Methods and compositions for inducing hematopoietic cell differentiation

Abstract

In various aspects, the invention provides culture platforms, cell culture media, and methods of differentiating pluripotent cells into hematopoietic cells. In certain aspects, the invention also provides pluripotent stem cell-derived hematopoietic cells generated using the media and methods disclosed herein. The pluripotent stem cell-derived permanent HE cells produced by the methods provided herein are capable of differentiating into hematopoietic lineage cells comprising T cell progenitor cells and T cells in addition to NK cell progenitor cells, NK cells, NKT cells, or B cells.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 375,680, filed on September 14, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to compositions and methods for making cells of all hematopoietic lineages from pluripotent stem cells.In a specific aspect, the present invention relates to an improved culture platform for making cells of all hematopoietic lineages from pluripotent stem cells, including human induced pluripotent stem cells. Background Art

[0004] Human induced pluripotent stem cell (hiPSC) technology represents a highly promising and potentially unlimited source of therapeutically viable hematopoietic cells for the treatment of a variety of hematological and non-hematological malignancies, including cancer. To advance the promise of hiPSC and genome-engineered hiPSC technology as an allogeneic source for hematopoietic cell therapy, it is imperative to be able to efficiently and reproducibly generate not only hematopoietic stem and progenitor cells (HSCs), but also immune effector populations, including distinct subsets of T, B, NKT, and NK lymphoid cells, and their progenitors.

[0005] During embryonic development, there are at least two different waves of blood formation in time and space: primitive hematopoiesis and permanent hematopoiesis, which makes it complicated to derive HSC with the potential to generate lymphocytes in vitro. Primitive hematopoiesis originates from the extraembryonic yolk sac and generates a transient and limited hematopoietic pool, mainly including primitive erythroid cells and myeloid cells. The nascent HSC with the potential to generate lymphocytes only appears later during the permanent wave from the specialized endothelial progenitor cells (called permanent hemogenic endothelial cells (HE)) in the arterial vasculature. Permanent HE then undergoes endothelial-hematopoietic transformation to produce HSC, which then eventually migrates to the bone marrow, where they maintain multi-lineage hematopoiesis throughout adult life, including T, B, NKT and NK lymphocytes. Therefore, the generation of HSC and subsequent lymphoid effector cells by pluripotent stem cells depends on the ability to accurately summarize the complex stages of early embryonic hematopoietic development to permanent programs by well-designed and validated methods and compositions.

[0006] A limited number of studies have described the in vitro directed differentiation of iPSCs to permanent HE. The main obstacle to using hiPSCs for therapeutic purposes has always been the need to co-culture such cells with mouse-derived or human-derived stromal cells to maintain pluripotency and induce differentiation in the presence of an ill-defined serum-containing medium. In addition, existing protocols have also adopted a strategy consisting of 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, for example, the aggregation of pluripotent cells by rotating to form clumps, allowing cells to precipitate and aggregate in wells, or allowing passive aggregation and formation of clumps during suspension culture. The formed EBs are maintained for a certain duration (usually seven to ten days) in a differentiation induction culture system to allow appropriate differentiation, and then the EBs are transferred to adhesion culture for further maturation or dissociation into single cells for cell type selection, so as to proceed with subsequent differentiation steps. (Kennedy et al., Cell Reports 2012: 1722-1735; Knorr et al., Stem Cells Translational Medicine 2013(2): 274-283). For example, Kennedy et al. teach the generation of EBs for iPSC differentiation, wherein pluripotent cells are treated with collagenase and trypsin to allow scraping of cells to form small aggregates, which are then cultured to form EBs. EB formation has been shown to promote pluripotent stem cell differentiation, however, the requirements for forming aggregates and subsequent EBs are labor intensive, with minimal increase in cell number during the process, and the cell content in the three-dimensional EB aggregates is inconsistent and unevenly exposed to culture medium factors, which produces heterogeneous cell products at variable differentiation stages and greatly hinders the scalability and repeatability of the manufacturing process that requires efficient and streamlined. Summary of the invention

[0007] In view of the above, there is a need for methods and compositions to efficiently and reliably differentiate stem cells into definitive hematopoiesis without the need for feeder layer co-culture or reliance on serum-containing medium, and without the need to form embryoid body aggregates as intermediates. The compositions and methods disclosed herein meet this need and also provide other advantages.

[0008] In various aspects, the present invention generally relates to cell culture conditions, culture media, culture platforms, methods for culturing and differentiating stem cells into a hematopoietic cell fate, and cell populations generated thereby.

[0009] In some aspects, the present invention provides methods and compositions, including identification of new cell surface markers of permanent hemogenic endothelial (HE) cells, for generating hematopoietic cell lineages derived from pluripotent stem cells (including iPSC) under serum-free / feeder-free conditions and in a scalable monolayer culture platform that does not require EB formation. The range of cells that can be differentiated according to the methods disclosed herein is pluripotent stem cells to progenitor cells that are specialized into specific terminally differentiated cells and transdifferentiated cells, and multiple lineage cells that are directly directed to hematopoietic fate without going through pluripotent intermediates. Similarly, the range of cells produced by stem cell differentiation is pluripotent stem cells or progenitor cells to terminally differentiated stem cells, and all intermediate hematopoietic cell lineages.

[0010] In one aspect, the present invention provides a cell population comprising cells having the following phenotypes: (i) CD82 + (ii) CD34 + CD82 + and / or (iii) CD34 + CD43 - CD82 + , wherein the cells comprise permanent hemogenic endothelial (HE) cells, and wherein the cells are derived from in vitro iPSC differentiation. In some embodiments of the cell population, permanent HE cells (i) are enriched; and / or (ii) are capable of differentiating into hematopoietic lineage cells comprising T cell progenitors and T cells in addition to NK cell progenitors, NK cells, NKT cells, or B cells. In some embodiments of the cell population, the iPSC is a clonal iPSC, a single cell dissociated iPSC, an iPSC cell line cell, or an iPSC master cell bank (MCB) cell. In some embodiments, wherein the iPSC is a naive iPSC. In various embodiments of the cell population, the iPSCs further comprise one or more genetic imprints introduced into the iPSCs by genome editing during or after reprogramming of the non-pluripotent cells to iPSCs, wherein the genetic imprint comprises (i) one or more gene modification patterns introduced by genomic insertions, deletions, or substitutions in the genome of the iPSCs; or (ii) one or more retainable therapeutic properties of source-specific immune cells specific for a donor, disease, or therapeutic response, and wherein the iPSCs are reprogrammed from the source-specific immune cells; and wherein the cells comprise the same one or more genetic imprints.

[0011] In various embodiments of the cell population, the iPSC differentiation to obtain the cell population comprises: (i) differentiating the iPSC to obtain hemogenic endothelial (HE) cells, and (ii) targeting CD82 + HE cells were sorted (e.g., by using an anti-CD82 antibody) to obtain cells expressing CD82. +Permanent HE cells with cell markers of , wherein the permanent HE cells are capable of differentiating into hematopoietic lineage cells. In some embodiments of the cell population, differentiating iPSCs to obtain HE cells further comprises: (a) differentiating iPSCs to obtain mesodermal progenitor cells; and (b) differentiating mesodermal progenitor cells to obtain HE cells.

[0012] In some embodiments of the cell population, the cell markers further include CD34 + 、CD43 - ,RUNX1 + or any combination thereof (e.g., obtained by sorting using anti-CD34 antibody and / or anti-CD43 antibody), wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + In various embodiments of the cell population, the iPSC differentiation comprises contacting the iPSC with: (i) a cytokine that results in a higher percentage of RUNX1 expressing cells compared to the absence of the cytokine; and / or (ii) a small molecule p38 MAPK (mitogen activated protein kinase) inhibitor that results in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor. In some embodiments, the cytokine comprises BMP4 and / or the small molecule p38 MAPK inhibitor comprises DBM1285. In some embodiments, at least 0.5%, at least 1%, or at least 2% of CD82 + The cells are permanent HE cells. In some embodiments, the cell population is a cell population having a phenotype (e.g., CD82 + 、CD34 + CD82 + 、CD34 + CD43CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43CD82 + RUNX1 + ) of a substantially pure cell population.

[0013] In another aspect, the present invention provides a composition comprising a cell population as described herein. In some embodiments, the composition further comprises a cryopreservation medium.

