematopoietic pluripotent progenitor cells further comprises contacting the 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 proliferate the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs.In some embodiments, the iPSC is a naive iPSC. In some embodiments, the iPSC contains one or more genetic imprints, and the one or more genetic imprints contained in the iPSC are retained in the pluripotent stem cell-derived hematopoietic pluripotent progenitor cells differentiated therefrom. In some embodiments, the differentiation of the pluripotent stem cell into hematopoietic pluripotent progenitor cells using the above method does not produce embryoid bodies and is performed in a monolayer culture format.

[0038] Further aspects of the present invention provide compositions comprising one or more cell populations produced from the culture platform disclosed herein: (i) pluripotent stem cell-derived CD34+ fate-determined hematopoietic endothelium (iCD34) having 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 being CD34+CD43-; (ii) pluripotent stem cell-derived fate-determined hematopoietic endothelium (iHE) being CD34+ and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) pluripotent stem cell-derived fate-determined HSCs being CD34+CD45+; (iv) CD34+CD4 (v) Pluripotent stem cell-derived hematopoietic pluripotent progenitor cells that are 5+; (v) Pluripotent stem cell-derived T cell progenitor cells that are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells that are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) Pluripotent stem cell-derived NK cell progenitor cells that are CD45+CD56+CD7+; (viii) Pluripotent stem cell-derived NK cells that are CD3-CD45+CD56+ and optionally further characterized by NKp46+, CD57+, and CD16+; (ix) Pluripotent stem cell-derived NKT cells that are CD45+Va24Ja18+CD3+; and (x) Pluripotent stem cell-derived B cells that are CD45+CD19+.

[0039] A further aspect of the present invention provides one or more cell lines or cloned cells produced using the methods disclosed herein: (i) pluripotent stem cell-derived CD34+ fate-determined hematopoietic endothelium (iCD34) having the ability to differentiate into pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, and NKT cells, and being CD34+CD43-; (ii) pluripotent stem cell-derived fate-determined hematopoietic endothelium (iHE) being CD34+ and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) pluripotent stem cell-derived fate-determined HSCs being CD34+CD45+; (iv) CD34+CD45+ Pluripotent stem cell-derived hematopoietic pluripotent progenitor cells (iMPP); (v) Pluripotent stem cell-derived T cell progenitor cells being CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells being CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) Pluripotent stem cell-derived NK cell progenitor cells being CD45+CD56+CD7+; (viii) Pluripotent stem cell-derived NK cells being CD3-CD45+CD56+ and optionally further characterized by NKp46+, CD57+, and CD16+; (ix) Pluripotent stem cell-derived NKT cells being CD45+Va24Ja18+CD3+; and (x) Pluripotent stem cell-derived B cells being CD45+CD19+.

[0040] Another aspect of the present invention provides a method for promoting self-renewal, reorganization, and engraftment of the hematopoietic system using one or more cell populations, cell lines, or cloned cells produced by the method of disclosure: (i) pluripotent stem cell-derived CD34+ fate-determined hematopoietic endothelium (iCD34) having the ability to differentiate into pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, and NKT cells, and being CD34+CD43-; (ii) pluripotent stem cell-derived fate-determined hematopoietic endothelium (iHE) being CD34+ and at least one of CD43-, CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) pluripotent stem cell-derived fate-determined HSCs that are CD34+CD45+; (i (v) Pluripotent stem cell-derived hematopoietic pluripotent progenitor cells that are CD34+CD45+; (v) Pluripotent stem cell-derived T cell progenitor cells that are CD34+CD45+CD7+ or CD34-CD45+CD7+; (vi) T cells that are CD45+CD3+CD4+ or CD45+CD3+CD8+; (vii) Pluripotent stem cell-derived NK cell progenitor cells that are CD45+CD56+CD7+; (viii) Pluripotent stem cell-derived NK cells that are CD3-CD45+CD56+ and optionally further characterized by NKp46+, CD57+, and CD16+; (ix) Pluripotent stem cell-derived NKT cells that are CD45+Va24Ja18+CD3+; and (x) Pluripotent stem cell-derived B cells that are CD45+CD19+.

[0041] Further aspects of the present invention provide a method for producing hematopoietic cells having enhanced therapeutic properties, the method comprising: obtaining iPSCs containing one or more genetic imprints; and inducing differentiation of the iPSCs into hematopoietic cells. The differentiation induction step further comprises: (i) obtaining mesodermal cells by contacting the pluripotent stem cells with a composition comprising a BMP pathway activator and optionally bFGF; and (ii) obtaining mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelial (HE) cells, which are capable of giving hematopoietic cells, by contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator. Preferably, the mesoderm cells and mesoderm cells having fate-determined HE differentiation potential are obtained in steps (i) and (ii) without embryoid body formation, and the resulting hematopoietic cells include fate-determined hematopoietic endothelial cells, hematopoietic stem cell / 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 cells retain the genetic imprint that was present in the iPSCs for differentiation induction.

[0042] In some embodiments, the differentiation induction step of the above method is to obtain fate-determined HE cells by contacting the mesoderm cells having the ability to differentiate into fate-determined HE cells with a composition comprising bFGF and a ROCK inhibitor; (ii) to obtain hematopoietic pluripotent progenitor cells (MPPs) by contacting the fate-determined HE cells with a composition comprising a BMP activator, optionally a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; and (iii) to obtain hematopoietic pluripotent progenitor cells (MPPs) by contacting the fate-determined HE cells with one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. (iv) to obtain pre-T cell progenitor cells, T cell progenitor cells, and / or T cells by contacting the fate-determined HE cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, and IL15, and optionally one or more BMP activators, ROCK inhibitors, VEGF, and bFGF.

[0043] One approach to obtaining iPSCs containing one or more genetic imprints may be to introduce one or more genetic imprints into iPSCs by gene editing during or after reprogramming from non-pluripotent cells, the imprints comprising one or more genetically modified modalities and introduced through intragenomic insertions, deletions, or substitutions in the genome of the iPSCs. Another approach may be to (i) introduce one or more genetic imprints into iPSCs by obtaining source-specific immune cells that are donor-specific, disease-specific, or treatment-response-specific and exhibit retrievable 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 the reprogramming of the source-specific immune cells into iPSCs. With respect to the genetically modified modalities contained in the source cells, iPSCs and / or iPSC-derived cells, they may include one or more of the following: safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or survival of iPSCs or their derived cells.

[0044] In some embodiments, the genetically modified modality is (i) deletion or reduction of the expression of B2M, TAP1, TAP2, 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 modalities include introducing or increasing the expression of surface-triggered receptors for R, CAR, TCR, or bispecific or multispecific engagers, one or more of these modalities. In some embodiments, the above-described genetically modified modalities are maintained in one or more of the following: fate-determined HE cells derived from pluripotent stem 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 engagers are specific to one or more lymphoid cell surface receptors and specific to one or more tumor-specific antigens on the surface of tumor cells. In certain embodiments, the surface-triggered receptors are universal in hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils. In certain embodiments, the universal surface-triggered receptors include an anti-epitope and a costimulatory domain, the anti-epitope being specific to the bispecific or multispecific engager. In some embodiments, the costimulatory domain includes IL2. In some embodiments, the lymphoid cell surface receptor comprises one or more of the modified modalities expressed on the surface, CD3, CD16, CD64, and CD89; while the tumor-specific antigen comprises one or more of the CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In some embodiments, the therapeutic properties of the source-specific immune cells comprises one or more of the following: (i) expression of antigen target receptors; (ii) presentation or absence of HLA; (iii) resistance to the intratumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0045] A further aspect of the present invention provides hematopoietic cells having enhanced therapeutic properties and containing the same genetic imprint that was present in the pre-derived pluripotent stem cells. In some embodiments, the genetic imprint of the pluripotent stem cells includes (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions, or substitutions in the genome of the pluripotent cells during or after reprogramming from non-pluripotent cells to iPSCs; or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor-specific, disease-specific, or therapeutic response-specific, wherein the pluripotent cells are reprogrammed from the source-specific immune cells. In some embodiments, the genetically modified modalities include one or more of the following: safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or survival of iPSCs or their derived cells.

[0046] In some embodiments, the genetically modified modality is (i) deletion or reduction of the expression of B2M, TAP1, TAP2, 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 invention includes one or more of the following: introduction or increase of the expression of surface-triggered receptors to R, CAR, TCR, or bispecific or multispecific engagers. In certain embodiments, the surface-triggered receptor is universal in hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils. In certain embodiments, the universal surface-triggered receptor comprises an anti-epitope and a costimulatory domain, the anti-epitope being specific to the bispecific or multispecific engager. In some embodiments, the costimulatory domain comprises IL2. In some further embodiments, the hematopoietic cells include source-specific immune cell therapeutic properties related to one or more of the following: (i) expression of antigen-targeted receptors; (ii) presentation or absence of HLA; (iii) resistance to the intratumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

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

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

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

[0050] In some embodiments, the co-stimulatory domain includes IL2.

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

[0052] In some embodiments, the hematopoietic cells include fate-determined hematopoietic endothelial cells, hematopoietic stem cell 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, the T cells include regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem). In certain embodiments, the NK cells include acquired NK cells.

[0053] In certain embodiments, hematopoietic cells include one or more bispecific or multispecific engagers for surface receptors. In some embodiments, the bispecific or multispecific engagers are a) hematopoietic cell type specific and specific to surface receptors including CD3, CD16, CD64, or CD89; or b) hematopoietic cell type independent and specific to the universal surface-triggered receptors.

[0054] Certain embodiments relate to methods for treating subjects in need of cell therapy, comprising administering a therapeutically sufficient number of T cell progenitor cells derived from induced pluripotent stem cell (iPSC) differentiation. In some embodiments, the subjects are (i) candidates for bone marrow transplantation or stem cell transplantation, or have previously received chemotherapy or radiotherapy; (ii) have previously received myeloablative or non-myeloablative chemotherapy or radiotherapy; (iii) have hematopoietic hyperproliferative disorders or cancers; (iv) have solid tumors; or (v) have viral infections or diseases associated with viral infections; and in vivo, the administered T cell progenitor cells activate the thymus to reconstitute T cells. In some embodiments, the method for treating a subject requiring cell therapy further comprises administering a pharmaceutical composition comprising a bispecific or multispecific engager that is a) specific to an effector cell type and specific to a surface receptor including CD3, CD16, CD64, or CD89; or b) independent of an effector cell type and specific to a universal surface-induced receptor contained in the T progenitor cells and T cells derived therefrom. In some embodiments, the genetic imprint from the source-specific immune cells comprises one or more of the following: (i) expression of antigen target receptors; (ii) presentation or absence thereof of HLA; (iii) resistance to the intratumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0055] Also provided are pharmaceutical compositions comprising a pharmaceutically acceptable culture medium and one or more hematopoietic cells having enhanced therapeutic properties prepared using the methods described herein. These hematopoietic cells having enhanced therapeutic properties include fate-determined hematopoietic endothelial cells, hematopoietic stem cell 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 include regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem). In some embodiments, the NK cells include acquired NK cells. Furthermore, therapeutic uses of the above-mentioned pharmaceutical composition are provided by introducing the composition into subjects suitable for adoptive cell therapy who have autoimmune disorders; hematopoietic malignancies; solid tumors; cancer; or infections associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0056] Another aspect of the present invention provides an antibody composition comprising one or more antibodies specific to at least one marker selected from the following: 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, Notch4, MSC, CD97, CD319, CD69, CD 338, podoplanin, CD111, CD304, CD326, CD257, CD100, CD32, CD253, CD79b, CD33, CD83, GARP, CD183, CD357, CD31, CD165, CD102, CD146, CD49c, CD13, CD58, integrin α9β1, CD51, CD10, CD202b, CD141, CD49a, CD9, CD201, CD47, CD262, CD109, CD39, CD317, CD143, integrin β5, CD105, CD155, SSEA-4, and CD93; these markers are for identifying fate-determined hematopoietic endothelium. In one embodiment, the antibody composition comprises an antibody specific to CD93; and cells bound to the CD93-specific antibody have one or more of the following phenotypes: CD34+, CD43-, CD73-, CXCR4-, and CD73-CXCR4-.

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

[0058] Furthermore, a method is provided for producing fate-determined hematopoietic endothelium derived from pluripotent stem cells, comprising culturing pluripotent stem cell-derived mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium (HE) in a medium containing a Wnt pathway agonist to obtain fate-determined HE cells. Obtaining fate-determined HE in the presence of a Wnt pathway activator increases the number and proportion of HE in the cell population; improves HE differentiation ability; and / or improves HE cellularity compared to culturing without a Wnt pathway activator. In some embodiments, the medium further comprises a ROCK inhibitor, as well as 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 naive iPSCs. In some embodiments, the iPSCs include one or more of the above genetic imprints, which are retained in the fate-determined HE derived from the pluripotent stem cells.

[0059] Certain aspects of the present invention relate to antigen-specific iPSCs or derived hematopoietic cells prepared by the method described herein. Certain aspects of the present invention relate to compositions comprising antigen-specific iPSCs or derived hematopoietic cells prepared by the method described herein. Some aspects of the present invention relate to pharmaceutical compositions comprising antigen-specific iPSCs or derived hematopoietic cells prepared by the method described herein and a pharmaceutically acceptable culture medium.

[0060] Furthermore, methods for producing antigen-specific induced pluripotent stem cells (iPSCs) and derived hematopoietic cells are provided herein, the methods comprising: (i) isolating primary antigen-specific T cells from a selected source that is donor-specific, disease-specific, or treatment response-specific; (ii) reprogramming the primary antigen-specific T cells to obtain pluripotent stem cells; and (iii) inducing differentiation of the pluripotent stem cells into hematopoietic cells. In some embodiments, the differentiation induction step comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP pathway activator and optionally bFGF to obtain mesodermal cells; and (ii) contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator to obtain mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium (HE) without embryoid body formation. In some embodiments, the isolated primary antigen-specific T cells are enriched by (i) co-culturing the primary antigen-specific T cells with tumor cells, non-transformed cells, dendritic cells, thymic epithelial cells, endothelial cells, or artificial antigen-presenting cells, particles, or peptides in plasma that express the antigen of interest; or by selecting the primary antigen-specific T cells using a T cell receptor-specific binder specific to the antigen of interest. In some embodiments, the primary antigen-specific T cells or enriched primary antigen-specific T cells can be rejuvenated by modifying them with transcription factors or small molecules.

[0061] Certain aspects of the present invention relate to antigen-specific iPSCs or derived hematopoietic cells prepared by the method described herein. Certain aspects of the present invention relate to compositions comprising antigen-specific iPSCs or derived hematopoietic cells prepared by the method described herein. Some aspects of the present invention relate to pharmaceutical compositions comprising antigen-specific iPSCs or derived hematopoietic cells prepared by the method described herein and a pharmaceutically acceptable culture medium.

[0062] In some embodiments, the antigen-specific pluripotent stem cells obtained by the above method may be further genetically modified during or after being reprogrammed to include a genetic imprint comprising one or more genetically modified modalities via intragenomic insertion, intragenomic deletion, or intragenomic substitution. In some embodiments, the genetically modified modalities may include one or more of the following: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, drug target candidates; cell surface proteins that transmit secondary or tertiary antigen specificity; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or survival of iPSCs or their derived cells. In some embodiments, the genetically modified modality is (i) deletion or reduction of the expression of B2M, TAP1, TAP2, 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 invention comprises one or more of the following: introduction or increase of the expression of surface-triggered receptors to R, CAR, TCR, or bispecific or multispecific engagers. In some embodiments, the bispecific or multispecific engager is hematopoietic cell type specific and specific to a surface receptor selected from CD3, CD16, CD64, or CD89. In some embodiments, the surface-triggered receptor is universal in hematopoietic cells, including T cells, NK cells, NKT cells, macrophages, and neutrophils. In some embodiments, the bispecific or multispecific engager is hematopoietic cell type independent and can bind to hematopoietic cells containing a compatible universal surface-triggered receptor. In some embodiments, the universal surface-triggered receptor includes an anti-epitope and a costimulatory domain, the anti-epitope being specific to an epitope contained in the bispecific or multispecific engager. In some embodiments, the co-stimulatory domain of the universal surface-triggered receptor contains all or part of IL2 for conotical or non-conotical cell activation and / or enhancement of effector cell function. In some embodiments, the bispecific or multispecific universal engager is specific to one or more tumor-specific antigens on the surface of tumor cells. In some embodiments, the tumor-specific antigens include one or more of CD19, CD20, CD30, EGFR, HER2 / ERBB2 / neu, EPCAM, EphA2, and CEA. In some embodiments, the bispecific or multispecific engager is specific to the universal surface-triggered receptor and is also specific to one or more tumor-specific antigens on the surface of tumor cells.

[0063] In some embodiments, the antigen-specific iPSC-derived hematopoietic cells include fate-determined hematopoietic endothelial cells (HE), hematopoietic stem cell / progenitor cells (HSC), hematopoietic pluripotent progenitor cells (MPP), 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.