[0014] In another aspect, the present invention provides a method for generating iPSC-derived permanent HE, wherein the method comprises differentiating iPSCs to obtain iPSC-derived hemogenic endothelial (HE) cells and targeting CD82 + HE cells are sorted (e.g., using antibodies comprising anti-CD82 antibodies) to obtain HE cells expressing CD82 + Permanent HE cells with cell markers of CD34+, wherein the permanent HE cells are capable of differentiating into hematopoietic lineage cells including T cell progenitor cells and T cells in addition to NK progenitor cells, NK cells, NKT cells or B cells. In various embodiments of the method, the sorting further comprises sorting for the following cells: CD34+ + 、CD43 - ,RUNX1 + or any combination thereof (e.g., obtained by sorting using anti-CD34 antibody and / or anti-CD43 antibody), wherein the obtained permanent HE cells contain CD34 + CD82 + or CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + In various embodiments of the method, the method further comprises: (i) contacting the iPSC with a medium comprising a BMP activator and bFGF, thereby differentiating the iPSC to obtain mesodermal progenitor cells; and (ii) contacting the mesodermal progenitor cells with a medium comprising a BMP activator, bFGF, VEGF, a Wnt pathway activator, and an optional p38 MAPK inhibitor, thereby differentiating the mesodermal progenitor cells to obtain HE cells. In various embodiments of the method, contact with a p38 MAPK inhibitor can increase the maintenance of CD82 expression in HE cells compared to the absence of a p38 MAPK inhibitor; the BMP activator comprises BMP4; and / or the Wnt pathway activator comprises a GSK3 inhibitor. In some embodiments, the p38 MAPK inhibitor comprises DBM1285; and / or the GSK3 inhibitor comprises CHIR99021.

[0015] In some embodiments of the method for generating iPSC-derived permanent HE, the iPSC comprises a naive iPSC, and / or is derived from an iPSC comprising one or more genetic imprints. In some embodiments, one or more genetic imprints contained in the iPSC are retained in the iPSC-derived permanent HE cells. In various embodiments, the method further comprises cryopreserving the permanent HE cells.

[0016] In another aspect, the present invention provides a composition for generating iPSC-derived permanent HE (hemogenic endothelial) cells, the composition comprising: a BMP activator, bFGF, VEGF, a Wnt pathway activator, and an optional p38 MAPK inhibitor. In various embodiments of the composition, (i) the composition does not contain a TGFβ receptor / ALK inhibitor; (ii) compared with differentiation without a BMP activator, the generated iPSC-derived permanent HE comprises increased RUNX1-expressing cells; and / or (iii) compared with differentiation without a p38 MAPK inhibitor, the generated iPSC-derived permanent HE comprises increased CD82-expressing cells. In some embodiments of the composition, the BMP activator comprises BMP4; and / or the p38 MAPK inhibitor comprises at least one of DBM1285, VX-745, VX-702, RO-4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RV568, CAS219138-24-6, SB203580, and SB242235. In some embodiments of the composition, the p38 MAPK inhibitor comprises DBM1285. In some embodiments, the composition further comprises iPSCs, mesodermal cells, or permanent HE cells.

[0017] In another aspect, the present invention provides a method for generating iPSC-derived permanent HE, the method comprising: (i) differentiating iPSCs to obtain mesodermal progenitor cells; (ii) differentiating mesodermal progenitor cells to obtain HE cells; and (iii) targeting CD82 + HE cells were sorted (e.g., by using an anti-CD82 antibody) to obtain cells expressing CD82. + Permanent hemogenic endothelial (HE) cells with cell markers of CD34+, wherein these permanent HE cells are capable of differentiating into hematopoietic lineage cells including T cell progenitors and T cells in addition to NK cell progenitors, NK cells, NKT cells or B cells. In various embodiments of the method, the sorting further comprises sorting for the following cells: CD34+ + 、CD43 - ,RUNX1+ or any combination thereof (e.g., obtained by sorting using anti-CD34 antibody and / or anti-CD43 antibody), and wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + In some embodiments of the method, the step (ii) of differentiating the mesodermal progenitor cells into HE comprises contacting the mesodermal progenitor cells with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor that results in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor. In some embodiments, the cytokine comprises BMP4 and / or the small molecule p38 MAPK inhibitor comprises DBM1285. In some embodiments, the method further comprises cryopreserving the obtained permanent HE cells.

[0018] In another aspect, the present invention provides a method for generating iPSC-derived hematopoietic lineage cells by differentiating the permanent HE cells described herein, wherein the method comprises contacting the permanent HE cells with a culture medium composition comprising SCF, Flt3L and IL7; and optionally one or more of a ROCK inhibitor, TPO and IL3, thereby obtaining iPSC-derived hematopoietic lineage cells comprising T cell progenitors and T cells in addition to NK cell progenitors, NK cells, NKT cells or B cells. In various embodiments of the method, the iPSC-derived hematopoietic lineage cells comprise NK cell progenitors and / or NK cells, and wherein (1) the culture medium composition further comprises IL15; and / or (2) the permanent HE cells comprise a gene insertion of a polynucleotide encoding a cytokine signaling complex comprising exogenous IL15 expressed on the cell surface and / or a portion or all of a peptide of its receptor. In some embodiments of the method, the culture medium composition does not contain OP9 stromal cells. In some embodiments, differentiation occurs in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4 (human DLL4 Fc chimeric recombinant protein).

[0019] In another aspect, the present invention provides a method for producing iPSC-derived hematopoietic lineage cells, the method comprising: differentiating iPSCs to obtain permanent hemogenic endothelial (HE) cells, wherein the permanent HE cells express a protein comprising CD82 + ; and differentiating permanent HE cells to obtain iPSC-derived hematopoietic lineage cells; wherein the iPSC-derived hematopoietic lineage cells further comprise T cell progenitors and T cells in addition to NK cell progenitors, NK cells, NKT cells or B cells. In some embodiments of the method, the iPSC comprises one or more genetic imprints introduced into the iPSC by genome editing during or after reprogramming the non-pluripotent cells into iPSCs, wherein the one or more genetic imprints comprise: (i) one or more gene modification patterns introduced by genomic insertions, deletions or substitutions in the genome of the iPSC; or (ii) one or more retentive therapeutic properties of source-specific immune cells specific for a donor, disease or treatment response, wherein the iPSC is reprogrammed by the source-specific immune cells, and wherein the one or more genetic imprints are retained in the iPSC-derived hematopoietic lineage cells. In some embodiments of the method, differentiating iPSCs to obtain definitive hemogenic endothelial (HE) cells comprises: (i) differentiating genetically engineered iPSCs to obtain mesodermal progenitor cells; (ii) differentiating mesodermal progenitor cells to obtain HE cells; and (iii) targeting CD82 + HE cells were sorted (e.g., by using an anti-CD82 antibody) to obtain cells expressing CD82. + In some embodiments, the sorting further comprises sorting for the following cells: CD34 + 、CD43 - ,RUNX1 + or any combination thereof (e.g., obtained by sorting using anti-CD34 antibody and / or anti-CD43 antibody), and wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + phenotype.

[0020] In various embodiments of the method, differentiating the mesodermal progenitor cells into HE cells comprises contacting the mesodermal progenitor cells with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor that results in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor. In some embodiments, the cytokine comprises a BMP activator (e.g., BMP4) and / or the small molecule p38 MAPK inhibitor comprises DBM1285. In some embodiments, the method further comprises cryopreserving permanent HE cells, wherein the cryopreserved permanent HE cells are thawed prior to their differentiation. In some embodiments, differentiating the permanent HE cells does not contain OP9 stromal cells. In some embodiments, differentiating the permanent HE cells occurs in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4.

[0021] In another aspect, the present invention provides a method for generating NK cells in a feeder-free environment, the method comprising: (a) differentiating iPSCs or permanent HE cells derived therefrom into NK lineage cells in a culture medium comprising one or more growth factors and cytokines (including SCF, Flt3L and IL7); wherein the culture medium does not contain OP9 stromal cells; and further, wherein: (i) the culture medium comprises IL15, and / or (ii) the permanent HE cells comprise a gene insertion of a polynucleotide encoding a cytokine signaling complex, the cytokine signaling complex comprising exogenous IL15 and / or part or all of its receptor peptides expressed on the cell surface; and (b) expanding and activating NK lineage cells to obtain NK cells that are cytotoxic to the target. In some embodiments, the culture medium further comprises one or more of a ROCK inhibitor, TPO and IL3. In some embodiments, the permanent HE cells comprise a phenotype comprising: (i) CD82 + (ii) CD34 + CD82 + (iii) CD34 + CD43 - CD82 + and / or (iv) CD34 + , and CD43 - 、CD93 - CXCR4 - 、CD73 - and RUNX1 + At least one of .

[0022] In some embodiments of the method of generating NK cells, differentiating iPSCs further comprises: (i) differentiating iPSCs to obtain mesodermal progenitor cells; (ii) differentiating mesodermal progenitor cells to obtain hemogenic endothelial (HE) cells; and (iii) differentiating iPSCs to obtain hemogenic endothelial (HE) cells. + HE cells are sorted (e.g., by using an anti-CD82 antibody) to obtain permanent HE cells, wherein the permanent HE cells express a protein comprising CD82 + In some embodiments of the method, the sorting further comprises sorting for the following cells: CD34 + 、CD43 - ,RUNX1 + or any combination thereof (e.g., obtained by sorting using anti-CD34 antibody and / or anti-CD43 antibody), wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + In some embodiments, differentiating mesodermal progenitor cells into HE cells comprises contacting the mesodermal progenitor cells with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor that results in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor. In some embodiments, the cytokine comprises BMP4 and / or the small molecule p38 MAPK inhibitor comprises DBM1285.

[0023] In some embodiments of the method, step (a) differentiation further comprises contacting the permanent HE cells with an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4; and / or step (b) expansion further comprises contacting the NK lineage cells with an expansion composition comprising nicotinamide. In some embodiments, step (b) expansion further comprises contacting the NK lineage cells with a small molecule AhR inhibitor, thereby modulating NK lineage cell activation. In some embodiments, the small molecule AhR inhibitor comprises CHIR223191, UM729, UM171, or SR1. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results show that the expression of RUNX1 in the control condition and in the presence of RUNX1 + Flow cytometric analysis of specialized cytokine-differentiated (cytokine-driven) D10 cells of HE.

[0025] Figure 2 A summary of candidate HE surface markers identified by the BioLegend LEGENDScreen is shown.

[0026] Figure 3A Shown is a UMAP visualization of curated gene expression used to identify cell clusters.

[0027] Figure 3B Transcriptome identification of the D10 cell population visualized by UMAP is shown. Each dot represents a cell.

[0028] Figure 3C Shown from Figure 3B Violin plots of CD82 expression within each cell cluster.

[0029] FIG. 4A to FIG. 4B Shown is flow cytometric analysis of cytokine-driven D10 cells comparing the expression of HE candidate markers with RUNX1 and CD82.

[0030] Figure 5 Flow cytometric analysis of cytokine-driven D10 cells showing CD82 + Cells in CD73 - CD93 - CXCR4 - Enrichment within the endothelial cell population. Cells were pre-gated for single / live events.

[0031] Figure 6 Shown are the frequencies of cytokine-driven HE within D10 populations that were subjected to fluorescence activated cell sorting (FAC) based on the indicated markers on the x-axis (mean ± SD).

[0032] Figure 7 Shown are flow cytometric analyses of D35 iT cells derived from the cytokine driven D10 population, which were FAC sorted based on the markers indicated in the flow plots above.

[0033] Figure 8 Shown are flow cytometric analyses of D30 iNK cells derived from the cytokine driven D10 population, which were FAC sorted based on the markers indicated in the flow plots above.

[0034] 9A to 9DShown is a comparison of expansion fold and iNK specification between cells differentiated on Retro / DLL4, irOP9-DLL4, and a commercial kit.

[0035] FIG. 10A to FIG. 10C Shown is a comparison of the fold expansion of progenitor iNK cells obtained using each of the three differentiation strategies when co-cultured with engineered feeder cells to obtain activated NK cells.

[0036] Fig.11A and Fig. 11B It was shown that antigen-dependent caspase 3 / 7 activity was comparable between mature iNK cells differentiated on Retro / DLL4 or irOP9-DLL4.

[0037] Fig. 12A and Fig. 12B showed that IFNγ and TNFα cytokine release was comparable between mature iNK cells differentiated on Retro / DLL4 or irOP9-DLL4.

[0038] Fig.13 We showed that iNK cells differentiated on Retro / DLL4 displayed antigen-dependent serial killing similar to that of cells differentiated on irOP9-DLL4.

[0039] Fig.14 showed that the addition of an AhR (aryl hydrocarbon receptor) inhibitor during the cell expansion phase of iPSC differentiation resulted in a greater expansion fold and yield of differentiated NK cells.

[0040] Fig.15 showed that cells treated with AhR inhibitors prior to cryopreservation displayed enhanced post-thaw antitumor efficacy over time. DETAILED DESCRIPTION

[0041] In various aspects, the present invention generally relates to methods and compositions for differentiating stem cells toward a definitive hematopoietic cell fate. In particular aspects, the present invention provides a multi-stage differentiation platform in which iPSCs or iPSC-derived cells at different developmental stages can be induced to assume a definitive hematopoietic phenotype, ranging from definitive hemogenic endothelial cells to fully differentiated hematopoietic cells, including T cells, B cells, NKT cells, and NK cells. Methods and compositions are provided for making cells more susceptible to assuming a definitive hematopoietic fate, e.g., CD34 + Definite hematopoietic stem cells. In some embodiments, the methods and compositions of the present invention generate definitive hemogenic endothelial cells (HE) from naive iPSCs in a scalable manner by avoiding the formation of EBs or aggregates.

[0042] definition

[0043] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those skilled in the art. In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0044] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents etc. described herein and may vary accordingly. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0045] As used herein, the articles "a", "an" and "the" herein refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0046] The use of alternatives such as "or" should be understood to mean any one or both of the alternatives or any combination thereof.

[0047] The term "and / or" should be understood to mean one or both of the alternatives.

[0048] Throughout this specification, unless the context requires otherwise, the word "comprising" should be understood to imply the inclusion of a stated step or element or a group of steps or elements, but not the exclusion of any other step or element or a group of steps or elements. In specific embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0049] The phrase “consisting of is intended to include and be limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or necessary, and that no other elements may be present.

[0050] By "consisting essentially of is meant to include any of the elements listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or function of the listed elements as specified in the present disclosure. Thus, the phrase "consisting essentially of indicates that the listed elements are required or necessary, but other elements are optional and may be present or absent depending on whether they affect the activity or function of the listed elements.

[0051] Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0052] As used herein, the term "about" or "approximately" refers to a range of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0053] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" or "essentially the same" refers to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weights, or lengths that are about the same as a reference amount, level, value, number, frequency, percentages, dimensions, sizes, amounts, weights, or lengths.

[0054] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition (e.g., a cell population or culture medium), refer to a composition that is free of a specified substance or a source thereof, e.g., 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable, as measured by conventional means. The term "free" or "essentially free" of a certain ingredient or substance in a composition also means that (1) the composition does not include such ingredient or substance at any concentration, or (2) the composition includes such ingredient or substance that is functionally inert but in low concentrations. Similar meanings can be applied to the term "lacking", which refers to the lack of a specific substance or source thereof in a composition.

[0055] The term "ex vivo" generally refers to an activity that occurs outside an organism, such as an experiment or measurement performed in or on a living tissue in an artificial environment outside an organism, preferably wherein the change in natural conditions is minimal. In a specific embodiment, an "ex vivo" procedure involves obtaining living cells or tissues from an organism and cultivating them in a laboratory device under sterile conditions, and typically cultivating for a few hours or up to about 24 hours, but including up to 48 hours or 72 hours or longer, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and thawed later to carry out ex vivo processing. Tissue culture experiments or procedures that use living cells or tissues for a duration longer than a few days are generally considered "in vitro", but in certain embodiments, this term can be used interchangeably with ex vivo.

[0056] The term "in vivo" generally refers to activities that take place inside an organism.

[0057] The term "effector cell" is generally applicable to certain cells that perform a specific activity in response to stimulation and / or activation in the immune system, or to cells that perform a specific function when activated. As used herein, the term "effector cell" includes immune cells, "differentiated immune cells", and primary or differentiated cells that are edited and / or regulated to perform a specific activity in response to stimulation and / or activation, and these terms are interchangeable in some cases. Non-limiting examples of effector cells include primary-derived or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0058] As used herein, the term "B lymphocyte" or "B cell" is used interchangeably and refers to a lymphocyte subgroup defined by the expression of a B cell receptor comprising heavy and light chains of immunoglobulins (BCR, Ig), CD19 or CD20 in the absence of a T cell receptor (CD3). As provided herein, B cells can also be derived from stem cells or progenitor cells via directed differentiation. B cells include the B cells of any subtype, and can be in any developmental stage, including but not limited to former B cells, pre-B cells, original B cells, B-1B cells, B-2B cells, marginal zone B cells, follicular B cells, memory B cells, plasmablasts, plasma cells, regulatory B cells.

[0059] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that completes maturation in the thymus and has a variety of roles in the immune system, including identifying specific foreign antigens in the body and activating and inactivating other immune cells in an MHC class I restricted manner. The T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. The T cell can be a CD3 + T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 +T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδT cells), etc. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). The term "T cell" can also refer to genetically engineered T cells, such as T cells that are modified to express T cell receptors (TCR) or chimeric antigen receptors (CAR). T cells or T cell-like effector cells can also be differentiated by stem cells or progenitor cells ("derived T cells" or "derived T cell-like effector cells", or collectively referred to as "derived T lineage cells"). Derived T cell-like effector cells may have T cell lineages in some aspects, but at the same time have one or more functional characteristics that are not present in primary T cells. In the present application, T cells, T cell-like effector cells, derived T cells, derived T cell-like effector cells or derived T lineage cells are collectively referred to as "T lineage cells".