[0064] Further aspects of the present invention provide a method for determining the clonality of induced pluripotent stem cells and their derived cells. The conventional method comprises reprogramming mature source T cells or B cells to obtain induced pluripotent stem cells (iPSCs); and detecting the presence of a specific V(D)J gene rearrangement identical to that contained in the mature T cells or B cells used to obtain the iPSCs in the iPSCs or hematopoietic cells derived therefrom. In some embodiments, the method further comprises isolating the iPSCs or hematopoietic cells containing the same V(D)J gene rearrangement as that of the mature source T cells or B cells. In some embodiments, the method comprises obtaining mature source T cells or B cells for reprogramming prior to the reprogramming of the source cells; and determining the V(D)J gene rearrangement contained in an immunoglobulin (Ig) or T cell receptor (TCR) specific to the mature source T cells or B cells.

[0065] A further aspect of the present invention provides a method for tracking adoptive cells in vivo in cell therapy, the method comprising: obtaining a blood, tissue, or tumor biopsy sample from a subject being given adoptive cells for treatment; isolating effector cells in the sample; and determining the V(D)J gene rearrangements of the effector cells; the adoptive cells being derived from pluripotent stem cells reprogrammed from mature source T cells or B cells; the mature source T cells or B cells containing a specific V(D)J gene rearrangement; and the presence of the original identical V(D)J gene rearrangement of the mature source T cells or B cells indicating adoptive cell homing, persistence, and / or proliferation.

[0066] Furthermore, a pharmaceutical composition is provided comprising a pharmaceutically acceptable culture medium and one or more of the antigen-specific hematopoietic cells produced using the above method, wherein the antigen-specific hematopoietic cells include fate-determined hematopoietic endothelial cells, hematopoietic stem cell / 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 include regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Tem). In some embodiments, the NK cells include acquired NK cells. Moreover, therapeutic uses of the pharmaceutical composition are provided by introducing the composition into subjects suitable for adoptive cell therapy who have autoimmune disorders; hematopoietic malignancies; solid tumors; cancer; or infections associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0067] In summary, the present invention provides a method and composition that enables the direct differentiation of pluripotent stem cells in a monolayer without embryoid body formation from pluripotent stem cells, thereby achieving differentiation and proliferation of mesodermal cells, fate-determined hematopoietic stem cells (HEs), and fate-determined hematopoietic stem cells (HSCs), from which other hematopoietic cells can be obtained with very high efficiency in a scalable and reliable manner. [Brief explanation of the drawing]

[0068] [Figure 1] Figure 1 shows a schematic diagram of the multi-step culture process for hematopoietic differentiation of induced pluripotent stem cells (iPSCs) into fate-determined hematopoietic endothelial cells (iHEs) and pluripotent progenitor cells (iMPPs). The culture medium can be converted to a fully defined state in which vitronectin is replaced with Matrigel®.

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

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

[0071] [Figure 4A] Figures 4A–4C show flow cytometry profiles illustrating the emergence of iHE over a 10-day period, as well as the production of iCD34 and iHE cells per iPSC differentiation. Calculations are based on snapshots of representative and unoptimized cultures. [Figure 4B] Same as above. [Figure 4C] Same as above.

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

[0073] [Figure 6A]Figures 6A and 6B show that HE on day 10 exhibits pluripotency and Notch signaling pathway-dependent secondary hematopoiesis. A) Morphological changes in the MPP assay over 7 days, accompanied by flow cytometry profiles of newly generated CD45 hematopoietic cells. B) iPSC-derived CD34+ cells generate Notch-dependent fate-determined CD45+ cells during the iMPP assay. [Figure 6B] Same as above.

[0074] [Figure 7A] Figures 7A and 7B show the effects of differentiation under hypoxic conditions on the development of iHE and iMPP hematopoietic progenitor cells. A) Monolayer differentiation under hypoxic conditions increases the proportion of both iCD34-positive cells and iHE cells on day 10. B) Day 10 iCD34+HE cells generated under hypoxic conditions can be further differentiated in the iMPP assay. [Figure 7B] Same as above.

[0075] [Figure 8-1] Figures 8A to 8D show the cryopreservation capacity and ability to maintain differentiation into the entire hematopoietic and lymphoid system of selected 10-day iCD34 cells. A) Cryopreserved 10-day iCD34 cells can survive thawing and exhibit a phenotype similar to fresh iCD34+ cells. B) Viability of selected 10-day cryopreserved CD34+ cells immediately after thawing. C) Selected 10-day cryopreserved iCD34+ cells can survive iMPP assays and produce CD45+ hematopoietic cells. D) Selected 10-day cryopreserved iCD34+ cells can survive and produce iT and iNK lymphocyte precursor cells. [Figure 8-2] Same as above.

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

[0077] [Figure 10-1] Figures 10A–10C show early CD34+CD7+ T cell progenitor cells and mature CD4+ and CD8+ T cell subsets derived from hiPSCs utilizing the CD45+CD56-gating strategy. A) Early T cell lineage markers label the presence of ipro-T cells defined by CD34+ / CD7+. B) Mature T cell markers label the presence of mature T cells defined by CD4+ or CD8+ cells. C) T cell differentiation over 5 days, comparing the differentiation potential of umbilical cord blood-derived CD34-positive cells and iCD34-positive cells to produce ipro-T cells. [Figure 10-2] Same as above.

[0078] [Figure 11A] Figures 11A–11C show early CD56+CD7+CD161+ NK cell progenitor cells and mature CD56+CD16+CD8+ NK cell subsets derived from hiPSCs utilizing a CD45+ gating strategy. A) Early NK lineage markers label the presence of ipro-NK cells defined by CD7 and CD56. B) Mature NK lineage markers label the presence of mature NK cells defined by CD57, CD16, CD94, and CD56. C) NK cell differentiation over 5 days, comparing the differentiation potential of umbilical cord blood-derived CD34-positive cells and iCD34-positive cells to produce ipro-NK cells. [Figure 11B] Same as above. [Figure 11C] Same as above.

[0079] [Figure 12A] Figures 12A–12C demonstrate that the monolayer hiPSC hematopoietic differentiation platform enables scalable proliferation strategies not seen during EB formation. A. hiPSCs were aggregated to form embryoid bodies and differentiated for 14 days, after which CD34 and CD43 expression was analyzed. B. hiPSCs were seeded in a monolayer and differentiated for 8 days, after which CD34, CD43, CXCR4, and CD73 were analyzed. C. CD34-positive cells were counted and plotted over time for both monolayer hematopoietic differentiation and EB-mediated hematopoietic differentiation. [Figure 12B] Same as above. [Figure 12C] Same as above.

[0080] [Figure 13] Figure 13 illustrates a schematic diagram of an extensible proliferation strategy for a monolayer hiPSC hematopoietic differentiation platform for off-the-shelf iNK and iT cell production. Calculations are based on snapshots of representative and unoptimized cultures.

[0081] [Figure 14] Figure 14 shows that hiPSC-derived CD34-positive cells possess immunoregulatory properties by suppressing CD3+ T cell survival.

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

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

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

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

[0086] [Figure 19] Astromal differentiation of iPSC-derived iCD34+ cells toward NK cells. Plate-bound DLL4 supports differentiation of CD56+CD7+CD161+ NK cell progenitor cells, but does not support differentiation of CD11b+ myeloid cells.

[0087] [Figure 20] Figure 20 shows the astrosomal differentiation of UCB CD34+ cells toward T cells.

[0088] [Figure 21] Figure 21 shows the astrosomal differentiation of iPSC-derived iCD34+ cells toward T cells.

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

[0090] [Figure 23]Figures 23A and 23B demonstrate that iPSC-derived NK cells secrete pro-inflammatory cytokines in response to cellular stimulation and possess cytotoxic function equivalent to that of peripheral blood NK cells and umbilical cord blood NK cells. A. iPSC-derived NK cells (iNK) or peripheral blood-derived NK cells (pbNK) were exposed to unstimulated (US) or 1:1 ratio feeder cell stimulation for 4 hours, then collected, stained for CD45, CD56, and TNF-α, and analyzed by flow cytometry. B. Cytotoxic function of iNK cells or umbilical cord blood-derived (CBNK) cells at effector cell-to-target cell ratios of 1:1, 3:1, and 10:1 was evaluated every 2 hours for 90 hours.

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

[0092] [Figure 25] Figure 25 shows the cell surface antibody screening of hiPSC-derived CD34+ cells using the CD34+CD43-gating strategy, and the workflow for hiPSC-derived CD34+ antibody screening, showing representative results for three distinct classifications. Class I markers include cell surface proteins expressed at less than 1% on hiPSC-derived CD34+ cells. Class II markers include cell surface proteins expressed at 1% to 99% on CD34+ cells. Class III markers include cell surface proteins expressed at more than 99% on CD34+ cells.

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

[0094] [Figure 27] Figure 27 shows the expression of the CD93 marker in the CD34+ population, as well as the expression of CXCR4 and CD73 in the CD34+CD93- fraction and the CD34+CD93+ fraction of CD34+ cells.

[0095] [Figure 28] Figures 28A and 28B show that the fate-determining hematopoietic differentiation potential of the CD34+ population is concentrated in the CD93- fraction. A. After 10 days of iNK cell differentiation from the CD34+CD93- population, the absolute number of CD45+ hematopoietic cells and CD45+CD7+ lymphocyte progenitor cells was evaluated. B. After another 10 days of iNK differentiation, the presence of iNK cells in the culture was evaluated by the expression of the CD56 marker and the NKp30 marker.

[0096] [Figure 29] Figures 29A and 29B show that regulation of WNT signaling improves the production of iCD34 cells. A. Cell cultures on day 6 were differentiated in the presence of the GSK3 inhibitor CHIR99021, a Wnt agonist, or the WNT inhibitor IWP2. B. CHIR99021 increased the number and proportion of CD34+CD43-CD93-HE phenotypes.

[0097] [Figure 30] Figures 30A and 30B show that regulation of WNT signaling improves the total hematopoietic and lymphoid production from iCD34 cells. A. CHIR99021 increased the production of CD45+ cells from regulated HE. B. CHIR99021 increased the production of NK progenitor cells from regulated HE.

[0098] [Figure 31] Figure 31 shows that the deletion of B2-microglobulin (B2M) in iPSCs resulted in the absence of HLA class I gene expression.

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

[0100] [Figure 33] Figure 33 shows that CD34+CD43-HE differentiated from B2M- / -iPSC remains HLA class I null.

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

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

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

[0104] [Figure 37A] Figures 37A and 37B show that iPSCs genetically modified to express high-affinity CD16 receptors and 41BBL co-stimulatory molecules retain expression throughout differentiation into iCD34 cells. A. Undifferentiated cells on day 0. B. Differentiated cells on day 10. [Figure 37B] Same as above.

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

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

[0107] [Figure 40]Figures 40A and 40B illustrate off-shelf targeting strategies for engaging T cells targeting cancer and other diseases with the entire effector repertoire. A. Illustration of iPSC-derived hematopoietic effector cells, each possessing lineage-specific inducing molecules capable of binding to engagers that recognize specific target cells. B. Illustration of modifications of universal engagers (specific and lineage-independent inducing molecules, including specific anti-epitope binding) that are ubiquitously expressed on all derived hematopoietic cells.

[0108] [Figure 41] Figure 41 shows that iPSCs genetically modified to express the HACD16 receptor retain its expression through differentiation into iNK cells on day 20. [Modes for carrying out the invention]

[0109] The present invention relates, as a whole, to methods and compositions for differentiating stem cells toward a predetermined fate to hematopoietic cells. More specifically, the present invention provides a multi-step differentiation platform in which iPSCs or iPSC-derived cells at various developmental stages can be induced to adopt a predetermined hematopoietic phenotype, ranging from predetermined hematopoietic endothelium to fully differentiated hematopoietic cells including T cells, B cells, NKT cells, and NK cells. That is, the present invention provides methods and compositions for making cells more likely to become predetermined hematopoietic fate (e.g., CD34+ predetermined hematopoietic stem cells). Alternatively, the methods and compositions of the present invention generate predetermined hematopoietic endothelium (HE) from naive iPSCs in an expandable manner by avoiding the formation of EBs or aggregates. A.Definition

[0110] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. For the purposes of this invention, the following terms are defined below: The articles “a,” “an,” and “the” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, “element (some element)” means one or more elements.

[0111] Please understand that the use of alternatives (e.g., "or") means one of the options, both, or any combination thereof.

[0112] Please understand that the terms "and / or" mean either one or both of the options.

[0113] As used herein, the terms “about” or “approximately” mean a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that differs by approximately 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “about” or “approximately” mean a range of quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% from the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0114] As used herein, the terms “substantially” or “essentially” mean a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “essentially identical” or “substantially identical” mean a range of quantities, levels, values, numbers, frequency, percentage, dimensions, size, volume, weight, or length that is substantially identical to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0115] As used herein, the terms "substantially free of" and "essentially free of" are used synonymously. When used to describe compositions such as cell populations or culture media, they refer to compositions that do not contain a particular substance or its source, for example, 95%, 96%, 97%, 98%, 99%, or are undetectable by conventional measurement of that substance or its source. The terms "free of" or "essentially free of" a particular component or substance in a composition mean that the component or substance is (1) not present in the composition at any concentration, or (2) present in the composition at a functionally inert low concentration. The same meaning applies to the term "absence of," which refers to the absence of a particular substance or its source in a composition.

[0116] As used herein, the term “appreciable” refers to a range of quantities, levels, values, numbers, frequencies, proportions, dimensions, sizes, amounts, weights, or lengths, or events, that are readily detectable by one or more standard methods. The terms “not-appreciable” and “not appreciable” and their synonyms refer to a range of quantities, levels, values, numbers, frequencies, proportions, dimensions, sizes, amounts, weights, or lengths, or events, that are not readily detectable or undetectable by standard methods. In one embodiment, an event is not appreciable if it occurs with a probability of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less.

[0117] Throughout this specification, unless the context requires a different interpretation, the terms “equipped with” and “equipped with” will be understood to mean that they encompass the steps or elements or groups of steps or elements described, but do not exclude other steps or elements or groups of steps or elements. In certain embodiments, the terms “equipped with,” “having,” “containing,” and “equipped with” are used synonymously.

[0118] The phrase "consists of..." means that everything listed before it is included and limited to it. Therefore, the phrase "consists of..." suggests that the listed elements are necessary or essential, and that other elements cannot exist.

[0119] "Essentially consisting of" means that this phrase includes all elements that are limited to the elements listed above and other elements that do not intervene in or contribute to the actions or functions identified in the disclosure to the listed elements. In other words, the phrase "essentially consisting of" requires or necessities the listed elements, but the other elements are optional and may or may not be present, depending on whether they affect the actions or functions of the listed elements.

[0120] Throughout this specification, any reference to “an embodiment,” “embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that the specific features, structures, or characteristics described in relation to that embodiment are included in at least one embodiment of the present invention. Therefore, although the above phrases appear in various places throughout this specification, they do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments in any preferred manner.

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

[0122] The term "in vivo" generally refers to activities that take place within an organism.

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

[0124] As used herein, the term “mesoderm” refers to one of the three germ layers that appear during early embryogenesis and give rise to various specialized cell types, including circulatory blood cells, muscle, heart, dermis, skeleton, and other supporting and connective tissues.

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

[0126] The term “hematopoietic stem cell” or “definitive hematopoietic stem cell” as used herein refers to CD34+ stem cells capable of giving rise to both mature myeloid cell types and mature lymphoid cell types, including T cells, natural killer cells, and B cells.

[0127] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased cell potency,” and “increased developmental potency” refer to methods of increasing the capacity of cells or dedifferentiating cells to a less differentiated state. For example, cells with increased cell potency have greater developmental plasticity (i.e., they can differentiate into more cell types) compared to the same cells in an unreprogrammed state. In other words, reprogrammed cells are cells that are less differentiated than the same cells in an unreprogrammed state.

[0128] As used herein, the term “differentiation” refers to the process by which unspecialized (“uncommitted”) or poorly specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or induced-differentiation cells are cells that have attained a more specialized (“determined”) position within the cell lineage. When applied to the process of differentiation, the term “committed” refers to cells that have progressed through the differentiation pathway to a point where, under normal circumstances, they will continue to differentiate into a particular cell type or subset of cell types, and under normal circumstances, they will not be able to differentiate into a different cell type or revert to a less differentiated cell type.

[0129] As used herein, the terms "differentiation marker gene" or "differentiation gene" refer to a gene that, through its expression, indicates that cell differentiation is occurring within a cell, such as a pluripotent stem cell. Examples of differentiation marker genes include, but are not limited to, the following: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH 11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D, FOXG1, LEFTY1, TUJ1, T gene (Brachyuri), ZIC1, GATA1, GATA2, HDAC4, HDAC5, HDAC7, HDAC9, NOTCH1, NOTCH2, NOTCH4, PAX5, RBPJ, RUNX1, STAT1 and STAT3.

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

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

[0132] As used herein, the term “pluripotency” refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryonic proper). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a range of developmental capabilities, from incomplete or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can produce a complete organism.

[0133] As used herein, the term “induced pluripotent stem cell,” or iPSC, means a stem cell produced from differentiated, mature neonatal or embryonic cells that has been induced or altered, i.e., reprogrammed, into cells capable of differentiating into tissues of all three germ layers or cortex (mesoderm, endoderm, and ectoderm). The produced iPSC does not refer to cells that exist naturally. As used herein, iPSC includes genome-modified iPSCs or iPSCs reprogrammed from immune cells of a preferred donor or patient, i.e., a distinctive genetic imprint is present in the iPSC and / or derived lymphoid effector cells.