[0060] "CD4 + "T cells" refers to a subpopulation of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a secretory profile upon stimulation that may include secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. The "CD4" molecule is a 55-kD glycoprotein that was originally defined as a differentiation antigen on T lymphocytes but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is indicated as a relevant recognition element in major histocompatibility complex (MHC) class II restricted immune responses. On T lymphocytes, it defines a subset of helper / inducer factors.

[0061] "CD8 + "T cells" refers to a subpopulation of T cells that express CD8 on their surface, are restricted to MHC class I, and act as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a relevant recognition element in major histocompatibility complex class I-restricted interactions.

[0062] As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). The NK cell can be any NK cell, such as a cultured NK cell, e.g., a primary NK cell, or an NK cell from a cultured or expanded NK cell or a cell line NK cell, e.g., NK-92, or an NK cell obtained from a healthy or diseased mammal. As used herein, the term "adaptive NK cell" is interchangeable with "memory NK cell" and refers to a subset of NK cells whose phenotype is CD3 - and CD56 + , expresses at least one of NKG2C and CD57 and optionally CD16, but lacks expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated CD56 + NK cell subsets include expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + Can be weaker or stronger expression.NK cell or NK cell-like effector cell can be differentiated by stem cell or progenitor cell (" derived NK cell " or " derived NK cell-like effector cell ", or be collectively referred to as " derived NK lineage cell "). Derived NK cell-like effector cell can have NK cell lineage in some aspects, but have one or more functional characteristics that are not present in primary NK cell at the same time. In the present application, NK cell, NK cell-like effector cell, derived NK cell, derived NK cell-like effector cell or derived NK lineage cell are collectively referred to as " NK lineage cell ".

[0063] As used herein, the term "NKT cell" or "natural killer T cell" or "NKT lineage cell" refers to a T cell restricted to CD1d that expresses a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d (a non-classical MHC molecule). Two types of NKT cells are recognized. Constant or type I NKT cells express a very limited TCR repertoire: a combination of a typical α chain (Vα24-Jα18 in humans) and a limited spectrum of β chains (Vβ11 in humans). The second NKT cell population, called non-classical or non-constant type II NKT cells, shows a more uneven TCRαβ utilization. Type I NKT cells are considered suitable for immunotherapy. Adaptive or constant (type I) NKT cells can be identified by the expression of one or more of the following markers: TCR Va24-Ja18, Vb11, CDld, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0064] As used herein, the term "permanent hemogenic endothelial cells" (HE) or "pluripotent stem cell-derived permanent hemogenic endothelial cells" (iHE) refers to a subpopulation of endothelial cells that give rise to hematopoietic stem cells and progenitor cells in a process known as the endothelial-to-hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially: from lateral plate mesoderm to angioblasts to permanent hemogenic endothelial cells and hematopoietic progenitor cells. In some embodiments, a population of iHE cells can be maintained, stored, and / or cryopreserved in multiple containers to reliably serve as a starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment.

[0065] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells" or "hematopoietic precursor cells" refer to cells that are specialized in the hematopoietic lineage but are capable of further differentiation into hematopoiesis, and include multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). As used herein, the term "permanent hematopoietic stem cell" refers to a CD34 + Hematopoietic cells, which are capable of giving rise to mature myeloid cell types and lymphocyte cell types, include cells of the T-lineage, NK-lineage, and B-lineage. Hematopoietic cells also include various subsets of primitive hematopoietic cells, which give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0066] As used herein, the term "embryonic stem cell" refers to a naturally occurring pluripotent stem cell in the inner cell mass of an embryonic blastocyst. Embryonic stem cells are pluripotent and produce all the derivative cells of the three primary germ layers: ectoderm, endoderm, and mesoderm during development. It does not contribute to the extraembryonic membranes or placenta and is not omnipotent to differentiate.

[0067] As used herein, the term "pluripotent stem cell" refers to a cell with the developmental potential to differentiate into one or more germ layers (ectoderm, mesoderm and endoderm) but not all three germ layers. Therefore, pluripotent cells may also be referred to as "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 cells can form many types of cells in a given lineage, rather than cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (erythrocytes, leukocytes, platelets, etc.), but they cannot form neurons. Therefore, the term "pluripotency" refers to a cell state in which the degree of developmental potential is lower than differentiation omnipotence and pluripotency.

[0068] As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of a body or soma (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells of each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuous developmental efficacy ranging from incomplete or partially pluripotent cells (e.g., ectoderm stem cells or EpiSCs) that cannot produce a complete organism to more primitive, more capable cells (e.g., embryonic stem cells) that can produce a complete organism.

[0069] As used herein, the term "induced pluripotent stem cell" or "iPSC" means a stem cell generated from a differentiated adult, neonatal, or fetal cell that has been induced or altered (i.e., reprogrammed) to be capable of differentiating into tissues of all three germ layers or dermis: mesoderm, endoderm, and ectoderm.

[0070] Pluripotency can be determined in part by assessing the pluripotency properties of the cells. Pluripotency properties include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60, TRA1-81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation composed of the three somatic cell lineages; and (vi) the formation of embryoid bodies composed of cells from the three somatic cell lineages.

[0071] Two types of pluripotency have been described previously: the "stimulated" or "metastable" pluripotency state is equivalent to the epiblast stem cells (EpiSCs) of the late blastocyst, and the "naive" or "basal" pluripotency state is equivalent to the inner cell mass of the early / preimplantation blastocyst. Although both pluripotency states exhibit the properties described above, the naive or basal state further exhibits: (i) pre-inactivation or reactivation of the X chromosome in female cells; (ii) improved clonality and survival during single-cell culture; (iii) overall reduction in DNA methylation; (iv) reduced deposition of the H3K27me3 repressive chromatin mark on the promoters of developmentally regulated genes; and (v) reduced expression of differentiation markers relative to pluripotent cells in the stimulated state. It is generally found that the standard method of cell reprogramming (in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells) has the characteristics of the stimulated state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the stimulated state unless exogenous transgene expression is maintained (in which the characteristics of the basal state are observed).

[0072] Pluripotency exists as a continuum, and induced pluripotent stem cells (iPSC) seem to exist in "excited" state and "original" state, and the cells in the original state may have greater differentiation potential. The induced pluripotent stem cells produced in conventional culture medium exist in an excited state and are more closely similar to the cells derived from the blastocyst after implantation, and the initial iPSC shows the pluripotency characteristics of the cells more closely similar to mouse embryonic stem cells or derived from the blastocyst before implantation. Excitation and initial cell states can be defined by various differences, including the difference in colony morphology, the inhibition of key signal transduction pathways or the cell response of activation, gene expression characteristics and the ability to reactivate genes related to extraembryonic cells. For example, the conventional iPSC representing the pluripotent state shows a flat colony morphology, and the initial iPSC shows a compact dome colony morphology similar to mouse embryonic stem cells. As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and compact shape, a high ratio of nucleus to cytoplasm, a significant presence of nucleolus, and typical intercellular spacing.

[0073] As used herein, the term "differentiation" is the process by which an unspecialized ("unspecialized") or weakly specialized cell acquires the characteristics of a specialized cell (e.g., a blood cell or a muscle cell). A differentiated cell or differentiation-induced cell is a cell that is already in a more specialized ("specialized") position within a cell lineage. The term "specialization" as applied to the differentiation process refers to a cell that has progressed in the differentiation pathway to a point where, under normal circumstances, it would continue to differentiate into a particular cell type or subset of cell types and, under normal circumstances, it is unable to differentiate into a different cell type or reverts to a more weakly differentiated cell type.

[0074] Differentiation of pluripotent stem cells needs to change culture system, such as changing the stimulant in culture medium or the physical state of cell.Most of conventional strategies use the formation of embryoid bodies (EB) as the common and key intermediate step of starting lineage-specific differentiation."Embryoid bodies" are three-dimensional clusters, which have been shown to simulate embryonic development because they produce multiple lineages in their three-dimensional regions.Through differentiation process, usually a few hours to a few days, simple EB (for example, through inducing differentiable aggregated pluripotent stem cells) continues to mature and develop into cystic EB, at this time, usually further processed for several days to a few weeks to continue differentiation.EB formation is started by making pluripotent stem cells close to each other in three-dimensional multilayer cell clusters.Usually, this is achieved by a method in several methods, including allowing pluripotent cells to settle in droplets, making cells settle in "U" shaped bottom well plates or by mechanical agitation.In order to promote EB development, pluripotent stem cell aggregates need to further differentiate prompts, because the aggregates maintained in pluripotent culture maintenance medium do not form suitable EBs. In the present invention, the pluripotent stem cell aggregates are transferred to the differentiation medium, and the differentiation medium provides the prompting to the selected pedigree. Through the appropriate proliferation in the EB cell cluster, the culture based on EB of the pluripotent stem cell usually causes the differentiated cell group (that is, ectoderm, mesoderm and endodermal germ layer) to produce. Although it has been confirmed that cell differentiation can be promoted, EB has produced heterogeneous cells with variable differentiation states, and the reason is that the cells in the three-dimensional structure are inconsistently exposed to the differentiation prompting in the environment. In addition, the formation of EB and maintenance trouble. In addition, the cell differentiation carried out by EB formation is accompanied by appropriate cell amplification, which also causes the differentiation efficiency to reduce.