[0134] As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferred therapeutic properties in a source cell or iPSC and is retainable in iPSCs derived from said source cell and / or hematopoietic cells derived from said iPSC. As used herein, “source cell” is a non-pluripotent cell that can be used to create iPSCs through reprogramming, and iPSCs derived from source cells can further differentiate into specific cell types encompassing any hematopoietic cell. Depending on the context, iPSCs derived from source cells and differentiated cells therefrom may be collectively referred to as “derived cells.” As used herein, genetic imprints that confer preferred therapeutic properties are incorporated into iPSCs through donor-specific, disease-specific, or treatment-response-specific reprogramming of selected source cells, or through the introduction of a mode of genetic modification into iPSCs using genome editing. In the context of source cells obtained from a specific donor, disease, or treatment situation, the genetic imprints contributing to preferred therapeutic properties may include any situation-specific genetic or epigenetic modifications that manifest a retrievable phenotype, i.e., preferred therapeutic property, inherited by derived cells of the selected source cells, regardless of whether the underlying molecular phenomena are identified. Donor-specific, disease-specific, or treatment-response-specific source cells may include retrievable genetic imprints in iPSCs and derived hematopoietic cells, but these genetic imprints may include, but are not limited to, a predetermined single-specific TCR, e.g., from virus-specific T cells or invariant natural killer T (iNKT) cells; a traceable and desirable genetic polymorphism, e.g., homozygous for a point mutation encoding a high-affinity CD16 receptor in the selected donor; and a predetermined HLA requirement, i.e., donor cells matched to the selected HLA, exhibiting a population-enhanced haplotype.As used herein, preferred therapeutic properties include improved transplantation, improved transport, improved homing, improved viability, improved self-renewal, improved persistence, improved control and regulation of the immune response, improved survival, and improved cytotoxicity of derived cells. Preferred therapeutic properties may also be associated with the expression of antigen-targeting receptors; HLA presentation or absence thereof; resistance to the intratumoral microenvironment; induction of bystander immune cells and immunomodulation; improved on-target specificity with reduced off-tumor effects; and resistance to treatments such as chemotherapy.

[0135] The term “enhanced therapeutic properties,” as used herein, refers to the enhanced therapeutic properties of a cell compared to a typical immune cell of the same normal cell type. For example, NK cells with “enhanced therapeutic properties” have enhanced, improved, and / or increased therapeutic properties compared to typical, unmodified, and / or spontaneously occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell transplantation, transport, homing, viability, self-renewal, persistence, control and regulation of immune responses, survival, and cytotoxicity. Therapeutic properties of immune cells can also be manifested by: antigen-targeting receptor expression; HLA presentation or absence; resistance to the tumor microenvironment; induction of bystander immune cells and immunomodulation; improved target specificity with reduced extratumor effects; and resistance to treatments such as chemotherapy.

[0136] As used herein, the term “engager” refers to a molecule, such as a fusion polypeptide, that is capable of ligation formation between immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils, and tumor cells, and activation of immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTE), bispecific killer cell engagers (BiKE), triplicate killer cell engagers, or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0137] As used herein, the term “surface triggering receptor” refers to a receptor capable of inducing or eliciting an immune response, such as a cytotoxic response. Surface triggering receptors may 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 the binding of bispecific or multispecific antibodies between effector cells and specific target cells (e.g., tumor cells), regardless of the innate receptors and cell type of the effector cells. Using this approach, iPSCs containing a universal surface triggering receptor may be generated and then differentiated into populations of various effector cell types expressing the universal surface triggering receptor. "Universal" means that the surface-triggered receptor can be expressed and activated in any effector cell, regardless of cell type, and that all effector cells expressing a universal receptor can be coupled to or ligated to an engager having the same epitope recognizable by the surface-triggered receptor, regardless of the engager's tumor-binding specificity. In some embodiments, an engager with the same tumor target specificity is used for coupling with a universal surface-triggered receptor. In some embodiments, an engager with different tumor target specificities is used for coupling with a universal surface-triggered receptor. Thus, by coupling one or more effector cell types, it may be possible to kill one specific type of tumor cell in some cases, and two or more types of tumors in some other cases. Surface-triggered receptors typically contain a costimulatory domain for effector cell activation and for anti-epitope specific to the engager's epitope. A bispecific engager is specific to the anti-epitope of a surface-triggered receptor at one end and specific to the tumor antigen at the other end.

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

[0139] As used herein, the term “pharmacoactive protein or peptide” refers to a protein or peptide capable of achieving biological and / or pharmaceutical effects on an organism. Pharmacoactive proteins may have a curative, therapeutic, or symptomatic nature to a disease and may be administered to improve, reduce, alleviate, reverse, or decrease the severity of the disease. Pharmacoactive proteins may also have a prophylactic nature and may be used to prevent the onset of a disease or, if the disease has already developed, to reduce the severity of such a disease or pathological condition. Pharmacoactive proteins include whole proteins or whole peptides or their pharmacoactive fragments. Pharmacoactive analogs of proteins or peptides, or analogs of protein or peptide fragments, are also included. The term pharmacoactive protein may also refer to multiple proteins or peptides that act synergistically or cooperatively to produce a therapeutic effect. Examples of pharmacokinetically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription factors and translation factors, tumor growth inhibitory proteins, antibodies or their fragments, growth factors, and / or cytokines.

[0140] As used herein, the term “signaling molecule” refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases intercellular communication. Signaling refers to the transmission of molecular signals in the form of chemical modifications, through the recruitment of protein complexes, along pathways that ultimately produce biochemical events in cells. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Toll gate signaling, ligand-opening ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0141] As used herein, the term “targeting modality” means a molecule, such as a polypeptide, that is genetically incorporated into a cell to enhance antigen specificity and / or epitope specificity, including, but not limited to, i) antigen specificity in the case of a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), ii) engager specificity in the case of a monoclonal antibody or bispecific engager, iii) targeting of transformed cells, iv) targeting of cancer stem cells, and v) other targeting strategies when there is no specific antigen or surface molecule.

[0142] As used herein, the term “pharmacokinetically active protein or peptide” refers to a protein or peptide capable of achieving 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.

[0143] As used herein, the term “signaling molecule” refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases intercellular communication. Signaling refers to the transmission of molecular signals in the form of chemical modifications, through the recruitment of protein complexes, along pathways that ultimately produce biochemical events in cells. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Toll gate signaling, ligand-opening ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0144] As used herein, the term “specific” may be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, in contrast to nonspecific or nonselective binding.

[0145] As used herein, the term “adoptive cell therapy” refers, as used herein, to cell-based immunotherapy relating to the transfusion of autologous or allogeneic lymphocytes, which are T cells or B cells, which have been grown in vitro before transfusion, and which may or may not be genetically modified.

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

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

[0148] As used herein, the term "multipotent stem cell" refers to a cell that has the developmental capacity to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, pluripotent cells may also be called "partially differentiated cells." Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells such as hematopoietic stem cells and neural stem cells. "Pluripotency" means that a cell can form many cell types in a given lineage, but not cells in other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but cannot form neurons. Therefore, the term "pluripotency" refers to a state in which the degree of developmental capacity of a cell is less than that of a totipotent or universally pluripotent cell.

[0149] Differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium or alterations in the physical state of the cells. In the most common strategy, embryoid body (EB) formation is utilized as a common and decisive intermediate to initiate lineage-specific differentiation. EBs are three-dimensional clusters that have been shown to mimic embryonic development by giving rise to numerous lineages within their three-dimensional region. Typically, through a differentiation process lasting several hours to several days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point they are further processed to continue differentiation, typically for several days to several weeks. EB formation is induced by bringing pluripotent stem cells closer together within a three-dimensional multilayer cell cluster, typically achieved by one of several methods, including settling the pluripotent cells in droplets and then settling the cells in a "U"-shaped bottom well plate, or by mechanical agitation. To promote EB development, pluripotent stem cell aggregates, maintained in pluripotent culture maintenance medium, require further differentiation cues because aggregates do not form appropriate EBs. Therefore, pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides induction cues toward the optimal lineage. EB-based pluripotent stem cell media typically result in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm) along with moderate proliferation within the EB cell aggregates. While known to promote cell differentiation, EBs produce heterogeneous cells with non-uniform differentiation states due to inconsistent cell exposure to environmental differentiation cues within a three-dimensional structure. Furthermore, EBs are cumbersome to produce and maintain. Additionally, EB-mediated cell differentiation involves moderate cell proliferation, which contributes to low differentiation efficiency.

[0150] In comparison, "aggregate formation," unlike "EB formation," can be used to proliferate cell populations derived from pluripotent stem cells. For example, during the proliferation of pluripotent stem cells based on aggregates, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of aggregates by forming larger aggregates, but cell proliferation in the culture can be maintained and the number of cells increased by periodically separating these aggregates into smaller aggregates mechanically or enzymatically. Unlike EB culture, cells cultured in aggregates in a maintenance culture medium retain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation signals to induce differentiation.

[0151] As used herein, “monolayer differentiation” refers to a differentiation method distinct from differentiation via three-dimensional multilayer cell clusters, i.e., “EB formation.” Monolayer differentiation does not require EB formation to induce differentiation, although other advantages are also disclosed herein. Since monolayer culture does not mimic embryonic development such as EB formation, differentiation toward a specific lineage is considered minimal compared to the differentiation of all three germ layers in EB.

[0152] Pluripotency can be determined in part by evaluating the pluripotent properties of cells. These properties include, but are not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers, but are 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 lines (ectoderm, mesoderm, and endoderm), (v) teratoma formation consisting of three somatic cell lines, and (vi) embryoid body formation consisting of cells from three somatic cell lines.

[0153] Two types of pluripotency were previously described: "first-stimulation" or "meta-stable" pluripotency, which is similar to the epiblast stem cells (EpiSCs) of a late-stage blastocyst, and "naive" or "basal" pluripotency, which is similar to the inner cell mass of an early / pre-implantation blastocyst. Both of these pluripotent states exhibit the characteristics described above, but the naive or basal state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and viability in single-cell culture, (iii) overall decrease in DNA methylation, (iv) decreased localization of H3K27me3 repressive chromatin labeling on developmentally regulatory gene promoters, and (v) reduced expression of differentiation markers compared to first-stimulation pluripotent cells. The standard methodology for cell reprogramming, which involves inducing exogenous pluripotent cells into somatic cells, expressing them, and then silencing or removing them from the resulting pluripotent cells, is generally considered to result in cells that retain the pluripotent characteristics of their initial state. Under standard pluripotent cell culture conditions, such cells remain in their initial state unless the expression of the exogenous transgene, which exhibits basal state characteristics, is maintained.

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

[0155] As used herein, “feeder cells” or “feeder” is a term describing a cell species that is co-cultured with a second cell species to provide an environment in which the second cell species can proliferate, by supplying growth factors and nutrients for the support of the second cell species. Feeder cells may be of a different species than the cells they support. For example, certain types of human cells, including stem cells, may be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. Feeder cells may typically be inactivated by irradiation or treatment with mitotic inhibitors such as mitomycin to prevent them from proliferating more than the cells they support when co-cultured with other cells. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell species may be a human feeder, such as human dermal fibroblasts. Another feeder cell species may be mouse embryonic fibroblasts (MEFs). Generally, by partially utilizing various feeder cells, it is possible to maintain pluripotency, direct differentiation towards a specific lineage, and promote maturation into specialized cell types such as effector cells.

[0156] As used herein, a “feeder-free (FF)” environment means a cell culture or medium environment that does not inherently contain feeder cells and / or has not been pre-conditioned by the growth of feeder cells. A “pre-conditioned” medium means a medium that has been collected after feeding cells have been grown in it for a period of time, for example, at least one day. A pre-conditioned medium contains numerous intermediary substances, including growth factors and cytokines, secreted by the feeder cells grown in the medium.

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

[0158] A "pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of cells, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of cells. Examples of pluripotency factors include transcription factors and small molecule reprogramming agents.

[0159] "Adhere" refers to cells adhering to a container, for example, cells adhering to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of a suitable culture medium. Certain cell classes will not survive or proliferate in culture unless they adhere to a cell culture vessel. Certain cell classes ("non-adherent cells") can survive and / or proliferate in culture without adhesion.

[0160] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in a tube environment. "Cell culture medium," "culture medium" (or simply "culture medium" in some cases), "supplement," and "culture medium supplement" refer to the nutrients used to cultivate cell cultures.

[0161] "Cultivate" refers to maintaining, reproducing, and / or differentiating cells that are outside of tissue or body, such as cells in a sterile plastic (or coated plastic) cell culture dish or flask. Culture media may be used as a source of nutrients, hormones, and / or other factors that help the cells reproduce and / or be maintained.

[0162] As used herein, “dissociated” cells mean cells that have been substantially separated or purified from other cells or a surface (e.g., the surface of a culture dish). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in a tube can be dissociated from each other, for example, by enzymatic or mechanical dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture dish or another surface. Therefore, dissociation may be involved in disrupting cell interactions between the extracellular matrix (ECM) and the culture substrate (e.g., the surface of the culture medium) or in disrupting the ECM between cells.

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

[0164] As used herein, the term “naive T cell” or Tn refers to a mature T cell that, unlike activated T cells or memory T cells, has not encountered its alloantigen in the periphery. Naive T cells are typically characterized by cell surface expression of L-selectin (CD62L); absence of the activation markers CD25, CD44, or CD69; and absence of the memory CD45RO isoform. Naive T cells also express a functional IL-7 receptor consisting of the IL-7 receptor-α (CD127) subunit and the common gamma chain (CD132) subunit. In the naive state, T cells are thought to be quiescent and non-dividing, requiring the common gamma chain cytokines IL-7 and IL-15 for homeostatic survival mechanisms.

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

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

[0167] As used herein, the term “NK cells” or “natural killer cells” 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). The NK cells provided herein may also be differentiated 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 whose phenotype is CD3- and CD56+, expressing NKG2C and CD57, and optionally CD16, but lacking the expression of one or more of the following (PLZF, SYK, FceRγ, and EAT-2). In some embodiments, the isolated CD56+ NK cell subpopulation includes the expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and repressing KIRs, NKG2A, and DNAM-1. CD56+ can be expressed in dim or bright conditions.

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

[0169] As used herein, the terms “B lymphocyte” or “B cell” are used synonymously and refer to a subset of lymphocytes that lack the T cell receptor (CD3) and are characterized by the expression of CD19 or CD20, which are B cell receptors (BCR, Ig) containing immunoglobulin heavy and light chains. As provided herein, B cells can also be derived from stem cells or progenitor cells through differentiation induction. B cells can be any developmental stage of B cells, encompassing all subtypes of B cells, including but not limited to pro-B cells, pre-B cells, naive B cells, B-1 B cells, B-2 B cells, marginal zone B cells, follicular B cells, memory B cells, plasmablastic cells, plasma cells, and regulatory B cells. B. Overview

[0170] The present invention relates, as a whole, to a multi-step process for differentiating naive pluripotent cells into non-pluripotent cells, or partially differentiated cells including mesodermal cells, fate-determined hematopoietic endothelial cells, fate-determined hematopoietic stem or progenitor cells, CD34+ cells, pluripotent progenitor cells (MPPs) (including neutrophil progenitor cells, capable of differentiating into myeloid cells), T cell progenitor cells, NK cell progenitor cells; or fully differentiated terminal hematopoietic cells such as T cells, B cells, NKT cells, or NK cells. The present invention also relates to compositions used in the disclosed method; and cell populations, cell lines, or cloned cells produced using the disclosed method.

[0171] Unlike methods used in the art, the present invention avoids EB formation in iPSC differentiation. As provided, hematopoietic cells derived from iPSCs are obtained by seeding clonal iPSC cells in TGFβ-free medium to maintain their pluripotent basal or naive state, differentiating the clonal iPSCs in a monolayer form without EB formation, and utilizing a stepwise strategy that applies appropriate combinations of small molecule chemicals, growth factors, and cytokines to the early and mid-stages of differentiation. Accordingly, the present invention enables the direct transfer of the proliferated clonal iPSCs to adherent culture in a monolayer form for immediate differentiation that does not require EB formation from the iPSCs.

[0172] In other words, the present invention provides a culture platform that enables highly efficient differentiation from stem cells to secondary hematopoiesis and functional hematopoietic cells without the use of TGFβ receptor / ALK inhibitors containing SB431532. Furthermore, unlike previous studies, the present invention also provides a culture platform using feeder-free, serum-free conditions that supports direct differentiation of iPSCs in monolayer culture without the need for EB or aggregates from iPSCs. C. Culture Platform

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

[0174] In certain embodiments, the feeder-free environment is essentially free of human feeder cells and is not pre-prepared with feeder cells, including but not limited to mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells. The feeder-free cell culture medium is suitable for use in pluripotent cell culture, cell reprogramming, single-cell culture of pluripotent cells, dissociation, and passage, pluripotent cell sorting, basal pluripotent cell development, maintenance of basal pluripotency, and induction of pluripotent cell differentiation. In certain embodiments, the feeder-free environment is used for inducing pluripotency, improving the efficiency of reprogramming, increasing or maintaining the differentiation potential of cells, and / or inducing differentiation. Furthermore, in certain embodiments, the feeder-free environment is substantially free of cytokines and growth factors, including bFGF.

[0175] In some embodiments of the present invention, one or more of the above iPSC differentiation steps may be performed under feeder-free conditions. Such feeder-free conditions may include, but are not limited to, monolayer culture and suspension culture. In one embodiment of the present invention, differentiation of pluripotent cells into mesodermal cells is performed under monolayer-free conditions. In another embodiment of the present invention, differentiation of mesodermal cells into fate-determined hematopoietic endothelial cells is performed under monolayer-free conditions. In yet another embodiment of the present invention, differentiation of fate-determined hematopoietic endothelial cells into hematopoietic stem cells is performed under monolayer-free conditions. In one embodiment of the present invention, differentiation of fate-determined hematopoietic stem cells into pluripotent progenitor cells, T cell progenitor cells, or NK cell progenitor cells is performed under suspension-free conditions, or under monolayer-free conditions followed by suspension-free conditions. In another embodiment of the present 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 suspension-free feeder conditions, or under monolayer-free feeder and subsequent suspension-free feeder conditions.

[0176] Any suitable container or cell culture vessel may be used as a support for cell culture in basic media and / or cell culture additives. In some embodiments, cell adhesion is promoted by coating the surface of the culture vessel with an adhesion-promoting matrix / sublayer (e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.), but in certain embodiments, the effects of the cell culture media and additives disclosed herein may be enhanced. Suitable culture media for cell culture and subculturing are known in the art and include, but are not limited to, mixtures of matrices produced by natural cell lines such as vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombospondin, Matrigel®, and synthetic or artificial surfaces such as polyamine monolayers and carboxylated monolayers. In some embodiments, preparation under feeder-free conditions includes culturing cells on a matrix-coated surface. In one embodiment, the culture platform contemplated herein includes a matrix / culture media containing Matrigel® or vitronectin. In some embodiments of the culture, Matrigel® is used, and therefore the culture is fully defined.

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

[0178] In further embodiments, one or more of the culture media of the culture platform are feeder-free environments and optionally substantially free of cytokines and / or growth factors. In other embodiments, the cell culture media contain additives such as serum, extracts, growth factors, hormones, and cytokines. Generally, the culture platform comprises one or more stage-specific feeder-free, serum-free media, each further comprising one or more of the following: nutrients / extracts, growth factors, hormones, cytokines, and medium additives. Suitable nutrients / extracts include, for example, DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), KOSR (Knockout Serum Substitute), L-glutamic acid (L-glut), and NEAA (Delegate Amino Acids), which are basic media widely used to support the growth of various mammalian cells. Other medium additives 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), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia suppressor 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 colony-stimulating factors (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), and other cytokines that act on stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines may be commercially available, for example from R&D Systems, Inc. (Minneapolis, Minnesota), and may be either naturally occurring 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-related tyrosine kinase 3 ligand (Flt3L); and one or more cytokines from leukemia suppressor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors / mitogenic factors and cytokines are stage-specific and / or cell-type-specific at concentrations determined experimentally or guided by established cytokine fields.

[0179] Generally, techniques for differentiating induced pluripotent cells involve direct or indirect modification of specific cellular pathways using polynucleotide-based approaches, polypeptide-based approaches, and / or small molecule-based approaches. The developmental potential of cells can be modified, for example, by contacting cells with one or more regulatory factors. "Contacting," as used herein, may include culturing cells in the presence of one or more factors (e.g., small molecules, proteins, peptides, etc.). In some embodiments, cell differentiation is induced by contact between cells and one or more agents. Such contact may occur, for example, by introducing the one or more agents into the cells in in vitro culture. That is, contact may occur by introducing the one or more agents into the cells in a vegetative cell culture medium. The cells may be maintained in the medium containing the one or more agents for a sufficient period of time for the cells to reach a 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 by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, vector systems having expression cassettes, or mRNA.

[0180] In other embodiments, one or more of the stage-specific feeder-free, serum-free media of the culture platform described herein further comprise one or more small molecules. In some embodiments, the culture platform comprises a cell culture medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, and does not comprise a small molecule inhibitor of the TGFβ / activin signaling pathway, comprising but not limited to a TGFβ receptor inhibitor or an ALK5 inhibitor.

[0181] The culture platform envisioned herein also offers numerous advantages by utilizing homogeneous populations of industrial-grade or clinical-grade pluripotent cells with reduced spontaneous differentiation and / or basal pluripotency. In one embodiment, the homogeneous iPSCs are maintained in a composition comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, but without a TGFβ receptor / ALK inhibitor. As used herein, the term “homogenous” refers to a population of cells in which each cell is identical or substantially identical to other cells in the population. In one embodiment, cells are identical to other cells in the population if each cell expresses one or more identical pluripotency markers envisioned herein (e.g., SSEA4 and TRA1-81). 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 its cells are identical or substantially identical to other cells in the population.

[0182] In various embodiments, the cell culture medium of the culture platform for generating hematopoietic cell lineages through fate-determined hematopoietic endothelium as described herein does not contain, or substantially does not contain, inhibitors of the TGFβ / activin signaling pathway, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In one embodiment, the culture platform includes a seeding medium for maintaining naive hiPSCs, comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor. While we do not wish to be bound by any particular theory, we have found that TGFβR / ALK5 inhibitors, while increasing the efficiency of reprogramming, interfere with the long-term maintenance, quality, and homogeneity of pluripotent cell populations. In other words, while inhibition of TGFβ pathway signaling improved the efficiency of cell reprogramming, reducing this inhibition contributes to the maintenance of the pluripotent cell population in subsequent in vitro culture systems, particularly in systems using single-cell enzymatic passage without feeder cells, where a homogeneous pluripotent population with reduced spontaneous differentiation and maintaining a “basal” or “naive” pluripotent state is preferred. As used herein, the term “long-term,” measured by passage number (but not limited to), often means at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more passages. As defined, “passaging” refers to the act of dividing and seeding cells into multiple cell culture surfaces or containers when the cells have grown to a desired extent. In addition, as disclosed herein, by culturing metastable pluripotent cells in a medium containing a GSK3 inhibitor and a MEK inhibitor, and optionally a ROCK inhibitor, but without a TGFβR / ALK5 inhibitor, transitional pluripotent cells achieve reduced spontaneous differentiation and / or basal pluripotency.

[0183] To obtain hematopoietic cells by differentiating iPSCs without forming EB intermediates, it is also important to achieve basal or naive pluripotency of the iPSCs. In addition, the differentiation efficiency of naive iPSCs into fate-determined HE cells is also greatly affected by the use of EB and its monolayer culture that does not form aggregates. In some embodiments, the culture platform comprises a medium that contains a ROCK inhibitor but does not contain, or is essentially not containing, a TGFβR / ALK5 inhibitor. In some other embodiments, the culture platform comprises a medium that contains a GSK3 inhibitor but does not contain a TGFβR / ALK5 inhibitor and promotes the development of fate-determined HE cells and / or fate-determined HSC cells by using the culture platform provided herein. 1. TGFβ receptor / ALK inhibitors

[0184] TGFβ receptor (e.g., ALK5) inhibitors may include antibodies against the TGFβ receptor (e.g., ALK5), dominant-negative variants of the TGFβ receptor (e.g., ALK5), and antisense nucleic acids that suppress the expression of the TGFβ receptor (e.g., ALK5). Examples of TGFβ receptor / ALK inhibitors include SB431542 (see, e.g., Inman, et al., Molecular Pharmacology 62(1):65-74 (2002)); A-83-01, also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (see, e.g., Tojo, et al., Cancer Science 96(11):791-800 (2005), and commercially available, e.g., from Tocris Bioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4-yl)-1,5-naphthiridine; Wnt3a / BIO (see, e.g., Dalton, et al., as incorporated herein by reference); See al., WO2008 / 094597); GW788388 (-{4-[3-(pyridine-2-yl)-1H-pyrazole-4-yl]pyridine-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-methylpyridine-2-yl)-4-(quinoxaline-6-yl)-1H-imidazole-2-yl)methyl)benzamide) (See, for example, Kim, et al., Xenobiotica See 38(3):325-339 (2008); GW6604 (2-phenyl-4-(3-pyridine-2-yl-1H-pyrazole-4-yl)pyridine) (e.g., de Gouville, et al.)See Drug News Perspective 19(2):85-90 (2006); SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazole-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, incorporated herein by reference). Furthermore, while it is not intended that nonspecific kinase inhibitors be included in the term "ALK5 inhibitor," it should be understood that "ALK5 inhibitors" include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as SB-431542 (see, for example, Inman, et al., J, Mol. Pharmacol. 62(1): 65-74 (2002)). Without intending to limit the scope of this invention, ALK5 inhibitors are thought to affect the mesenchymal epithelial transition (MET) process. The TGFβ / activin pathway is the driver of epithelial-mesenchymal transition (EMT). Therefore, inhibition of the TGFβ / activin pathway may facilitate the MET (i.e., reprogramming) process.

[0185] Data demonstrating the effects of ALK5 inhibition suggest that inhibition of the TGFβ / activin pathway produces a similar ALK5 inhibitory effect. That is, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with, or instead of, the ALK5 inhibitors described in each subsection herein. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, SMAD2 / 3 phosphorylation inhibitors, SMAD2 / 3 and SMAD4 interaction inhibitors, and SMAD6 and SMAD7 activators / agonists. Furthermore, the following classifications are for organizational purposes only, and those skilled in the art will know that a compound may affect one or more points in the pathway, and therefore a compound may function in more than two of the defined classifications.

[0186] TGFβ receptor (TGFβR) inhibitors may include antibodies against the TGFβ receptor, dominant-negative variants of the TGFβ receptor, and siRNAs or antisense nucleic acids that target the TGFβ receptor. Specific examples of TGFβ receptor inhibitors include SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazole-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192); GC-1008;ID11;AP-12009;AP-11014;LY550410;LY580 276;LY364947;LY2109761;SB-505124;SB-431542;SD-208;SM16;NPC-30345;Ki26894;SB-203580;SD-093;Gleevec;3,5,7,2',4'-pentahydroxyflavone (Morin);Activin-M108A;P144;Soluble TBR2-Fc;and antisense-transmitted tumor cells targeting the TGFβ receptor (e.g., Wrzesinski, Examples include, but are not limited to, et al., Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., Acta Biochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiers in Bioscience 12:4393-4401 (2007).

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

[0188] Inhibitors of SMAD2 / 3 and SMAD4 interactions may 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 SMAD2 / 3 and bSMAD4 interactions include, but are not limited to, Trx-SARA, Trx-xFoxH1b, and Trx-Lef1 (see, e.g., Cui, et al., Oncogene 24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell, 17:3819-3831 (2006)).

[0189] Activators / agonists of SMAD6 and SMAD7 include, but are not limited to, antibodies against dominant-negative variants of SMAD6 or SMAD7, and antibodies against antisense nucleic acids targeting SMAD6 or SMAD7. Specific examples of inhibitors include, but are not limited to, Smad7-as-PTO-oligonucleotides (see, for example, Miyazono, et al., US6534476 and Steinbrecher, et al., US2005119203, both incorporated herein by reference). 2. WNT pathway agonists

[0190] As used herein, the terms “Wnt signaling promoter,” “Wnt pathway activator,” “Wnt pathway activating agent,” or “Wnt pathway agonist” refer to agonists of the Wnt signaling pathway and include, but are not limited to, one or more agonists of Wntl, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wntl0b, Wnt11, Wnt14, Wnt15, and Wnt16. Wnt pathway agonists further include, but are not limited to, one or more of the following polypeptides or fragments: Dkk polypeptide, crescent polypeptide, cerberus polypeptide, axin polypeptide, Frzb polypeptide, T-cell factor polypeptide, or dominant negative disheveled polypeptide.

[0191] Non-limiting examples of Wnt pathway agonists further include one or more of the following: nucleic acids comprising a nucleotide sequence encoding a Wnt polypeptide; polypeptides comprising the amino acid sequence of a Wnt polypeptide; nucleic acids comprising a nucleotide sequence encoding an activated Wnt receptor; polypeptides comprising the amino acid sequence of an activated Wnt receptor; small organic molecules that promote Wnt / β-catenin signaling; small organic molecules that inhibit the expression or activity of a Wnt antagonist; antisense oligonucleotides that inhibit the expression of a Wnt antagonist; ribozymes that inhibit the expression of a Wnt antagonist; RNAi constructs, siRNAs, or shRNAs that inhibit the expression of a Wnt antagonist; antibodies that bind to a Wnt antagonist and inhibit its activity; nucleic acids comprising a nucleotide sequence encoding a β-catenin polypeptide; polypeptides comprising the amino acid sequence of a β-catenin polypeptide; nucleic acids comprising a nucleotide sequence encoding a Lef-1 polypeptide; and polypeptides comprising the amino acid sequence of a Lef-1 polypeptide.

[0192] Wnt pathway agonists further include GSK3 inhibitors, such as nucleic acids containing nucleotide sequences encoding dominant-negative GSK-3, GSK3α, or GSK3 polypeptides; polypeptides containing amino acid sequences of dominant-negative GSK-3, GSK3α, or GSK3 polypeptides; small organic molecules that bind to GSK-3, GSK3α, or GSK3 and inhibit their expression or activity; RNAi constructs, siRNAs, or shRNAs that bind to GSK-3, GSK3α, or GSK3 and inhibit their expression and / or activity; antisense oligonucleotides that bind to GSK-3, GSK3α, or GSK3 and inhibit their expression; antibodies that bind to GSK-3, GSK3α, or GSK3 and inhibit their expression and / or activity; ribozymes that bind to GSK-3, GSK3α, or GSK3 and inhibit their expression; and any GSK-3-independent reagents that activate β-catenin target genes with effects similar to GSK-3 inhibition. 3. GSK3 inhibitors

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

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

[0195] In certain exemplary embodiments, the GSK3 inhibitor is CHIR99021, BIO, or Kaempaulon.

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

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

[0198] ERK / MEK inhibitors suitable for use in the compositions intended herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ERK / MEK inhibitors intended herein may reduce the expression and / or activity of MEK or ERK. Examples of MEK / ERK inhibitors intended herein include, but are not limited to, anti-MEK antibodies or anti-ERK antibodies, dominant-negative MEK variants or ERK variants, and MEK or ERK-targeting siRNAs, shRNAs, miRNAs, and antisense nucleic acids.

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

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

[0201] Further examples of MEK / ERK inhibitors include the following compounds: 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide, 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-(tetrahydropyran-2-ylmeethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo-2 -Chlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethylethoxy)amide, 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-(tetrahydrofuran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2- Hydroxy-ethoxy)amide, 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)amide, 6-(2,4-dichlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)amide, 6-(4-bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carb (2-hydroxyethoxy)amide, hereafter 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 (hereafter referred to as MEK inhibitor 2); 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide; and pharmaceutically acceptable salts thereof.

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

[0203] Rho-related kinase (ROCK) is a serine-threonine kinase that acts as a downstream effector of Rho kinase (three isoforms exist: RhoA, RhoB, and RhoC). ROCK inhibitors suitable for use in the compositions envisioned herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ROCK inhibitors envisioned herein may reduce ROCK expression and / or ROCK activity. Examples of ROCK inhibitors envisioned herein include, but are not limited to, anti-ROCK antibodies, dominant-negative ROCK variants, ROCK-targeting siRNAs, shRNAs, miRNAs, and antisense nucleic acids.

[0204] Examples of ROCK inhibitors intended herein include, but are not limited to, thiazovibin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, as well as ROCK inhibitors disclosed in U.S. Patent No. 8,044,201, which is incorporated herein by reference in its entirety.

[0205] In one embodiment, the ROCK inhibitor is thiazovibin, Y27632, or pyrintegrin (pyrintegrin).

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

[0207] The content of these small molecules in the compositions and cell culture media intended herein can be varied and may be optimized depending on the specific molecules and combinations used, the type of cells cultured in the medium, and the specific culture conditions encompassing the specific application. In one embodiment, the small molecules are present in the composition at concentrations sufficient to induce pluripotency, improve the efficiency of reprogramming, increase or maintain the differentiation ability of cells, or induce or maintain basal pluripotency.

[0208] Another aspect of the present invention relates to Notch activators used in conjunction with the present invention. Notch encompasses all members of the Notch receptor family, including but not limited to Notch1. Notch activators include, but are not limited to, agonists of the Notch receptor. The Notch agonists bind to the Notch receptor and further induce or mediate Notch receptor-related signaling events, such as cleavage of the intracellular domain of Notch and translocation to the nucleus. Notch activators include, but are not limited to, Jag1, Jag2, DLL-1, DLL-3, and DLL-4. Notch activators include, but are not limited to, those disclosed in EP2606884, US6689744, and US5780300, whose disclosures are incorporated herein by reference. In some embodiments, one or more of the 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 for immobilizing Notch ligands can be agarose beads, magnetic beads, and latex beads. In one embodiment, the Notch ligand peptide is bound / immobilized to the beads. In another embodiment, the Notch ligand peptide is bound / immobilized to the surface of a polystyrene plate. In some embodiments, the immobilization of the Notch ligand is non-covalent. In some embodiments, the Notch ligand peptide is provided by cells.