[0075] In contrast, "aggregate formation" different from "EB formation" can be used to expand pluripotent stem cell derived cell populations. For example, during the pluripotent stem cell expansion based on aggregates, a culture medium that can maintain proliferation and pluripotency is selected. Cell proliferation usually increases the size of aggregates, thereby forming larger aggregates, which can be dissociated into smaller aggregates by mechanical or enzymatic means, thereby maintaining cell proliferation in culture and increasing cell number. Different from EB culture, cells cultured in aggregates maintaining culture medium maintain pluripotency markers. Pluripotent stem cell aggregates require further differentiation prompts to induce differentiation.

[0076] As used herein, "monolayer differentiation" is a term for a differentiation method that is different from the differentiation performed by three-dimensional multilayer cell clusters, i.e., "embryoid bodies," "EBs," or "EB formation." In addition to other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Because monolayer culture does not simulate embryonic development, such as in the case of EB formation, differentiation to a specific lineage is considered to be minimal compared to all three germ layer differentiations in EB formation.

[0077] "Cultivation" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "medium" (in each case, the singular form "medium"), "supplement" and "medium supplement" refer to a nutrient composition for growing cell cultures.

[0078] As used herein, "feeder cells" or "feeder layers" are terms describing a type of cell that is co-cultured with cells of a second type to provide an environment in which cells of the second type can grow, expand or differentiate, because feeder cells provide stimulation, growth factors and nutrition to support the second cell type. Feeder cells are optionally from species different from the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. When feeder cells are co-cultured with other cells, they can be inactivated by irradiation or treatment with anti-mitotic agents (such as mitomycin) to prevent them from growing beyond the cells they support. Feeder cells can include endothelial cells, stromal cells (such as epithelial cells or fibroblasts) and leukemia cells. Not limited to the foregoing, a specific feeder cell type can be a human feeder layer, such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEF). In general, a variety of feeder cells can be used in part to maintain pluripotency, directed differentiation toward a certain lineage, enhance proliferation capacity, and promote maturation into specialized cell types (eg, effector cells).

[0079] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, which is substantially free of feeder layers or stromal cells, and / or has not been preconditioned by cultivating feeder cells. "Preconditioned" culture medium refers to a culture medium collected after feeder cells have been cultivated in the culture medium for a certain period of time (e.g., at least one day). The preconditioned culture medium contains a variety of mediator substances, including growth factors and cytokines secreted by feeder cells cultivated in the culture medium. In some embodiments, the feed-free environment does not contain feeder layers or stromal cells, and is not preconditioned by cultivating feeder cells. Feeder cells include, but are not limited to, stromal cells, mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells.

[0080] "Cultivating" or "maintaining" refers to the maintenance, propagation (growth) and / or differentiation of cells outside of a tissue or body (e.g., in a sterile plastic (or coated plastic) cell culture dish or flask). "Cultivating" or "maintaining" can use a culture medium as a source of nutrients, hormones and / or other factors that aid in the propagation and / or maintenance of cells.

[0081] As used herein, "passage" refers to the behavior of dividing the cultured cells by subdividing the cells and plating them on multiple cell culture surfaces or containers when the cells have proliferated to the desired extent. In some embodiments, "passage" refers to subdividing the cells, diluting and plating them. When cells are passaged from the primary culture surface or container to a subsequent set of surfaces or containers, the subsequent culture may be referred to as "subculture" or "first passage" in this article. Each behavior of subdividing and plating in a new culture container is considered to be a passage. In some embodiments, the cultured cells are passaged once every 1, 2, 3, 4, 5, 6, 7 days or more. In some embodiments, the iPSC initially selected after reprogramming is passaged once every 3 days-7 days.

[0082] As used herein, "dissociated cells" or "single dissociated cells" refer to cells that have been substantially separated or purified from other cells or surfaces (e.g., culture plate surfaces). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in vitro can be enzymatically or mechanically dissociated from each other, such as by dissociating into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells can be dissociated from a culture plate or other surface. Thus, dissociation may involve disruption of cellular interactions with the extracellular matrix (ECM) and substrate (e.g., culture surface), or disruption of the ECM between cells.

[0083] As used herein, the term "separation" etc. refers to a cell or cell group that has been separated from its initial environment, that is, the environment of the separated cell is substantially free of at least one component found in the environment of the "unseparated" reference cell. The term includes cells removed from some or all components, as they are found in their natural environment, such as separated from tissues or biopsy samples. The term also includes cells removed from at least one, some or all components, because the cell is present in a non-naturally occurring environment, such as separated from cell culture or cell suspensions. Therefore, "separated cells" are partially or completely separated from at least one component (including other substances, cells or cell groups), as they are found in nature or as they grow, store or survive in a non-naturally occurring environment. Specific examples of separated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in non-naturally occurring culture media. Separated cells can be obtained by separating the desired cell or its group from other substances or cells in the environment or by removing one or more other cell groups or subgroups from the environment.

[0084] As used herein, the term "purified" and the like refers to increased purity. For example, the purity of a particular cell type within a cell population can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

[0085] As used herein, a "master cell bank" or "MCB" refers to a clonal master engineered iPSC line, which is a clonal population of iPSCs that have been engineered to include one or more therapeutic attributes, have been characterized, tested, qualified, and expanded, and have been shown to reliably function as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing setting. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic variation and / or potential contamination by reducing and / or eliminating the total number of passages, thawing, or handling of the iPS cell line during the manufacturing process.

[0086] As used herein, the term "reprogramming" or "dedifferentiation" or "improving cell efficacy" or "improving developmental efficacy" refers to a method of improving cell efficacy or dedifferentiating cells into a lower differentiation state. For example, cells with improved cell efficacy have greater developmental plasticity (i.e., can be differentiated into more cell types) than the same cells in a non-reprogrammed state. In other words, reprogrammed cells are cells with a lower differentiation state than the same cells in a non-reprogrammed state. In contrast to "reprogrammed cells", "reprogrammed cells" refer to non-pluripotent cells that undergo reprogramming / dedifferentiation into a pluripotent state, presenting a transitional morphology (i.e., morphological changes) but without the markers of pluripotent cells, including pluripotent stem cell morphology or stable endogenous pluripotency gene expression, such as OCT4, NANOG, SOX2, SSEA4, TRA181, CD30 and / or CD50. The transitional morphology of "reprogrammed cells" distinguishes cells from the initial non-pluripotent cells before reprogramming induction and from reprogrammed cells with embryonic stem cell marker morphology. For example, when fibroblasts are reprogrammed, the morphological changes of reprogrammed cells include MET (mesenchymal to epithelial transition). Those skilled in the art will readily understand and identify this transitional morphology of various types of somatic cells for induction of reprogramming. In some embodiments, reprogrammed cells are induced to reprogram at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or more days, but no more than 21 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days, 35 days, 40 days or any days therebetween intermediate cells, wherein the cell has not yet entered self-sustaining or self-sustaining pluripotent state. When cells are introduced with one or more reprogramming factors, non-pluripotent cell reprogramming is induced. The reprogrammed cells induced to reprogram 1, 2, 3 or 4 days are cells (transduction day is day 0) after reprogramming factor transduction 1, 2, 3 or 4 days. Unlike somatic cells prior to exposure to exogenous expression of reprogramming factors, "reprogrammed cells" can progress during the reprogramming process to reach a stable pluripotent state, and become "reprogrammed cells" even in the absence of exogenous expression of reprogramming factors, given a sufficient period of time.

[0087] "Pluripotency factor" or "reprogramming factor" refers to an agent or combination of agents used to induce or improve the developmental efficacy of a cell. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can improve the developmental efficacy of a cell. Exemplary pluripotency factors include, for example, transcription factors OCT4 and SOX2, and small molecule reprogramming agents, such as, for example, TGFβ inhibitors, GSK3 inhibitors, MEK inhibitors, and ROCK inhibitors.

[0088] As used herein, "genetic modification" refers to gene editing, including (1) those naturally derived from rearrangements, mutations, genetic imprints and / or epigenetic modifications that occur in cells or during cell development, or (2) those obtained by genome engineering through cell manipulation, which cell manipulation includes but is not limited to insertions, deletions or substitutions in the cell genome. Genetic modification as used herein also includes one or more retentive therapeutic properties of source-specific immune cells specific to donors, diseases or therapeutic responses. Genetically modified cells are cells that contain genetic modifications (e.g., gene editing) compared to corresponding wild-type cells without such genetic modifications.