[0209] Further aspects of the present invention relate to BMP pathway activators, including, for example, agents disclosed in the following publications, the disclosure of which is incorporated herein by reference: International Publication No. 2014011540, International Publication No. 2014062138, and International Publication No. 2005117994. Examples of BMP pathway activators used in the present invention include, but are not limited to, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In one non-limiting embodiment of the present invention, the BMP pathway activator is BMP-4. BMPs are multifunctional cytokines and members of the transforming growth factor β superfamily. Bone morphogenetic protein (BMP) receptors mediate BMP signaling through the activation of Smad. BMP ligands bind to the BMP receptors BMPRI and BMPRII. Phosphorylation of BMPRII is followed by activation of BMPRI. Phosphorylated BMPRI then phosphorylates the receptor-activating Smad protein (R-Smad), which binds to the common intermediary -Smad (co-Smad) and enters the nucleus, thereby regulating gene expression. In one embodiment, the BMP pathway activator is BMP4.

[0210] The present invention provides compositions for obtaining hematopoietic cells from iPSCs via fate-determined hematopoietic endothelial cells differentiated from iPSCs, or via fate-determined hematopoietic endothelial cells differentiated from iPSCs, each of which approaches does not result in EB formation from iPSCs for desired cell differentiation. 6. Platform for hiPSC differentiation I. iCD34 Platform

[0211] One aspect of the present invention provides a culture platform for obtaining fate-determined hematopoietic endothelium using pluripotent stem cells. As used herein, fate-determined hematopoietic endothelium is a population of hematopoietic cells oriented towards secondary hematopoiesis, having the ability to produce all hematopoietic cells, including but not limited to fate-determined HSCs, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, and / or B cells.

[0212] In one embodiment, a culture platform for obtaining fate-determined hematopoietic endothelium using pluripotent stem cells containing iPSCs comprises a seeding medium containing MEKi, GSKi, and ROCKi. In some embodiments, the seeding medium does not contain, or is essentially without, a TGFβ receptor / ALK inhibitor. In one embodiment, the combination of small molecules in the seeding medium of the present invention is shown in Table 9 as Fate Maintenance Medium (FMM). The components of the medium may be present in the medium in amounts within the concentration range shown in Table 1. In one embodiment, the iPSCs used to obtain fate-determined hematopoietic endothelium were cell lines that were generated by using Fate Reprogramming Medium (FRM) and further maintained in FMM, thereby establishing and maintaining a basal or naive state of the iPSC cell line and being suitable for stage-specific differentiation as described herein. The basal or naive iPSCs thus obtained are suitable for cryopreservation. In the present invention, a conserved iPSC cell line or clonal iPSCs can be seeded in FMM for subsequent differentiation into hematopoietic endothelial cells with determined fate. [Table 1]

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

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

[0215] One aspect of the present invention provides a medium for obtaining mesodermal cells capable of differentiating from pluripotent stem cells including iPSCs into fate-determined hemogenic endothelium. In some embodiments, the iPSC is a naive iPSC. In one embodiment, the medium comprises a BMP activator, a Wnt pathway activator, and bFGF. In one embodiment, the Wnt pathway activator is a GSK3 inhibitor. In one embodiment, the medium containing a GSK3 inhibitor is only applied after the determination of mesodermal cell differentiation, in order to acquire the ability to differentiate into fate-determined HE. In one embodiment, the medium comprising a BMP activator, a GSK3 inhibitor, and bFGF further comprises a CD34 basal medium containing small molecules in the combination shown in Table 3. In one embodiment, the aforementioned medium does not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the medium comprises an extracellular matrix protein. In other embodiments, the medium herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 11. In some embodiments, the medium is completely defined, with vitronectin replaced by Matrigel™. [Table 3]

[0216] One aspect of the present invention provides a medium for obtaining fate-determined hematopoietic endothelium from mesoderm cells. In one embodiment, the medium comprises a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In one embodiment, the medium comprises a CD34 basic medium containing VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor, as well as small molecules in the combinations shown in Table 4. In one embodiment, the medium comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11 further comprises one or more of a Wnt pathway activator, IGF, and EPO. In some embodiments, a medium for generating fate-determined HE from mesoderm cells comprises a ROCK inhibitor, a Wnt pathway activator, VEGF, bFGF, SCF, IL6, and IL11, as well as 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 medium comprising VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor does not include one or more of the Wnt pathway activator, TGFβ receptor / ALK inhibitor, IGF1, and EPO. In other embodiments, the medium as described herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 4. [Table 4]

[0217] One aspect of the present invention provides a culture platform for obtaining pluripotent progenitor cells (MPPs) from fate-determined hematopoietic endothelium. The MPPs can be further differentiated into myeloid cells comprising neutrophil progenitor cells. In one embodiment, the culture platform comprises (i) a 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, and suitable for differentiation from fate-determined hematopoietic endothelium to pre-HSCs (Table 5). In another embodiment, the culture platform comprising a medium for differentiating fate-determined hematopoietic endothelium to pre-HSCs further comprises (ii) a medium containing a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but without a ROCK inhibitor, and suitable for differentiation from pre-HSCs to pluripotent progenitor cells. In some embodiments, the ROCK inhibitor is thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium as described herein contains small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 5. [Table 5] II. iNK / iT Platform

[0218] One aspect of the present invention provides a culture platform for generating T cell progenitor cells or T cells from fate-determined hematopoietic endothelium. In one embodiment, the culture platform comprises (i) a culture medium suitable for differentiation of fate-determined hematopoietic endothelium into pre-T cell progenitor cells (pre-iproT), and / or (ii) a culture medium suitable for differentiation of the pre-T cell progenitor cells into T cell progenitor cells (pre-iproT), comprising (i) one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor (Table 6). In some embodiments, the medium for differentiating fate-determined HE cells into pre-iproT cells comprises a ROCK inhibitor, SCF, Flt3L, TPO, and IL7; but does not contain a BMP activator. In some embodiments, the medium for differentiating pre-iproT cells into T cell progenitor cells or T cells comprises SCF, Flt3L, and IL7. In some embodiments, the ROCK inhibitor is thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the medium as described herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 6. [Table 6]

[0219] One aspect of the present invention provides a culture platform for generating NK cell progenitor cells or NK cells from fate-determined hematopoietic endothelium. In one embodiment, the culture platform comprises (i) a culture medium suitable for differentiation from fate-determined hematopoietic endothelium to pre-NK cell progenitor cells (pre-iproNK), comprising (i) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator; and (ii) a culture medium suitable for differentiation of the pre-NK cell progenitor cells into NK cell progenitor cells or NK cells, comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor. In some embodiments, the medium for differentiating fate-determined HE cells into pre-iproNK cells comprises a ROCK inhibitor, SCF, Flt3L, TPO, IL3, IL7, and IL15; but does not contain a BMP activator. In some embodiments, the medium for differentiating pre-iproNK cells into NK cell progenitor cells or NK cells comprises SCF, Flt3L, and IL15. In some embodiments, the ROCK inhibitor is thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the medium as described herein comprises small molecules, growth factors, and / or cytokines in the concentration ranges shown in Table 7. [Table 7]

[0220] 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 medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not comprising, or essentially not comprising, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors, and suitable for the differentiation of pre-T cell progenitor cells into T cell progenitor cells or T cells; (ii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally comprising a BMP activator, and suitable for the differentiation of fate-determined hematopoietic endothelium into pre-T cell progenitor cells. A culture medium comprising (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, and suitable for the differentiation and proliferation of fate-determined hematopoietic endothelium from mesoderm cells; (iv) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and suitable for obtaining the ability to differentiate into fate-determined hematopoietic endothelium in mesoderm cells; (v) a culture medium comprising a BMP activator and optionally bFGF, and suitable for the development and proliferation of mesoderm cells from iPSCs; and (vi) a culture medium comprising MEKi, GSKi, and ROCKi, and not comprising or essentially not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and proliferation of naive iPSCs. In some embodiments, all of the above culture media are not comprising or essentially not comprising a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the ROCK inhibitor is thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0221] In one embodiment, the culture platform for generating T cell progenitor cells or T cells comprises (i) a medium that includes SCF, Flt3L, and IL7, and does not include, or essentially does not include, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors, and is suitable for the differentiation of pre-T cell progenitor cells into T cell progenitor cells or T cells. In another embodiment, the culture platform for generating T cell progenitor cells or T cells, comprising medium (i), further comprises (ii) a medium that includes a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator; and is suitable for the differentiation of fate-determined hematopoietic endothelium into pre-T cell progenitor cells. In another embodiment, the culture platform for generating T cell progenitor cells or T cells, comprising media (i) and (ii), further comprises (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; optionally a Wnt pathway activator, and a medium suitable for differentiation and proliferation of fate-determined hematopoietic endothelium from mesodermal cells. In yet another embodiment, the culture platform for generating T cell progenitor cells or T cells, comprising media (i), (ii), and (iii), further comprises (iv) a medium suitable for obtaining differentiation ability into fate-determined hematopoietic endothelium in mesodermal cells. In yet another embodiment, the culture platform for generating T cell progenitor cells or T cells, comprising media (i), (ii), (iii), and (iv), further comprises (v) a BMP activator and optionally bFGF, and is suitable for the development and proliferation of 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 medium comprising MEKi, GSKi, and ROCKi, and not comprising or essentially not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and proliferation of naive iPSCs. In some embodiments, all of the above media do not comprise or essentially comprise 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 thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, Notch factor is used in the culture platform for generating T cell progenitor cells or T cells. In some embodiments, Notch factors comprising Jag1, Jag2, DLL-1, DLL-3, and DLL-4 can be introduced as soluble peptides, bead-bound peptides, face-bound peptides, or cell-presented peptides.

[0222] Another aspect of the present invention provides a culture platform for obtaining NK cell progenitor cells or NK cells comprising one or more of the following: (i) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising, or essentially not comprising, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors, and suitable for the differentiation of pre-NK cell progenitor cells into NK cell progenitor cells or NK cells; (ii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally comprising a BMP activator, and for the differentiation of fate-determined hematopoietic endothelium into pre-NK cell progenitor cells. A suitable medium comprising: (iii) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; and suitable for the differentiation and proliferation of fate-determined hematopoietic endothelium from mesoderm cells; (iv) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and suitable for obtaining the ability to differentiate into fate-determined hematopoietic endothelium in mesoderm cells; (v) a medium comprising a BMP activator and optionally bFGF, and suitable for the development and proliferation of mesoderm cells from iPSCs; (vi) a medium comprising MEKi, GSKi, and ROCKi, and not comprising or essentially not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and proliferation of naive iPSCs. In some embodiments, all of the above mediums do not comprise or essentially comprise a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the ROCK inhibitor is thiazovibin 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 introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells to stimulate NK proliferation, development, and maturation.

[0223] In one embodiment, the culture platform for generating NK cell progenitor cells or NK cells comprises a medium that (i) contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and does not contain, or essentially does not contain, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors, and is suitable for differentiation of pre-NK cell progenitor cells into NK cell progenitor cells or NK cells. In some embodiments, NK maturation is performed using one or more artificial antigens introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells to stimulate NK proliferation, development, and maturation. In another embodiment, the culture platform for generating NK cell progenitor cells or NK cells, comprising medium (i), further comprises (ii) one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator; and a medium suitable for differentiation of fate-determined hematopoietic endothelium into pre-NK cell progenitor cells. In another embodiment, the culture platform for generating NK cell progenitor cells or NK cells, comprising mediums (i) and (ii), further comprises (iii) one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; and a medium suitable for differentiation and proliferation of fate-determined hematopoietic endothelium from mesodermal cells. In yet another embodiment, the culture platform for generating NK cell progenitor cells or NK cells, comprising media (i), (ii), and (iii), further comprises (iv) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and is suitable for obtaining the ability to differentiate mesodermal cells into fate-determined hematopoietic endothelium. In yet another embodiment, the culture platform for generating NK cell progenitor cells or NK cells, comprising media (i), (ii), (iii), and (iv), further comprises (v) a medium comprising a BMP activator, and optionally bFGF, and is suitable for the development and proliferation of 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 medium comprising MEKi, GSKi, and ROCKi, and not comprising or essentially not comprising a TGFβ receptor / ALK inhibitor, and suitable for seeding and proliferation of naive iPSCs. In some embodiments, all of the above media do not comprise or essentially comprise 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 thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0224] One aspect of the present invention provides a culture platform for generating fate-determined hematopoietic endothelium, the culture platform comprising one or more of the following: (i) a medium for differentiation and proliferation of fate-determined hematopoietic endothelium from mesodermal cells, comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator; (ii) a medium for obtaining differentiation ability into fate-determined hematopoietic systems in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a medium for differentiation and proliferation of mesodermal cells from naive iPSCs, comprising a BMP activator, and optionally bFGF; and (iv) a seeding and proliferation medium for naive iPSCs, comprising MEKi, GSKi, and ROCKi, wherein the seeding medium does not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the fate-determined hematopoietic endothelium is CD34+. In some embodiments, all of the above culture media do not contain, or essentially do not contain, a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0225] In one embodiment, the culture platform for obtaining fate-committed hemogenic endothelium comprises: (i) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally, a Wnt pathway activator; and a medium suitable for the differentiation and proliferation of fate-committed hemogenic endothelium from mesodermal cells. In one embodiment, the culture platform comprising said medium (i) further comprises (ii) a medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, which is suitable for obtaining differentiation potential toward the fate-committed hematopoietic lineage in mesodermal cells. In another embodiment, the culture platform comprising said media (i) and (ii) further comprises (iii) a medium comprising a BMP activator, and optionally bFGF, which is suitable for the differentiation and proliferation of mesodermal cells from naive iPSCs. In still another embodiment, the culture platform comprising said media (i), (ii) and (iii) further comprises (iv) a medium comprising MEKi, GSKi, and ROCKi, which does not or essentially does not comprise a TGFβ receptor / ALK inhibitor, and is suitable for seeding and proliferation of naive iPSCs. In some embodiments, all of the above media do not or essentially do not comprise a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0226] One aspect of the present invention provides a culture platform for generating CD34+ fate-determined hematopoietic endothelium, the culture platform comprising one or more of the following: (i) a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, thereby differentiating fate-determined hematopoietic endothelium from mesodermal cells, including CD34+ fate-determined hematopoietic endothelium. (ii) a medium for proliferation; (ii) a medium for obtaining differentiation ability into fate-determining hematopoiesis in mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor; (iii) a medium for differentiation and proliferation of mesodermal cells from naive iPSCs, comprising a BMP activator and optionally bFGF; and (iv) a seeding or proliferation medium for naive iPSCs, comprising MEKi, GSKi, and ROCKi, wherein the seeding medium does not contain or is essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, all of the above mediums do not contain or are essentially free of a TGFβ receptor / ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovibin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

[0227] One aspect of the present invention provides a culture platform for generating mesodermal cells, the culture platform comprising one or more of the following: (i) a medium for differentiation and proliferation of mesodermal cells from naive iPSCs comprising a BMP activator and optionally bFGF; and (ii) a seeding or proliferation medium for naive iPSCs comprising MEKi, GSKi, and ROCKi, wherein the seeding medium is free from or essentially free from TGFβ receptor / ALK inhibitors. In some embodiments, all of the above media are free from or essentially free from 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 thiazovibin 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 include (iii) a medium for obtaining differentiation ability into deterministic hematopoiesis in the mesodermal cells, comprising a BMP activator, bFGF, and a GSK3 inhibitor. In some embodiments, the medium comprising the BMP activator, bFGF, and a GSK3 inhibitor does not include a TGFβ receptor / ALK inhibitor. C. Methods for obtaining fate-determined hematopoietic endothelial cells, pluripotent progenitor cells, T cell progenitor cells, or NK cell progenitor cells, T cells, and / or NK cells.

[0228] The present invention provides a method for producing fate-determined hematopoietic cells derived from pluripotent stem cells using a multi-step culture platform comprising one or more culture media. The method is suitable for feeder-free conditions. The method is also suitable for monolayer culture and, therefore, does not require EB formation or aggregation intermediates for differentiation of pluripotent stem cells compared to methods known in the art. The provided method generates and simultaneously proliferates fate-determined hematopoietic endothelial cells (iHE), CD34+HE (iCD34), which are capable of further differentiation into pluripotent progenitor cells (iMPP), natural killer cell progenitor cells (ipro-NK), T cell progenitor cells (ipro-T), mature NK cells (iNK), and mature T cells (iT). Further aspects of the present invention also provide a method for producing myeloid cells differentiated from pluripotent stem cell-derived CD34+, HE, HSC, and / or MPP.

[0229] In one embodiment, the present invention provides a method for differentiating and proliferating hematopoietic cells from pluripotent stem cells in monolayer culture, the method comprising: contacting the pluripotent stem cells with a BMP pathway activator and optionally bFGF to obtain and proliferate pluripotent stem cell-derived mesoderm cells without embryoid body formation from the pluripotent stem cells; and then contacting the pluripotent stem cell-derived mesoderm cells with deterministic hematopoietic endothelial (HE) differentiation potential without embryoid body formation from the pluripotent stem cells. Subsequent contact with bFGF, and optionally a ROCK inhibitor and / or WNT pathway activator, causes the mesoderm cells with deterministic HE differentiation potential to differentiate into deterministic HE cells, which also proliferate during differentiation.

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

[0231] As detailed below, the present invention provides a method for obtaining hematopoietic cells from pluripotent stem cells through the acquisition of fate-determined hematopoietic endothelium. In particular, the present invention provides a method for inducing differentiation of hematopoietic cells from pluripotent stem cells without EB formation for differentiation. I. iCD34 Platform 1. Derivation and proliferation of fate-determining iHE

[0232] One aspect of the present invention provides a method for producing fate-determined hematopoietic endothelium (iHE) using an optimized multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are seeded and proliferated. The pluripotent stem cells are then differentiated into mesodermal cells, which proliferate during this step. The proliferated mesodermal population is then differentiated into a mesodermal population having the ability to differentiate into fate-determined hematopoietic endothelium, which is subsequently differentiated and proliferated into fate-determined hematopoietic endothelium. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The present invention further provides a method for generating and proliferating fate-determined hematopoietic endothelium (iHE), the method comprising differentiating and proliferating pluripotent stem cell-derived mesodermal cells, and, after obtaining mesodermal cells having the ability to differentiate into fate-determined iHE, differentiating these into iHE. Alternatively, the present invention provides a method for generating and proliferating fate-determined hematopoietic endothelium, the method comprising differentiating pluripotent stem cell-derived mesodermal cells having fate-determined hematopoietic endothelial cell differentiation ability into fate-determined iHE. The method disclosed herein utilizes an optimized monolayer iCD34 culture platform in which no EB is formed and which does not contain or is essentially free from TGFβ receptor / ALK inhibitors.

[0233] In one embodiment of a method for producing fate-determined hematopoietic endothelium (iHE) from pluripotent stem cells, the method comprises: (1) differentiating and proliferating a mesodermal population from pluripotent stem cells by contacting cells with a culture medium containing a BMP activator and optionally bFGF; (2) differentiating and proliferating the mesodermal population by contacting cells with a culture medium containing a BMP activator, a Wnt pathway activator and bFGF to obtain the ability to differentiate the mesodermal cells into fate-determined HE cells; and (3) differentiating and proliferating the mesodermal cells having the ability to differentiate into fate-determined HE cells by contacting cells with a culture medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained by the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, the sorting uses CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity. In some other embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD93-. In some other embodiments, the sorting uses CD34 positivity and CD93 negativity. In some embodiments, the culture medium in the above method does not contain, or is essentially without, a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, contact between the culture medium containing the GSK3 inhibitor and the cells is limited to after the differentiation determination of the mesodermal cells in order to acquire fate-determining HE differentiation ability.In some embodiments, the above method further comprises exposing the seeded iPSCs and / or mesodermal cells to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the above method further comprises seeding pluripotent stem cells by bringing the pluripotent stem cells into contact with a culture medium on which the pluripotent stem cells grow, comprising MEKi, GSKi, and ROCKi.

[0234] In one embodiment of a method for producing fate-determined hematopoietic endothelium (iHE) from seeded pluripotent stem cells, the method comprises: (1) differentiating and proliferating pluripotent stem cells into mesodermal cells by contacting pluripotent stem cells with a medium containing a BMP activator and optionally bFGF; (2) obtaining mesodermal cells having the ability to differentiate into fate-determined iHE by contacting the mesodermal cells with a medium containing a BMP activator, a Wnt pathway activator and bFGF; and (3) differentiating and proliferating fate-determined HE cells from the iHE-differentiating mesodermal cells by contacting the mesodermal cells with a medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained by the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the above method further includes sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, the sorting uses CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD93 negativity. In some embodiments, the sorting uses CD34 positivity and CD93 negativity. In some embodiments, the culture medium in the above method does not contain, or is essentially without, a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes exposing seeded iPSCs and / or mesodermal cells to a hypoxic partial pressure of about 2% to about 10%.

[0235] In one embodiment of a method for producing fate-determined hematopoietic endothelial cells (iHE) from pluripotent stem cell-derived mesodermal cells, the method comprises (1) contacting mesodermal cells with a medium containing a BMP activator, a Wnt pathway activator, and bFGF to obtain mesodermal cells having the ability to differentiate into fate-determined HE cells; and (2) contacting mesodermal cells with a medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator to differentiate and proliferate fate-determined HE cells from the mesodermal cells having the ability to differentiate into fate-determined HE cells. 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 naive 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 screening uses CD34-positive and CD43-negative cells. In some embodiments, the screening uses CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the screening uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the screening uses CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the screening uses CD34-positive and CD93-negative cells. In some embodiments, the culture medium in the above method does not contain or is essentially devoid of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes exposing the mesodermal cells to a hypoxic partial pressure of about 2% to about 10%.

[0236] In one embodiment of a method for obtaining fate-determined hematopoietic endothelial (iHE) differentiation potential in pluripotent stem cell-derived mesoderm cells, the method comprises contacting mesoderm cells with a medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator, and growing the mesoderm cells in the medium. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the medium in the above method does not contain, or is essentially without, a TGFβ receptor inhibitor. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further includes exposing the mesoderm cells to a hypoxic partial pressure of about 2% to about 10%. 2. Derivation and proliferation of pluripotent stem cell-derived mesodermal cells with the ability to differentiate into hematopoietic endothelium with a determined fate.

[0237] One aspect of the present invention provides a method for producing pluripotent stem cell-derived mesoderm cells using an optimized multi-step process. Generally, the method begins with seeding of pluripotent stem cells. The seeded pluripotent stem cells are then developed into mesoderm and further differentiated into mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived mesoderm cells, comprising differentiating seeded pluripotent stem cells into mesoderm and then differentiating the mesoderm into mesoderm cells capable of differentiating into fate-determined hematopoietic lineage. The present invention further provides a method for producing pluripotent stem cell-derived mesoderm cells capable of differentiating into fate-determined hematopoietic lineage (HE) mesoderm, the method comprising differentiating pluripotent stem cell-derived mesoderm into mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that is free from or essentially free from TGFβ receptor / ALK inhibitors.

[0238] In one embodiment of a method for obtaining the ability to differentiate into fate-determined hematopoietic endothelial cells in mesodermal cells derived from pluripotent stem cells, the method comprises (1) differentiating and proliferating mesodermal cells from pluripotent stem cells by contacting the pluripotent stem cells with a medium containing a BMP activator and optionally bFGF; and (2) obtaining the ability to differentiate into fate-determined hematopoietic endothelial cells in the mesodermal cells by contacting the cells with a 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 naive iPSCs. In some embodiments, the medium in the method does not contain or essentially does not contain a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the method further comprises exposing the pluripotent stem cells to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the above method further includes seeding pluripotent stem cells by contacting the cells with a culture medium containing MEKi, GSKi, and ROCKi.

[0239] In one embodiment of a method for producing pluripotent stem cell-derived mesoderm cells capable of differentiating from pluripotent stem cell-derived mesoderm into mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium, the method comprises differentiating the mesoderm into mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium by contacting the cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC. In some embodiments, the culture medium in the method does not contain or essentially does not contain a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the method further comprises exposing the mesoderm cells to a hypoxic partial pressure of about 2% to about 10%. 3. Derivation and proliferation of mesoderm from pluripotent stem cells

[0240] One aspect of the present invention provides a method for producing pluripotent stem cell-derived mesoderm using an optimized multi-step process. Generally, the method begins with a first step in which pluripotent stem cells are seeded, and the seeded cells are then differentiated into mesoderm in a second step. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The method disclosed herein utilizes an optimized iCD34 culture platform that does not contain, or is essentially free of, TGFβ receptor / ALK inhibitors.

[0241] In one embodiment of a method for producing pluripotent stem cell-derived mesoderm from pluripotent cells, the method comprises differentiating and growing mesodermal cells from seeded pluripotent stem cells by contacting the cells with a culture medium containing a BMP activator and optionally bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the culture medium in the method does not contain or is essentially devoid of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the method is BMP4. In some embodiments, the method further comprises exposing the seeded iPSCs to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the method further comprises seeding and growing the iPSCs by contacting the pluripotent cells with a culture medium containing MEKi, GSKi, and ROCKi. 4. Derivation of hematopoietic pluripotent progenitor cells (iMPPs)

[0242] One aspect of the present invention provides a method for producing pluripotent progenitor cells (iMPPs) derived from pluripotent stem cells using an optimized multi-step process. Generally, the method begins with seeding of pluripotent stem cells. The seeded cells are proliferated and differentiated into mesodermal cells. The mesoderm is proliferated and differentiated into mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium, and then differentiated into fate-determined hematopoietic endothelium. The fate-determined HE cells are proliferated and differentiated into pre-HSCs, and then differentiated into pluripotent progenitor cells capable of differentiating into myeloid cells containing neutrophil progenitor cells. Alternatively, the present invention provides a method for producing pluripotent progenitor cells (iMPPs) derived from pluripotent stem cells, comprising differentiating seeded pluripotent cells into mesoderm, differentiating the mesoderm into mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium, then differentiating the mesodermal cells into fate-determined iHE cells, and then differentiating them into iMPPs. The present invention further provides a method for producing pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived mesoderm into mesoderm cells capable of differentiating into deterministic hematopoietic endothelium, then differentiating the mesoderm cells into deterministic iHEs, and then differentiating them into iMPPs. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived mesoderm cells into deterministic iHEs, and then differentiating them into iMPPs. Furthermore, the present invention provides a method for producing pluripotent stem cell-derived iMPPs, comprising differentiating pluripotent stem cell-derived iHEs into iMPPs. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized monolayer iCD34 culture platform in which no EBs are formed and which do not contain or are essentially devoid of TGFβ receptor / ALK inhibitors.

[0243] In one embodiment of a method for producing hematopoietic pluripotent progenitor cells (iMPPs) from pluripotent stem cells, the method involves (1) differentiating and proliferating pluripotent stem cells into mesodermal cells by contacting pluripotent stem cells with a culture medium containing a BMP activator and optionally bFGF; (2) obtaining the ability to differentiate into fate-determined hematopoietic endothelium in mesodermal cells by contacting mesodermal cells with a culture medium containing a BMP activator, a Wnt pathway activator and bFGF; and (3) growth factors and cytokines selected from the group consisting of ROCK inhibitors, VEGF, bFGF, SCF, IL6, and IL11. The method comprises (4) differentiating the fate-determined HE cells from mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium by contacting the cells with a culture medium containing one or more of the following; and optionally a Wnt pathway activator; and differentiating the fate-determined HE cells into iMPPs by contacting the HE cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the method further comprises seeding and growing pluripotent stem cells by contacting the cells with a culture medium containing MEKi, GSKi, and ROCKi. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-HSCs by contacting the HE cells with a 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. In other embodiments, the method comprising differentiating the fate-determined HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a 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, but not containing or essentially not containing a ROCK inhibitor.In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the culture medium in the above method does not contain or is essentially devoid of a TGFβ receptor inhibitor. In some embodiments, the above method further includes exposing seeded pluripotent stem cells, mesoderm cells having the ability to differentiate into mesoderm and / or fate-determined hematopoietic endothelium under a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34-positive and CD43-negative. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In some embodiments, the screening uses CD34-positive, CD43-negative, and CD93-negative samples. In some embodiments, the screening uses CD34-positive and CD93-negative samples. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin.

[0244] In one embodiment of a method for producing pluripotent stem cell-derived pluripotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesoderm, the method involves (1) contacting mesodermal cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF to obtain differentiation ability into fate-determined hematopoietic endothelium in pluripotent stem cell-derived mesodermal cells; (2) one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator. (3) Differentiating and proliferating fate-determined HE cells from mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium by contacting the cells with a culture medium containing a activator; (4) Differentiating the fate-determined HE cells into iMPPs by contacting the HE cells with 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, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the fate-determined HE cells into pre-HSCs by contacting the HE cells 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 TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method comprising differentiating the fate-determined HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and not containing or essentially containing a ROCK inhibitor. In some embodiments, the medium in the above method does not contain or essentially contains a TGFβ receptor inhibitor.In some embodiments, the above method further includes exposing mesoderm cells having the ability to differentiate into mesoderm and / or fate-determining hematopoietic endothelium to a hypoxic partial pressure of about 2% to about 10%.

[0245] In one embodiment of a method for producing pluripotent stem cell-derived pluripotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium, the method comprises (1) differentiating and proliferating fate-determined HE cells from the pluripotent stem cell-derived mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium by contacting the cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11, a ROCK inhibitor, and optionally a Wnt pathway activator; and (2) differentiating the fate-determined HE cells into iMPPs by contacting the HE cells 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, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-HSCs by contacting the HE cells with a 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. In other embodiments, the method, which comprises differentiating the fate-determined HE cells into pre-HSCs, further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a 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, but not containing or essentially not containing a ROCK inhibitor. In some embodiments, the medium in the method does not contain or essentially does not contain a TGFβ receptor inhibitor. In some embodiments, the above method further includes exposing mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium to a hypoxic partial pressure of about 2% to about 10%.

[0246] In one embodiment of a method for producing pluripotent progenitor cells (iMPPs) derived from pluripotent stem cells (HE) cells with determined fate, the method further comprises differentiating the HE cells into iMPPs by contacting them with 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, Flt3L, and IL11, and optionally a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the 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 selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. In other embodiments, the method, which includes differentiating the fate-determined HE cells into pre-HSCs, further includes differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a 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 which does not contain or is essentially free of ROCK inhibitors. In some embodiments, the medium in the above method does not contain or is essentially free of TGFβ receptor inhibitors. In some embodiments, the above method further includes exposing mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium under a hypoxic partial pressure of about 2% to about 10%. 5. Obtaining pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells - iCD34 platform and iT platform

[0247] One aspect of the present invention provides a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells using an optimized multi-step process. Generally, the method begins with pluripotent stem cells, from which mesodermal cells are differentiated and proliferated. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The mesoderm is then differentiated into mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium. The mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium are then differentiated into fate-determined hematopoietic endothelium and simultaneously proliferated in the culture medium. The fate-determined HE cells can then be differentiated into pre-iproT, then into T cell progenitor cells (pro-T), and subsequently into T cells in the same culture medium. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells, comprising differentiating seeded pluripotent stem cells into mesoderm, differentiating the mesoderm into mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium, then differentiating the mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium into iHE, and then differentiating them into T cell progenitor cells or T cells. The present invention further provides a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium, then differentiating them into iHE, and then differentiating them into ipro-T or T cells. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived T cell progenitor cells or pluripotent stem cell-derived T cells, comprising differentiating pluripotent stem cell-derived mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium into iHE, and then differentiating these into ipro-T or T cells. Furthermore, the present invention provides a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived 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 naive iPSCs.The methods disclosed herein utilize an optimized iCD34 culture platform that is free from or essentially free from 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, face-bound peptides, or cell-presented peptides.

[0248] In one embodiment of a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells (iT) from pluripotent stem cells, the method comprises: (1) differentiating the seeded pluripotent stem cells into mesoderm by contacting the cells with a culture medium containing a BMP activator and optionally bFGF; (2) differentiating the mesoderm into mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium by contacting the cells with a culture medium containing a BMP activator, a Wnt pathway activator and bFGF; and (3) proliferation selected from the group consisting of ROCK inhibitors, VEGF, bFGF, SCF, IL6, and IL11. The method comprises (4) differentiating mesodermal cells capable of differentiating into fate-determined hematopoietic endothelial cells into fate-determined HE cells by contacting the cells with a 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 selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors, and differentiating the fate-determined HE cells into ipro-T or iT. In some embodiments, the method further comprises seeding and growing pluripotent stem cells by contacting the cells with a medium containing MEKi, GSKi, and ROCKi. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the fate-determined HE cells into pre-iproT by contacting the HE cells with a medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator.In other embodiments, the method, which includes differentiating the fate-determined HE cells into pre-iproT, further includes differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not containing, or essentially not containing, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method includes pluripotent stem cell-derived pro-T with one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, bead-bound peptides, face-bound peptides, or cell-presented peptides. In some embodiments, the method further comprises exposing seeded pluripotent stem cells, mesoderm cells having the ability to differentiate into mesoderm and / or fate-determined hematopoietic endothelium, to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the method does not contain, or is essentially without, a TGFβ receptor inhibitor.

[0249] In one embodiment of a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells from pluripotent stem cell-derived mesoderm, the method comprises: (1) differentiating the mesoderm into mesodermal cells capable of differentiating into fate-determining hematopoietic endothelium by contacting the cells with a culture medium containing a BMP activator, a Wnt pathway activator, and bFGF; (2) one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally Wnt The method comprises (3) differentiating mesodermal cells having the ability to differentiate into the fate-determined hematopoietic endothelium into fate-determined HE cells by contacting the cells with a culture medium containing a pathway activator, and differentiating the fate-determined HE cells into ipro-T or iT by contacting the 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 selected from the group consisting of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-iproT by contacting the HE cells with a culture medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator. In other embodiments, the method, which includes differentiating the fate-determined HE cells into pre-iproT, further includes differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not containing, or essentially not containing, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method includes pluripotent stem cell-derived pro-T with one or more Notch factors.In some embodiments, the Notch factor is Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides bound to beads, peptides bound to faces, or peptides presented by cells. In some embodiments, the method further comprises exposing mesodermal cells having the ability to differentiate into mesoderm and / or fate-determined hematopoietic endothelium to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the method does not contain, or is essentially without, a TGFβ receptor inhibitor.