[0089] As used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, "function" refers to (1) at the genetic level - successful knock-in, knock-out, reduced gene expression, transgenic or controlled gene expression, such as inducible or transient expression at a desired cell developmental stage, achieved by direct genome editing or modification or by "transfer", via differentiation or reprogramming of the starting cell that was initially genome engineered; or (2) at the cellular level - successful removal, addition or alteration of cellular function / property, achieved by: (i) in said cell Gene expression modifications obtained by direct genome editing, (ii) gene expression modifications maintained in the cell by "transfer" through differentiation or reprogramming from the starting cell that was originally genome engineered; (iii) downstream gene regulation in the cell as a result of gene expression modifications that only occurred at an earlier developmental stage of the cell or only occurred in the starting cell that generated the cell through differentiation or reprogramming; or (iv) enhanced or newly acquired cellular functions or properties exhibited in a mature cell product that was originally derived from genome editing or modification performed at an iPSC, progenitor cell, or dedifferentiated cell source.

[0090] As used herein, the term "genetic imprint" refers to the genetic or epigenetic information that contributes to the preferred and / or enhanced therapeutic properties of source cells or iPSC, and can be retained in the iPSC derived from the source cell and / or the hematopoietic lineage cells derived from iPSC. As used herein, "source cell" is a non-pluripotent cell that can be used to produce iPSC by reprogramming, and the source cell derived iPSC can be further differentiated into a specific cell type, including any hematopoietic lineage cells. Depending on the context, source cell derived iPSC and its differentiated cells are sometimes collectively referred to as "derived (derived or derivative) cells". For example, as used throughout this application, derived effector cells or derived NK cells or derived T lineage cells are cells differentiated by iPSC compared to their primary corresponding cells obtained from natural / primitive sources (such as peripheral blood, umbilical cord blood or other donor tissues). As used herein, the genetic imprints that confer preferred and / or enhanced therapeutic attributes are incorporated into iPSCs by reprogramming selected source cells that are specific to donors, diseases, or therapeutic responses or by introducing genetic modification patterns into iPSCs using genome editing. In the aspect of source cells obtained from a particularly selected donor, disease, or therapeutic background, the genetic imprints that contribute to the preferred therapeutic attributes may include any background-specific genes or epigenetic modifications that manifest a phenotype that can be retained, i.e., a preferred therapeutic attribute, which is transmitted to cells derived from iPSCs of selected source cells, regardless of whether the underlying molecular events are identified or not. Donor, disease or treatment response specific source cells may include genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including but not limited to pre-arranged monospecific TCRs, such as from virus-specific T cells or constant natural killer T (iNKT) cells; trackable and desired genetic polymorphisms, such as homotypic for point mutations encoding high-affinity CD16 receptors in selected donors; and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibit haplotypes as the population increases. As used herein, preferred and / or enhanced therapeutic attributes include transplantation, transport, homing, vitality, self-renewal, retention, immune response regulation and regulation, survival and cytotoxicity improvements of derived cells. Preferred therapeutic attributes may also involve antigen-targeted receptor expression; HLA presentation or its lack; resistance to tumor microenvironment; induction and immunomodulation of neighboring immune cells; improved on-target specificity as off-tumor effects decrease; resistance to treatments such as chemotherapy. Derived cells having one or more therapeutic properties are also referred to as "synthetic cells" when such derived cells are obtained by differentiating iPSCs having a genetic imprint that confers a preferential therapeutic property that was integrated into the iPSCs.Typically, when compared to the closest corresponding primary cell, the synthetic cell has one or more non-native cell functions, whether the synthetic cell is differentiated from an engineered pluripotent cell or obtained by engineering primary cells from a natural / native source (such as peripheral blood, umbilical cord blood or other donor tissue). For example, as used throughout this application, a synthetic effector cell or a synthetic NK cell or a synthetic T cell is a cell differentiated from a genome-modified iPSC compared to its primary corresponding cell obtained from a natural / native source (such as peripheral blood, umbilical cord blood or other donor tissue). In some embodiments, a synthetic cell has one or more non-native cell functions when compared to its closest corresponding primary cell.

[0091] As used herein, the term "exogenous" is intended to mean that a reference molecule or reference activity is introduced into a host cell, or is non-native to a host cell. Exogenous molecules can be introduced, for example, by introducing a coding nucleic acid into a host genetic material, such as by integration into a host chromosome, or as a non-chromosomal genetic material, such as a plasmid. Therefore, the term, when used with respect to the expression of a coding nucleic acid, refers to the introduction of a coding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in a host cell. Similarly, the term, when used with respect to the expression of a coding nucleic acid, refers to the expression of a coding nucleic acid contained in a cell rather than introduced exogenously.

[0092] "Construct" refers to a macromolecule or molecular complex containing a polynucleotide to be delivered to a host cell in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, in which the nucleic acid construct can be replicated and / or expressed. Therefore, the term "vector" includes the construct to be delivered. The vector can be a linear or circular molecule. The vector can be an integrating or non-integrating vector. The main types of vectors include, but are not limited to, plasmids, free vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0093] So-called "integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, that is, covalently linked to the nucleic acid sequence in the cell chromosome DNA. So-called "targeted integration" means that the nucleotides of the construct are inserted into the cell chromosome or mitochondrial DNA at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that involves inserting one or more exogenous sequences or nucleotides of the construct into the integration site when the endogenous sequence or nucleotide is missing or not missing. In the case of a missing insertion site, "integration" may also include replacing the missing endogenous sequence or nucleotide with one or more inserted nucleotides.

[0094] As used herein, the term "encoding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in a biological process, which have a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties obtained therefrom. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is consistent with the mRNA sequence and is usually provided in the sequence table) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as "encoding" a protein or other product of the gene or cDNA.

[0095] As used herein, a "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that is transcribed into RNA in vivo and, in some cases, translated into a polypeptide when placed under the control of appropriate regulatory sequences. Genes or polynucleotides of interest may include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode a miRNA, shRNA, a native polypeptide (i.e., a polypeptide found in nature) or fragments thereof; a variant polypeptide (i.e., a mutant of a native polypeptide having less than 100% sequence identity with a native polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

[0096] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or its analogs. A polynucleotide sequence is composed of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) (when the polynucleotide is RNA, uracil replaces thymine). A polynucleotide may include a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. "Polynucleotide" also refers to double-stranded and single-stranded molecules.

[0097] "Operably linked (operably linked / operatively linked and operably connected / operatively connected are used interchangeably)" refers to the association of a nucleic acid sequence on a single nucleic acid fragment (or amino acids in a polypeptide having multiple domains) such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA when it is capable of affecting the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. As another example, a receptor binding domain can be operably linked to an intracellular signaling domain such that binding of a receptor to a ligand transduces a signal in response to the binding.

[0098] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules in which amino acid residues are covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and the maximum number of amino acids of a polypeptide is not limited. As used herein, the terms refer to short chains (also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (commonly referred to in the art as polypeptides or proteins). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins and others. The polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides or combinations thereof.

[0099] As used herein, "fusion protein" or "chimeric protein" is a protein produced by genetic engineering, used to connect two or more partial or complete polynucleotide sequences encoding separate proteins, and the expression of these connected polynucleotides produces a single peptide or multiple polypeptides with functional properties derived from each of the original proteins or their fragments. Between two adjacent polypeptides of different origin in a fusion protein, a linker (or spacer) peptide may be added.

[0100] As used herein, the term "adapter" refers to a molecule, such as a fusion polypeptide, that enables a connection between an immune cell (e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil) and a tumor cell; and activates the immune cell. Examples of adaptors include, but are not limited to, bispecific T cell adaptors (BiTEs), bispecific killer cell adaptors (BiKEs), trispecific killer cell adaptors (TriKEs), or multispecific killer cell adaptors, or universal adaptors compatible with a variety of immune cell types.

[0101] As used herein, the term "surface triggering receptor" refers to a receptor that can trigger or initiate an immune response (e.g., a cytotoxic reaction). Surface triggering receptors can be engineered and can be expressed on effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils). In some embodiments, surface triggering receptors promote bispecific or multispecific antibody engagement between effector cells and specific target cells (e.g., tumor cells), independent of the natural receptors and cell types of effector cells. Using this method, iPSCs containing universal surface triggering receptors can be produced, and then such iPSCs are differentiated into populations of various effector cell types expressing universal surface triggering receptors. "Universal" means that surface triggering receptors can be expressed in any effector cell and activate any effector cell (regardless of the cell type), and all effector cells expressing universal receptors can be coupled or connected to adaptors recognizable by surface triggering receptors (regardless of the tumor binding specificity of the adaptor). In some embodiments, adaptors with the same tumor targeting specificity are used to couple with universal surface triggering receptors. In some embodiments, adaptors with different tumor targeting specificities are used to couple with universal surface triggering receptors. Thus, one or more effector cell types can be engaged, thereby killing one specific type of tumor cell in some cases and killing two or more types of tumors in other cases. Surface triggering receptors typically contain a co-stimulatory domain for effector cell activation and an anti-epitope specific for an epitope of the adaptor. Bispecific adaptors have specificity for an anti-epitope of a surface triggering receptor at one end and specificity for a tumor antigen at the other end.