[0250] In one embodiment of a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells (iT) from pluripotent stem cell-derived mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium, the method comprises (1) differentiating the mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium into fate-determined HE cells by contacting the cells with a culture medium containing one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator; and (2) differentiating the fate-determined HE cells into ipro-T or iT by contacting the HE cells with a culture medium containing one or more factors selected from the group consisting of a ROCK inhibitor, Flt3L, and IL7; and optionally one or more factors selected from the group consisting of a VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-iproT by contacting the HE cells with a medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator. In other embodiments, the method, which comprises differentiating the fate-determined HE cells into pre-iproT, further comprises differentiating the pre-iproT into ipro-T or iT by contacting the pre-iproT cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not containing, or essentially not containing, one or more of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the method further comprises pluripotent stem cell-derived pro-T with one or more Notch factors. In some embodiments, the Notch factor is Jag1, Jag2, DLL-1, DLL-3, or DLL-4.In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, peptides bound to beads, peptides bound to a surface, or peptides presented by cells. In some embodiments, the method further comprises exposing the mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the method express CD34. In some embodiments, the method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the method does not contain, or is essentially without, a TGFβ receptor inhibitor.

[0251] In one embodiment of a method for producing pluripotent stem cell-derived T cell progenitor cells (ipro-T) or pluripotent stem cell-derived T cells (iT) from pluripotent stem cell-derived HE cells, the method comprises differentiating the fate-determined HE cells into ipro-T or T by contacting the 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 selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-iproT by contacting the HE cells with a culture medium containing a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL7, VEGF, and bFGF; and optionally a BMP activator. In other embodiments, the method, which includes differentiating the fate-determined HE cells into pre-iproT, further includes differentiating the pre-iproT into iproT or iT by contacting the pre-iproT cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and not containing, or essentially not containing, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method includes pluripotent stem cell-derived pro-T with one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, bead-bound peptides, face-bound peptides, or cell-presented peptides. In some embodiments, the above method further includes exposing the pluripotent stem cell-derived HE cells to a hypoxic partial pressure 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 comprises sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the above method does not contain, or is essentially without, a TGFβ receptor inhibitor. 6. Obtaining pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells - iCD34 platform and iNK platform

[0252] One aspect of the present invention provides a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) using an optimized multi-step process. Generally, the method begins with pluripotent stem cells, and in some embodiments, pluripotent stem cells are seeded. The pluripotent stem cells are developed into mesodermal cells, proliferated, and then differentiated into mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium. Fate-determined hematopoietic endothelium is then differentiated and proliferated from the mesodermal cells capable of differentiating into fate-determined hematopoietic endothelium. The HE cells can differentiate into pre-iproNK and then into NK cell progenitor cells (pro-NK), and can be continuously differentiated into NK cells in the same culture medium. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells, comprising differentiating seeded pluripotent stem cells into mesoderm, differentiating the mesoderm into mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium, then differentiating the mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium into iHE, and then differentiating them into NK cell progenitor cells. The present invention further provides a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium, differentiating the mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium into iHE, and then differentiating them into ipro-NK or iNK. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived NK cell progenitor cells or iNK, comprising differentiating pluripotent stem cell-derived mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelium to iHE, and then differentiating these to ipro-NK cells or iNK. Furthermore, the present invention provides a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells, comprising differentiating pluripotent stem cell-derived iHE to ipro-NK or iNK. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is a naive iPSC.In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises NK cells being derived by contact with pro-NK cells and one or more artificial antigens introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells, for stimulating NK proliferation, development, and maturation. The methods disclosed herein utilize an optimized iCD34 culture platform that does not contain, or is essentially free of, TGFβ receptor / ALK inhibitors.

[0253] In one embodiment of a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from seeded iPSCs, the method involves (1) differentiating and proliferating pluripotent stem cells into mesodermal cells by contacting the cells with a culture medium containing a BMP activator and optionally bFGF; (2) obtaining the ability to differentiate into fate-determined hematopoietic endothelium in the mesodermal cells by contacting the mesodermal cells with a culture medium containing a BMP activator, a Wnt pathway activator and bFGF; and (3) a growth factor selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11, and (4) Differentiating and proliferating fate-determined HE cells from mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium by contacting the cells with a culture medium containing one or more itokines; and optionally a Wnt pathway activator; and differentiating the fate-determined HE cells into ipro-NK or iNK by contacting the HE 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; and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the fate-determined HE cells into pre-iproNK by contacting the HE cells 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, which includes differentiating the fate-determined HE cells into pre-iproNK, further includes differentiating the pre-iproNK into pro-iNK or iNK by contacting the pre-iproNK cells with a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising, or essentially not comprising, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises pro-NK cells obtained by contacting pro-NK cells with one or more artificial antigens for stimulating the proliferation, development, and maturation of NK, which are introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells. In one embodiment, the method further includes seeding and growing naive pluripotent cells by contacting pluripotent cells with a medium comprising MEKi, GSKi, and ROCKi. In some embodiments, the above method further includes exposing seeded iPSCs, mesoderm cells having the ability to differentiate into mesoderm and / or fate-determined hematopoietic endothelium to a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34-positive and CD43-negative cells. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the sorting uses CD34-positive and CD93-negative cells. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor.In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the above method does not contain, or is essentially without, a TGFβ receptor inhibitor.

[0254] In one embodiment of a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from pluripotent stem cell-derived mesoderm, the method comprises: (1) differentiating the mesoderm by contacting it with a medium containing a BMP activator, a Wnt pathway activator, and bFGF to obtain mesoderm cells capable of differentiating into fate-determined hematopoietic endothelium; (2) one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally the Wnt pathway The method includes (3) differentiating mesodermal cells having the ability to differentiate into the fate-determined hematopoietic endothelium by contacting cells with a culture medium containing an activator, thereby obtaining fate-determined HE cells; and differentiating the fate-determined HE cells to obtain ipro-NK or iNK by contacting HE 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, and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further includes differentiating the fate-determined HE cells to pre-iproNK by contacting HE cells 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 the fate-determined HE cells into pre-iproNK further comprises differentiating the pre-iproNK cells into iproNK or iNK by contacting the pre-iproNK cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising, or essentially not comprising, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors.In some embodiments, the culture platform for obtaining NK cell progenitor cells includes NK cells being derived by contact with pro-NK cells and one or more artificial antigens introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells, to stimulate the proliferation, development, and maturation of NK cells. In some embodiments, the above method further includes exposing mesodermal cells having the ability to differentiate into mesoderm and / or fate-determined hematopoietic endothelium under a hypoxic partial pressure of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34-positive and CD43-negative. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the sorting uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative. In some embodiments, the screening uses CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the screening uses CD34-positive and CD93-negative cells. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the method does not contain, or is essentially without, a TGFβ receptor inhibitor.

[0255] In one embodiment of a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from pluripotent stem cell-derived mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium, the method comprises (1) differentiating and proliferating fate-determined HE cells by contacting mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium with a medium containing one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, VEGF, bFGF, SCF, IL6, and IL11; and optionally a Wnt pathway activator; and (2) differentiating the fate-determined HE cells into ipro-NK or iNK by contacting the HE cells with a medium containing one or more factors selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15; and optionally VEGF, bFGF, BMP activator, and ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-iproNKs by contacting the HE cells with a 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 the fate-determined HE cells into pre-iproNKs further comprises differentiating the pre-iproNKs into iproNKs or iNKs by contacting the pre-iproNK cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, but not containing, or essentially not containing, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors.In some embodiments, the culture platform for obtaining NK cell progenitor cells includes NK cells being derived by contact with pro-NK cells and one or more artificial antigens introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells, to stimulate the proliferation, development, and maturation of NK cells. In some embodiments, the above method further includes exposing seeded pluripotent stem cells, mesoderm, and / or mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium to about 2% to about 10% hypoxic partial pressure. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further includes sorting the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34-positive and CD43-negative. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative. In some other embodiments, the screening uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the screening uses CD34-positive, CD43-negative, and CD93-negative cells. In some embodiments, the screening uses CD34-positive and CD93-negative cells. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the method does not contain, or is essentially without, a TGFβ receptor inhibitor.

[0256] In one embodiment of a method for producing pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) or pluripotent stem cell-derived NK cells (iNK) from pluripotent stem cell-derived HE cells, the method comprises differentiating the fate-determined HE cells into ipro-NK or iNK by contacting the HE 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, and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the fate-determined HE cells into pre-iproNK by contacting the HE cells 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, which includes differentiating the fate-determined HE cells into pre-iproNK, further includes differentiating the pre-iproNK into iproNK or iNK by contacting the pre-iproNK cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and not comprising, or essentially not comprising, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK cell progenitor cells comprises iproNK cells being derived by contacting iproNK cells with one or more artificial antigens for stimulating the proliferation, development, and maturation of NK, which are introduced in the form of bead conjugates, plasma membrane particles, and / or antigen-presenting cells. In some embodiments, the above method further includes exposing seeded pluripotent stem cells, mesoderm, and / or mesoderm cells having the ability to differentiate into fate-determined hematopoietic endothelium to a hypoxic partial pressure 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 the obtained iHE cells using CD34, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the sorting uses CD34-positive and CD43-negative cells. In some embodiments, the sorting uses CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting uses CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some other embodiments, the sorting uses CD34-positive, CD43-negative, and CD93-negative cells. In some other embodiments, the sorting uses CD34-positive and CD93-negative cells. In some embodiments, the BMP activator of the method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovibin. In some embodiments, the culture medium in the above method does not contain or is essentially without a TGFβ receptor inhibitor.

[0257] In light of the foregoing, one of the advantages of the culture platform intended herein is the absence of EB formation for differentiation of pluripotent stem cells and the enhanced viability and survival of single pluripotent cells during culture, passage, and dissociation. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs are genome-modified. In some embodiments, the iPSCs are reprogrammed from immune cells of a specific donor or patient. Dissociation of cells into single cells, such as a single-cell suspension, can be achieved by enzymatic or mechanical means. Any enzymatic agent known in the art for dissociating cells into single cells may be used in the method of the present invention. In one embodiment, the dissociation agent is selected from trypsin / EDTA, TrypLE-Select, collagenase IV, and dispase. When dissociating cells according to the methods intended herein, chelating agents such as EDTA, Accutase, or AccuMax may be used alone or in combination with enzymes. To promote dissociation into single cells, the dissociating agent may be dissolved in calcium and magnesium-free PBS. To enhance cell viability during and after dissociation, in some embodiments, prostibules, such as one or more growth factors, inhibitors of cellular pathways involved in cell death and apoptosis, or conditional media are added. In one embodiment, the prostibules are ROCK inhibitors, including but not limited to thiazovibin.

[0258] In some embodiments, the iPSCs for differentiation include a genetic imprint. In some embodiments, the genetic imprint of the pluripotent stem cell includes (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions, or substitutions in the genome of the pluripotent cell during or after reprogramming from a non-pluripotent cell to an iPSC; or (ii) one or more retainable therapeutic properties of a source-specific immune cell that are donor-specific, disease-specific, or treatment response-specific, wherein the pluripotent cell is reprogrammed from the source-specific immune cell, the iPSC retains the source therapeutic properties, and the source therapeutic properties are also included in hematopoietic cells derived from the iPSC. In some embodiments, the genetically modified modality includes one or more of the following: safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or survival of iPSCs or their derived cells. In some other embodiments, the genetically modified modality includes one or more of the following: (i) deletion or reduction of the expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) introduction or increase of the expression of surface-induced receptors for HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, TCR, or bispecific or multispecific engagers. In some embodiments, the surface-triggered receptor is universal, i.e., compatible with any effector cell type, and effector cells expressing this universal surface-triggered receptor can bind to the same bispecific or multispecific engager regardless of their cell type. In some embodiments, the universal surface-triggered receptor includes an anti-epitope and a costimulatory domain, and the anti-epitope is specific to the epitope of the bispecific or multispecific engager.In some embodiments, the co-stimulatory domain of the universal surface-induced receptor includes IL2 for standard or non-cononical cell activation and enhancement of effector cell function. In several other embodiments, the hematopoietic cells include source-specific immune cell therapeutic properties related to one or more of the following: (i) expression of antigen target receptors; (ii) presentation or absence of HLA; (iii) resistance to the intratumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

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

[0260] In some embodiments, the iPSC and its derived hematopoietic cells are B2M null, HLA-E / G, PDL1, A 2A These cells contain one or more of the following: R, CD47, LAG3 null, TIM3 null, TAP1 null, TAP2 null, Tapacin null, NLRC5 null, PD1 null, RFKANK null, CITTA null, RFX5 null, and RFXAP null. These cells, possessing modified HLA class I and / or modified HLA class II, exhibit improved in vivo persistence due to increased resistance to immunodetection. Furthermore, such cells avoid the HLA compatibility requirement in adoptive cell therapy, providing a source for universal, ready-made therapeutic dosing regimens.

[0261] In some embodiments, the iPSCs and their derived hematopoietic cells include one or more of HACD16, 41BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells have enhanced immunoeffector activity.

[0262] In some embodiments, the iPSCs and their derived hematopoietic cells contain surface-triggered receptors for binding to bispecific or multispecific engagers. Such cells have enhanced tumor target specificity.

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

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

[0265] In this invention, strategies for enriching cell populations with distinct characterization are provided at various stages of the method. In one embodiment, a method for enriching pluripotent stem cells from a cell population comprises preparing a single-cell suspension by dissociating the cells in the population and resuspending the cells. The dissociated cells can be resuspended in any suitable solution or medium for cell maintenance or for performing cell sorting. In a particular embodiment, the pluripotent single-cell suspension comprises a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, but does not contain a TFGβ inhibitor. In a particular embodiment, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and / or the Rock inhibitor is thiazovibin.

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

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

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

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

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

[0271] Accordingly, one aspect of the present invention provides a composition comprising one or more cell populations, cell lines, or cloned cells: (i) pluripotent stem cell-derived CD34+ HE cells (iCD34) having the ability to differentiate into pluripotent progenitor cells and being CD34+CD43-; (ii) pluripotent stem cell-derived fate-determined hematopoietic endothelial cells (iHE) being CD34+ and at least one of CD93-, CXCR4-, CD73-, and CXCR4-CD73-; (iii) CD34+CD45+ Pluripotent stem cell-derived pluripotent progenitor cells (iMPP); (v) Pluripotent stem cell-derived T cell progenitor cells (ipro-T) that are CD34+CD45+CD7+; (iv) Pluripotent stem cell-derived T cells (iT) that are CD45+CD4+CD3+ or CD45+CD8+CD3+; (vi) Pluripotent stem cell-derived NK cell progenitor cells (ipro-NK) that are CD45+CD56+CD7+CD3-; and (vii) Pluripotent stem cell-derived NK cells (iNK) that are CD45+CD56+NKp46+. In some embodiments, the above compositions, cell populations, cell lines, or cloned cells can be cryopreserved. In some embodiments, the compositions, cell populations, cell lines, or cloned cells can be stored in ambient conditions for more than 12 hours, more than 24 hours, more than 36 hours, or more than 48 hours, but not for more than 3 days, more than 4 days, more than 5 days, more than 6 days, or more than 1 week.

[0272] Another aspect of the present invention provides a mixture comprising one or more of the following: (i) pluripotent stem cell-derived CD34+ HE cells (iCD34), and one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) pluripotent stem cell-derived fate-determined hematopoietic endothelium (iHE), and one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) pluripotent stem cell-derived fate-determined HSCs, and iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2. (iv) One or more media selected from K-B2; (v) One or more media selected from pluripotent stem cell-derived pluripotent progenitor cells (iMPP) and iMPP-A; (v) One or more media selected from pluripotent stem cell-derived T cell progenitor cells (ipro-T), and iTC-A2 and iTC-B2; (vi) One or more media selected from pluripotent stem cell-derived NK cell progenitor cells (ipro-NK), and iNK-A2 and iNK-B2; and / or (viii) One or more media selected from pluripotent stem cell-derived NK cells (iNK) and iNK-B2. In some embodiments, the media: a. iCD34-C comprises one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator; but does not contain TGFβ receptor / ALK inhibitors; b. iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; c. iTC-A2 comprises a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7; and optionally, a BMP activator; d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; 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 selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally a BMP activator. f. iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15. Therapeutic applications of E. iPSC-derived immune cells

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

[0274] In some embodiments, the iPSCs for differentiation include a genetic imprint that transmits a desired therapeutic property in effector cells, and the genetic imprint is retained and functional in the post-differentiated hematopoietic cells derived from the iPSCs.

[0275] In some embodiments, the genetic imprint of the pluripotent stem cell includes (i) one or more genetically modified modalities obtained through intragenomic insertions, deletions, or substitutions in the genome of the pluripotent cell during or after reprogramming from non-pluripotent cells to iPSCs; or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor-specific, disease-specific, or treatment response-specific, wherein the pluripotent cells are reprogrammed from the source-specific immune cells, the iPSCs retain the source therapeutic properties, and the source therapeutic properties are also included in hematopoietic cells derived from the iPSCs.

[0276] In some embodiments, the genetically modified modality includes one or more of the following: safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, drug target candidates; or proteins that promote the transplantation, transport, homing, viability, self-renewal, persistence, control and regulation of immune responses, and / or survival of iPSCs or derived cells. In some other embodiments, the genetically modified modality includes one or more of the following: (i) deletion or reduction of the expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP; (ii) introduction or increase in the expression of surface-induced receptors for binding to HLA-E, HLA-G, HACD16, 41BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A2AR, CAR, TCR, or bispecific or multispecific engagers.

[0277] In several other embodiments, the hematopoietic cells include source-specific immune cell therapeutic properties related to one or more of the following: (i) expression of antigen target receptors; (ii) presentation or absence of HLA; (iii) resistance to the intratumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

[0278] In some embodiments, the iPSCs and their derived hematopoietic cells include one or more of the following: B2M null, HLA-E / G null, PDL1 null, A2AR null, CD47 null, LAG3 null, TIM3 null, TAP1 null, TAP2 null, Tapasin null, NLRC5 null, PD1 null, RFKANK null, CITTA null, RFX5 null, and RFXAP null. These cells having modified HLA class I and / or modified HLA class II exhibit improved in vivo persistence due to increased resistance to immunodetection. Furthermore, such cells avoid the HLA compatibility requirement in adoptive cell therapy, thus providing a source for universal, ready-made therapeutic dosing plans.

[0279] In some embodiments, iPSCs and their derived hematopoietic cells include one or more of HACD16, 41BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells have enhanced immunoeffector activity.

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

[0281] By introducing the immune cells of the present invention into subjects suitable for adoptive cell therapy, various diseases can be improved. Examples of diseases include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), certain juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, certain myocarditis, multiple sclerosis, pemphigus / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic erythrorhizosis, lupus erythematosus, certain thyroiditis, certain uveitis, vitiligo, and granulomatosis (Wegener) with polyangiitis; various autoimmune disorders; acute leukemia and Hematopoietic malignancies, including but not limited to diffuse leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome; solid tumors, including but not limited to tumors of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, or esophagus; and infections, including but not limited to HIV-related disorders, RSV-related disorders, EBV-related disorders, CMV-related disorders, adenovirus-related disorders, and BK polyomavirus-related disorders.

[0282] Certain embodiments of the present invention relate to methods of treating subjects in need of treatment by administering compositions comprising any of the cells described herein to the subject. In certain embodiments, the terms “to treat,” “to cure,” etc., are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. Such effect may be prophylactic in terms of complete or partial prevention of disease, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects resulting from said disease. “Cure,” as used herein, encompasses all treatment of disease in mammals, including preventing the onset of said disease in subjects susceptible to said disease but not yet diagnosed as having said disease; suppressing said disease, i.e., inhibiting its development; or mitigating said disease, i.e., causing a regression of said disease. The therapeutic agent or composition may be administered before, during, or after the onset of disease or injury. Treatment of an ongoing disease to stabilize or reduce undesirable clinical symptoms in a patient is of particular importance.

[0283] In certain embodiments, the subjects have a disease, condition, and / or injury that can be treated, remitted, and / or improved by cell therapy. In some embodiments, the subjects requiring cell therapy are subjects having a certain injury, disease, or condition, thereby enabling cell therapy (e.g., therapy in which cellular material is administered to the subjects) to treat, remit, improve, and / or reduce the severity of at least one symptom associated with the injury, disease, or condition. In certain embodiments, subjects requiring cell therapy are intended to include, but are not limited to, candidates for bone marrow or stem cell transplantation, subjects who have undergone chemotherapy or radiotherapy, subjects who have or are at risk of developing hyperproliferative disorders or cancer (e.g., hyperproliferative disorders or cancer of the hematopoietic system), subjects who have or are at risk of developing tumors (e.g., solid tumors), subjects who have or are at risk of developing viral infections or diseases associated with viral infections.

[0284] Accordingly, the present invention further provides a pharmaceutical composition comprising pluripotent stem cell-derived hematopoietic cells prepared by the methods and compositions disclosed herein, and further comprising a pharmaceutically acceptable culture medium. In one embodiment, the pharmaceutical composition comprises pluripotent stem cell-derived T cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent stem cell-derived NK cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent stem cell-derived CD34HE cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises pluripotent stem cell-derived HSC cells prepared by the methods and compositions disclosed herein.

[0285] In addition, the present invention provides therapeutic uses of the above-mentioned pharmaceutical composition by introducing the composition into subjects suitable for adoptive cell therapy who have autoimmune disorders; hematopoietic malignancies; solid tumors; or infections associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0286] The isolated pluripotent stem cell-derived hematopoietic cells may contain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% T cells, NK cells, NKT cells, CD34+HE cells, or HSCs. In some embodiments, the isolated pluripotent stem cell-derived hematopoietic cells contain 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 containing purified T cells, NK cells, NKT cells, CD34+HE cells, or HSCs, such as compositions containing an isolated population of about 95% T cells, NK cells, NKT cells, CD34+HE cells, or HSCs, for treating subjects requiring cell therapy.

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

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

[0289] As will be understood by those skilled in the art, both autologous and allogeneic immune cells can be used in cell therapy. Autologous cell therapy may reduce infection, lower the probability of GvHD, and accelerate immune reconstitution. Allogeneic cell therapy may have an immune-mediated graft-versus-tumor (GVM) effect and lower recurrence rates. Based on the specific conditions of the patient or subject requiring cell therapy, those skilled in the art can determine which particular type of therapy to administer.

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

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

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

[0293] In some embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is approximately 1 × 10⁶ 6 cells ~ approx. 3 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 5 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 10×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 20×10 6 cells, approximately 10 x 10 6 Cells ~ approx. 30 x 10 6 Cells, or approximately 20 x 10 6 Cells ~ approx. 30 x 10 6 It is a cell.

[0294] In some other embodiments, the number of derived hematopoietic cells in the pharmaceutical composition is about 1 × 10⁶ 6 Cells ~ approx. 30 x 10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 20×10 6 Cells; approx. 1.0×10 6 Cells ~ approx. 10×10 6 cells, approximately 2.0 x 10 6 Cells ~ approx. 30 x 10 6 cells, approximately 2.0 x 10 6 Cells ~ approx. 20×10 6 Cells, or approximately 2.0 × 10⁻⁶ 6 Cells ~ approx. 10×10 6 It is a cell.

[0295] In yet another embodiment, the number of derived hematopoietic cells in the pharmaceutical composition is approximately 1 × 10⁶ 6 cells, approximately 2 x 10 6 cells, approximately 5 x 10 6 cells, approximately 7 x 10 6 cells, approximately 10 x 10 6 cells, approximately 15 x 10 6cells, about 17×10 6 cells, about 20×10 6 cells, about 25×10 6 cells, or about 30×10 6 cells.

[0296] In one embodiment, the number of derived hematopoietic cells in the pharmaceutical composition is the number of immune cells in part or one umbilical cord blood, that is, at least 0.1×10 5 cells / kg body weight, at least 0.5×10 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 cells / kg body weight, at least 0.75×10 6 cells / kg body weight, at least 1×10 6 cells / kg body weight, at least 1.25×10 6 cells / kg body weight, at least 1.5×10 6 cells / kg body weight, at least 1.75×10 6 cells / kg body weight, at least 2×10 6 cells / kg body weight, at least 2.5×10 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 cells / kg body weight, or at least 30×10 6 cells / kg body weight.

[0297] The derived hematopoietic cells provided by the present invention can be administered to a subject without prior growth in ex vivo or in vitro. In certain embodiments, an isolated population of derived hematopoietic cells is modified and treated ex vivo with one or more agents for obtaining immune cells with improved therapeutic efficacy. The treatment agents can be removed by washing the modified derived hematopoietic cell population, and the improved population is administered to the patient without further in vitro growth of the population.

[0298] In other embodiments, the present invention provides an isolated population of derived hematopoietic cells that are proliferated prior to the regulation of an isolated population or subpopulation of T lymphocytes with one or more agents. The isolated population of derived hematopoietic cells can be prepared by recombination to express TCR, CAR or other proteins.

[0299] For genetically modified derived hematopoietic cells expressing recombinant TCR or CAR, whether before or after the genetic modification of the cells, said cells are, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; and It can be activated and propagated using the methods described in Patent Nos. 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0300] In certain embodiments, primary and co-stimulatory signals to derived genetically modified hematopoietic cells can be delivered by various protocols. For example, each signaling agent may be in solution or conjugated to a surface. If conjugated to a surface, the agents may be conjugated to the same surface (i.e., a "cis" structure) or to separate surfaces (i.e., a "trans" structure). Alternatively, one agent may be conjugated to a surface and the other to a solution. In one embodiment, the co-stimulatory signaling agent may be conjugated to the cell surface, and the primary activation signaling agent may be in solution or conjugated to the surface. In certain embodiments, both agents may be in solution. In another embodiment, these agents, after being made soluble, can be cross-linked to the surface of cells expressing Fc receptors or antibodies or other conjugates that bind to these agents, for example, artificial antigen-presenting cells (aAPCs) disclosed in U.S. Patent Application Publications 20040101519 and 20060034810 are intended for use in the activation and proliferation of T lymphocytes in the present invention.

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

[0302] A sterile, therapeutically acceptable composition suitable for administration to a patient may comprise one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable media, e.g., cell culture media) or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition administered and the specific method used to administer the therapeutic composition. Accordingly, a wide variety of suitable formulations of the therapeutic compositions of the present invention exist (see, for example, Remington's Pharmaceutical Sciences, 17th ed. 1985, the entire disclosure of which is incorporated herein by reference).

[0303] In certain embodiments, the therapeutic cell composition comprising an isolated population of derived hematopoietic cells also comprises a pharmaceutically acceptable cell culture medium. The therapeutic compositions comprising the derived hematopoietic cell population disclosed herein may be administered alone or in combination with other suitable compounds by enteral or parenteral administration to achieve a desired therapeutic objective.

[0304] The pharmaceutically acceptable carrier and / or diluent must be sufficiently pure and sufficiently toxic to be suitable for administration to the human subject being treated. This should further maintain or increase the stability of the therapeutic composition. The pharmaceutically acceptable carrier may be liquid or solid and is selected considering the intended mode of administration so as to provide the desired bulk, consistency, etc., when combined with the other components of the therapeutic composition of the present invention. For example, the pharmaceutically acceptable carriers may, but are not limited to, the following: binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), excipients (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), or wetting agents (e.g., sodium lauryl sulfate, etc.). Other pharmaceutically acceptable carriers suitable for the compositions of the present invention may, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0305] Such carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer refers to a solution or liquid having a chemical composition that neutralizes an acid or base without significantly changing the pH. Examples of buffers envisioned in the present invention include, but are not limited to, Dulbecco's phosphate-buffered saline (PBS), Ringer's solution, 5% glucose aqueous solution (D5W), and physiological saline (0.9% NaCl).

[0306] These pharmaceutically acceptable carriers and / or diluents may be present in amounts sufficient to maintain a pH of about 3 to about 10 of the therapeutic composition. That is, the buffer may be about 5% of the total composition on a weight-to-weight basis. Electrolytes such as sodium chloride and potassium chloride may also be included in the therapeutic composition, but are not limited to these. In one embodiment, 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 includes a buffer having a pH within one of the above pH ranges. In yet 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.

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

[0308] A suspension is a dispersion (mixture) of fine chemical species combined with other chemical species, the former of which is very finely divided and mixed and therefore does not settle rapidly. Suspensions can be prepared using a vehicle such as a liquid culture medium containing the 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 culture medium or solution (e.g., saline or serum-free medium) and not attached to a solid phase. In everyday life, the most common suspension is a suspension of solids in liquid water. Acceptable diluents that can be used (e.g., vehicles and solvents) include water, Ringer's solution, isotonic sodium chloride (saline) solution, and serum-free cell culture medium. In some embodiments, hypertonic solutions are used to prepare suspensions. In addition, sterile non-volatile oils have conventionally been used as solvents or suspension media. For parenteral administration, particularly preferred vehicles consist of a solution, preferably an oily or aqueous solution, and a suspension, emulsion, or implant. The aqueous suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. In some embodiments, the infusion is isotonic with respect to the target tissue. In some embodiments, the infusion is hypertonic with respect to the target tissue.

[0309] The pharmaceutically acceptable carriers, diluents, and other components comprising the administerable pharmaceutical composition of the present invention are obtained from US pharmaceutical-grade reagents that enable the use of the therapeutic composition in clinical therapy. Typically, these final reagents, comprising any culture medium, solution, or other pharmaceutically acceptable carrier and / or diluent, are sterilized by methods practiced in the art, such as filtration sterilization, and tested for various undesirable contaminants, such as mycoplasma contamination, endotoxin contamination, or viral contamination, before use. The pharmaceutically acceptable carriers, in one embodiment, are substantially free of human or animal-derived native proteins and are suitable for preserving cell populations of the pharmaceutical composition, including hematopoietic stem cells and progenitor cells. Since the pharmaceutical composition is intended for administration to human patients, it is substantially free of cell culture medium components such as bovine serum albumin, horse serum, and fetal bovine serum.

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

[0311] The pharmaceutical composition may include a serum-free medium suitable for preserving isolated populations of regulated derived hematopoietic cells. In various embodiments, the serum-free medium may be animal-free and optionally protein-free. Optionally, the medium may contain biopharmaceutically acceptable recombinant proteins. An animal-free medium refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace the animal proteins originally present in the animal-free medium, and these nutrients are obtained from synthetic, plant, or microbial sources. A protein-free medium, in contrast, is defined as substantially protein-free.

[0312] Those skilled in the art will understand that the above examples of culture media are illustrative and do not limit the formulation of culture media suitable for use in the present invention, and that there are many suitable culture media known and available to those skilled in the art.

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

[0314] With respect to mycoplasma and microbial contamination, “substantially free” means, as used herein, that the measured value in a generally accepted test known to those skilled in the art is negative. For example, mycoplasma contamination is determined by subculturing a sample of the therapeutic composition in broth medium with appropriate positive and negative controls and seeding it onto agar plates at 37°C on days 1, 3, 7, and 14. The appearance of the sample is compared with the appearance of the positive and negative controls under a 100× microscope. Furthermore, seedings of the indicator cell culture are incubated for 3 and 5 days and examined for the presence or absence of mycoplasma by epifluorescence microscopy using a DNA-binding fluorescent dye at 600×. A sample is considered satisfactory if the agar and / or broth medium procedure, as well as the indicator cell culture procedure, shows no evidence of mycoplasma contamination.

[0315] Organic solvents, or preferred organic solvents, generally relate to carbon-containing liquids or gases that dissolve solid, liquid, or gaseous solutes, yielding a solution. Preferred organic solvents are suitable for ex...

Claims

1. A method for obtaining fate-determined hematopoietic endothelial (HE) cells or a population thereof, comprising differentiating inducible pluripotent stem cells (iPSCs) containing one or more genetic imprints into fate-determined HE cells, (a) Differentiation induction is (i) The iPSC is contacted with a composition containing a BMP pathway activator and optionally bFGF to obtain mesodermal cells. (ii) Contact the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator to obtain mesodermal cells having the ability to differentiate into fate-determined hematopoietic endothelial cells (HE) that can provide hematopoietic cells, and (iii) A method for obtaining fate-determined HE cells by contacting cells having the ability to differentiate into fate-determined HE cells with a composition comprising bFGF and a ROCK inhibitor, (b) The mesodermal cells having the ability to differentiate into fate-determined HE cells are obtained in steps (a)(i) and (a)(ii) without forming embryoid bodies. (c) The fate-determined HE cells include the one or more genetic imprints contained in the iPSC, (d) The fate-determined HE cells can differentiate into hematopoietic cells, (e) The one or more genetic imprints include at least one deletion or reduced expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP. method.

2. The method according to claim 1, wherein the one or more genetic imprints include gene modifications that knock out one or more of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, and RFXAP.

3. The method according to claim 1, wherein the hematopoietic cells include hematopoietic stem cell / progenitor cells (HSC), hematopoietic pluripotent progenitor cells (MPP), 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.

4. The method according to claim 3, wherein the T cells include regulatory T cells (Treg), central memory T cells (Tcm), stem cell memory T cells (Tscm), and / or effector memory T cells (Temp).

5. The method according to claim 3, wherein the NK cells include acquired NK cells.

6. The method according to any one of claims 1 to 5, further comprising preparing a composition comprising the hematopoietic cells.

7. The method according to claim 6, wherein the composition comprises a pharmaceutically acceptable medium.

8. The method according to claim 1, wherein the WNT pathway activator is a GSK3 inhibitor, and optionally the GSK3 inhibitor is CHIR99021.

9. The method according to claim 8, wherein the BMP pathway activator is BMP4.

10. The method according to claim 1, wherein the fate-determined HE cells are cryopreserved, and the cryopreserved HE cells survive after thawing and retain the one or more genetic imprints.

11. The method according to claim 1, wherein the hematopoietic cells comprising the one or more genetic imprints further comprises effector cells comprising the one or more genetic imprints, the effector cells comprising one or more enhanced therapeutic properties selected from (i) expression of antigen target receptors; (ii) absence of HLA presentation; (iii) resistance to the intratumor microenvironment; (iv) induction of bystander immune cells and immunomodulation; (iv) improved target specificity with reduced extratumor effects; (v) resistance to treatments such as chemotherapy; and (vi) improved homing, persistence, and cytotoxicity.

12. The method according to claim 1, further comprising obtaining an iPSC containing one or more genetic imprints by an intragenomic insertion or deletion that deletes or reduces the expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5, or RFXAP.

Images