[0102] As used herein, the term "safety switch protein" refers to an engineered protein that is designed to prevent the potential toxicity of cell therapy or otherwise prevent side effects. In some cases, the expression of the safety switch protein is conditionally controlled to address the safety issues of the engineered cells that have been transplanted, and the gene encoding the safety switch protein has been permanently incorporated into its genome. This conditional regulation can be variable and may include control by small molecule-mediated post-translational activation and tissue-specific and / or temporal transcriptional regulation. The safety switch protein can mediate the induction of apoptosis, inhibition of protein synthesis or DNA replication, growth arrest, transcription and post-transcriptional genetic regulation, and / or antibody-mediated depletion. In some cases, the safety switch protein is activated by an exogenous molecule such as a prodrug, which triggers apoptosis and / or cell death of the treated cells when activated. Examples of safety switch proteins include, but are not limited to, suicide genes, such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0103] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide that can achieve a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative or palliative properties for a disease and can be administered to improve, alleviate, slow down, reverse or reduce the severity of a disease. Pharmaceutically active proteins also have preventive properties and are used to prevent the onset of disease or to reduce the severity of such diseases or pathological conditions when they manifest. "Pharmaceutically active proteins" include complete proteins or peptides or pharmaceutically active fragments thereof. The term also includes pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term pharmaceutically active protein also refers to a variety of proteins or peptides that act in a collaborative or synergistic manner to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors and / or cytokines.

[0104] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces or increases cell signal transduction. "Signal transduction" refers to the transmission of molecular signals in a chemically modified form, which is achieved by recruiting protein complexes along the path that ultimately triggers biochemical events in cells. Signal transduction 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, TG point signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0105] The term "ligand" refers to a substance that forms a complex with a target molecule to generate a signal by binding to a site on the target. A ligand can be a natural or artificial substance that can specifically bind to a target. A ligand can be in the form of a protein, peptide, antibody, antibody complex, conjugate, nucleic acid, lipid, polysaccharide, monosaccharide, small molecule, nanoparticle, ion, neurotransmitter, or any other molecular entity that can specifically bind to a target. The target bound to the ligand can be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes the function of the target to trigger a signal transduction reaction is referred to as "agonism" or "agonist". A ligand that binds to a target and blocks or reduces a signal transduction reaction is referred to as "antagonism" or "antagonist".

[0106] As used herein, the terms "specific" or "specificity" may be used to refer to the ability of a molecule (eg, a receptor, antibody, or adaptor) to selectively bind to a target molecule, as compared to non-specific or non-selective binding.

[0107] As used herein, the term "targeting modality" refers to the genetic incorporation of molecules (e.g., polypeptides) into cells to promote antigen and / or epitope specificity, which includes but is not limited to i) antigen specificity (when it involves a unique chimeric antigen receptor (CAR) or T cell receptor (TCR)); ii) adaptor specificity (when it involves a monoclonal antibody or a bispecific adaptor); iii) targeting transformed cells; iv) targeting cancer stem cells, and v) other targeting strategies in the absence of specific antigens or surface molecules.

[0108] "HLA deficiency", including HLA class I deficiency, or HLA class II deficiency, or both, refers to the lack or no longer maintaining the surface expression of a complete MHC complex comprising HLA class I protein heterodimers and / or HLA class II heterodimers, or the reduced level of the surface expression, so that the weakened or reduced level is lower than the level that can be detected naturally by other cells or can be detected by synthetic methods. HLA class I deficiency can be achieved by causing any region of the HLA class I locus (chromosome 6p21) to be functionally lost or HLA class I-related genes (including but not limited to β-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and TAP-related protein) to be lost or the expression level is reduced. HLA class II deficiency can be achieved by causing HLA-II-related genes (including but not limited to RFXANK, CIITA, RFX5 and RFXAP) to be functionally lost or reduced. It was previously unclear whether iPSCs lacking or altered HLA complexes have the ability to enter development, maturation and produce functional differentiated cells while maintaining regulatory activity. Furthermore, it was previously unclear whether HLA complex-deficient differentiated cells could be reprogrammed into iPSCs and maintained as pluripotent stem cells while possessing HLA complex deficiency. Unexpected failures during cell reprogramming, maintenance of pluripotency and differentiation may involve aspects including, but not limited to, developmental stage-specific gene expression or its lack, the requirement for HLA complex presentation, protein shedding of introduced surface expression patterns, the need for proper and efficient clonal reprogramming, and the need for remodeling of differentiation protocols.

[0109] As used herein, "modified iPSC lacking HLA" refers to iPSC lacking HLA, which is additionally modified by introducing a gene expressing a protein associated with, but not limited to, improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, retention, immune escape, suppression resistance, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3z, 4-1BBL, CD47, CD113 and PDL1. "Modified" cells lacking HLA also include cells other than iPSC.

[0110] The term "antibody" is used in the broadest sense herein, and generally refers to a molecule that produces an immune response containing at least one binding site that specifically binds to a target, wherein the target can be an antigen or a receptor that can interact with certain antibodies. For example, NK cells can be activated by binding of an antibody or an antibody's Fc region to its Fc-γ receptor (FcγR), thereby triggering ADCC (antibody-dependent cellular toxicity)-mediated effector cell activation. The specific fragment or part of an antigen or receptor or target that binds to an antibody is generally referred to as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, native antibodies and variants thereof, fragments of native antibodies and variants thereof, peptide bodies and variants thereof, and antibody mimics that simulate the structure and / or function of antibodies or their specific fragments or parts (including single-chain antibodies and fragments thereof). Antibodies can be mouse antibodies, human antibodies, humanized antibodies, camel IgG, single variable new antigen receptors (VNAR), shark heavy chain antibodies (Ig-NAR), chimeric antibodies, recombinant antibodies, single domain antibodies (dAb), anti-idiotypic antibodies, bispecific antibodies, multispecific antibodies or multimeric antibodies, or their antibody fragments. Anti-idiotypic antibodies are specific for binding to the idiotype of another antibody, where the idiotype is an antigenic determinant of the antibody. Bispecific antibodies can be BiTEs (bispecific T cell engagers) or BiKEs (bispecific killer cell engagers), and multispecific antibodies can be TriKEs (trispecific killer cell engagers). Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single-chain variable region fragments (scFv), tandem scFv (scFv)2, single-chain Fab (scFab), disulfide-stabilized Fv (dsFv), minibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single domain antigen binding fragments (sdAb), camelid heavy chain IgG and Fragments, recombinant antibodies with only heavy chains (VHH) and other antibody fragments that maintain the binding specificity of the antibody.

[0111] "Fc receptors" (abbreviated as FcR) are classified based on the type of antibody they recognize. For example, the receptors that bind the most common class of antibodies (IgG) are called Fc-gamma receptors (FcγRs), the receptors that bind IgA are called Fc-alpha receptors (FcαRs) and the receptors that bind IgE are called Fc-epsilon receptors (FcεRs). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγRs) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), which have different affinities for their antibodies due to their different molecular structures.

[0112] FcγR receptor CD16 has been identified as having two isomers: Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells, which binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high-affinity CD16", "non-cleavable CD16" or "non-cleavable high-affinity CD16 (abbreviated as hnCD16)" refers to natural or non-natural CD16 variants. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, which is a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes after NK cell activation. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants that change or eliminate cleavage sites (positions 195-198) in regions close to the membrane (positions 189-212) do not experience shedding. The cleavage site and the region close to the membrane are described in detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. The CD16 S197P variant is an engineered non-cleavable version of CD16. The CD16 variant comprising F158V and S197P has high affinity and is non-cleavable. Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 comprising an extracellular domain derived from one or more of the three exons of the CD64 extracellular domain.

[0113] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy that, as used herein, involves the infusion of autologous or allogeneic lymphocytes, such as CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, or immunomodulatory cells, whether genetically modified or not, which have been expanded ex vivo prior to said infusion.

[0114] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0115] As used herein, the terms "treat", "treatment", and the like, when used to refer to a subject in need of therapeutic treatment, refer to obtaining the desired pharmacological and / or physiological effect, including but not limited to achieving amelioration or elimination of disease symptoms. With respect to a disease and / or adverse effects attributable to the disease, the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of achieving amelioration or elimination of symptoms, or providing a partial or complete cure. The term "treatment" includes any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the onset of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, or arresting its development; (c) alleviating the disease, or causing regression of the disease, or completely or partially eliminating the symptoms of the disease; and / or (d) restoring an individual to a pre-disease state, such as reconstructing the hematopoietic system.

[0116] As used herein, "therapeutically sufficient amount" includes within its meaning a non-toxic but sufficient and / or effective amount of the specific therapeutic agent and / or pharmaceutical composition to which it refers for providing the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's general health, the patient's age, and the stage and severity of the condition being treated. In some embodiments, a "therapeutically sufficient amount" is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0117] A. Compositions and methods for generating and differentiating induced pluripotent stem cells

[0118] In some aspects, the present invention generally relates to a multi-stage process for differentiating naive pluripotent cells into non-pluripotent cells or partially differentiated cells, including mesodermal progenitor cells, mesodermal cells, definitive hemogenic endothelial cells, definitive hematopoietic stem or progenitor cells, CD34 +Cells, multipotent progenitor cells (MPP) (capable of differentiating into bone marrow cells including neutrophil progenitor cells), T cell progenitor cells, NK cell progenitor cells; or fully differentiated terminal hematopoietic cells, such as, for example, T cells, B cells, NKT cells or NK cells. In various embodiments, such original pluripotent cells can be obtained by reprogramming source non-pluripotent cells into induced pluripotent stem cells (iPSCs), wherein iPSCs retain one or more therapeutic properties of source cells. In some aspects, the present invention relates to compositions used in the disclosed methods; and cell colonies, cell lines, cloned cells or master cell banks generated using the disclosed methods.

[0119] Existing methods for culturing pluripotent cells (such as iPSC) rely heavily on feeder cells or culture media pretreated with feeder cells and containing fetal bovine serum; however, such environments may not be suitable for producing cells for clinical and therapeutic purposes. For example, cells cultured in such xeno-contaminated environments are generally considered unsuitable for human cell transplantation because exposure to animal components may pose a serious risk of immune rejection and spread unknown pathogens to the treated patient, and may potentially reactivate animal retroviruses. The culture system contemplated herein using animal-free and feeder-free culture media promotes the manufacture of clinical-grade cell lines, particularly ESC, iPSC, and pluripotent stem cell-derived T, B, NKT, or NK cell lines.

[0120] In some embodiments, the feeder-free environment is substantially free of human feeder cells and is not pretreated with feeder cells, including but not limited to mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells. In some embodiments, the feeder-free environment is also free of stromal cells, such as OP9 stromal cells. The feeder-free cell culture medium is suitable for culturing pluripotent cells, single cell culture, dissociation, and passage of pluripotent cells; cell sorting of pluripotent cells; generating basal state pluripotent cells; maintaining basal state pluripotency; inducing pluripotent cell differentiation; and maturing effector cells differentiated from pluripotent cells.

[0121] In contrast to the methods used in the art, various aspects of the present invention avoid the formation of EB during differentiation. As provided in various embodiments, hematopoietic lineage cells derived from iPSC are obtained in the following manner: cloned iPSC cells are inoculated in a culture medium without TGFβ to maintain the basis or original state of its pluripotency, cloned iPSC is differentiated in a monolayer form without forming EB, and a step-by-step strategy is used to apply a suitable combination of small molecules, growth factors and / or cytokines in the early and mid-stages of differentiation. Therefore, aspects of the present invention enable the cloned iPSC of amplification to be directly transferred to adherent culture in a monolayer form to differentiate immediately, without the need to form EB from iPSC.

[0122] Compositions provided herein can be used in part to produce industrial or clinical grade pluripotent cells, which have a reduced spontaneous differentiation compared to the cells produced or cultured in the absence of compositions. In one embodiment, non-pluripotent cells are induced into pluripotent cells and cultured to maintain pluripotency for a long time. In another embodiment, non-pluripotent cells are induced into pluripotent cells and cultured to achieve and / or maintain the reduced spontaneous differentiation compared to the cells cultured in the absence of compositions. In another embodiment, non-pluripotent cells are induced into pluripotent cells and cultured to achieve and / or maintain basic state pluripotency (see, e.g., compositions in Tables 1 and 2).

[0123] In various embodiments, the compositions provided herein maintain basal state pluripotency, normal karyotype, and genomic stability of one or more pluripotent cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, or more passages, including any intermediate number of passages. In other embodiments, the compositions provided herein (see, e.g., Table 2) maintain reduced spontaneous differentiation in one or more pluripotent cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, or more passages, including any intermediate number of passages.

[0124] In various embodiments, the culture medium provided herein can include any defined basal medium suitable for supporting the maintenance and / or growth of stem cells, such as conventional human embryonic stem cell culture medium. Examples of defined basal culture media that can be used according to embodiments of the present invention include, but are not limited to: Dulbecco's Modified Eagle's Medium ("DMEM"), Basic Eagle's Medium (BME), DMEM / F-12 (1:1 DMEM and F-12, volume:volume); Medium 199; F-12 (Ham) Nutrient Mixture; F-10 (Ham) Nutrient Mixture; Minimal Essential Medium (MEM), -34, Williams medium E; and RPMI 1640, all of which are available from Gibco-BRL / Life Technologies, Inc., Gaithersburg, Md., etc. Several versions of many of these culture media are available, including but not limited to: DMEM 11966, DMEM 10314, MEM11095, Williams medium E 12251, Ham F12 11059, MEM-α12561, and medium-199 11151 (Gibco-BRL / Life Technologies). In various embodiments, the culture medium may include, for example, one or more of the following: amino acids, vitamins, organic salts, inorganic salts, trace elements, buffer salts, sugars, ATP, etc.

[0125] Small molecules and their types used in cell culture medium according to embodiments of the present invention are described more fully below. In one embodiment, the composition comprises cell culture medium and one or more of the following: TGFβ family proteins, Rho kinase inhibitors (ROCKi), and MEK inhibitors (MEKi) and WNT activators. In various embodiments, the composition does not include TGFβ inhibitors (TGFβi). In various embodiments, one or more of TGFβ family proteins, ROCKi, MEKi and WNT activators can be added at one or more specific stages during iPSC generation, maintenance and / or differentiation of a predetermined duration. Such specific stages during iPSC generation (reprogramming) include but are not limited to somatic cell transfection (day 0), exogenous gene expression, heterochromatin increase, somatic cell identity loss and iPSC colony formation. Specific stages during iPSC maintenance include, but are not limited to, single cell dissociation of iPSC colonies, single cell sorting of dissociated iPSCs, iPSC single cell clonal expansion, clonal iPSC master cell bank (MCB) cryopreservation, thawing of iPSC MCBs, and optional additional cryopreservation-thawing cycles of iPSC MCBs.

[0126] Table 1: Exemplary culture media for iPSC reprogramming and maintenance

[0127]

[0128]

[0129] Table 2 - Exemplary stage-specific culture media for iPSC reprogramming and maintenance

[0130]

[0131] Suitable nutrients / extracts may include, for example, KOSR (knockout serum replacement rate); L-glut; and NEAA (non-essential amino acids). Other media additives may include, but are not limited to, MTG, ITS, βME, antioxidants (e.g., ascorbic acid), and nicotinamide (NAM). In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial cell growth factor (VEGF), transferrin, various interleukins (such as IL-1 to IL-18), various colony stimulating factors (such as granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (such as IFN-γ), and other cytokines that have effects on stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines can be obtained commercially, for example, from R&D Systems (Minneapolis, Minnesota), and can be natural or recombinant. In some other embodiments, the culture medium of the present invention comprises one or more of the following: bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factor (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (Flt3L); and one or more cytokines, for example, leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11 and IL15. In some embodiments, growth factors / mitogens and cytokines are stage and / or cell type specific, and their concentrations are determined empirically or under the guidance of established cytokine technology. In some embodiments, cytokines are added at a specific stage of differentiation.

[0132] Any suitable vessel or cell culture container can be used as a support for cell culture in the basal medium and / or cell culture supplement. However, in some embodiments, coating the surface of the culture container with a matrix / substrate that promotes adhesion (e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.) can promote cell attachment, and in some embodiments can enhance the effect of the cell culture medium and supplements disclosed herein and / or provide feeder-free differentiation. Suitable matrices for culturing and passage cells are known in the art and include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombospondin, a mixture of matrices produced by naturally occurring cell lines (such as Matrigel TM ) and synthetic or artificial surfaces (such as polyamine monolayers and carboxyl-terminated monolayers). In some embodiments, providing feeder-free conditions includes culturing cells on a matrix-coated surface / substrate. In some embodiments, the matrix is ​​an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4. In some embodiments, the recombinant human fibronectin is In other embodiments, the matrix comprises Matrigel TM or vitronectin.

[0133] ROCK inhibitors

[0134] Rho-associated kinase (ROCK) is a serine / threonine kinase that acts as a downstream effector of Rho kinase (of which there are three isoforms - RhoA, RhoB and RhoC). ROCK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides and small molecules. ROCK inhibitors contemplated herein (also referred to as "ROCKi") can reduce ROCK expression and / or ROCK activity. Exemplary ROCK inhibitors include, but are not limited to, antibodies to ROCK, dominant negative ROCK variants, and siRNA and antisense nucleic acids that inhibit ROCK expression. Other exemplary ROCK inhibitors include, but are not limited to, thiabendazole, Y27632, Fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridinyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridinyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridinyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.

[0135] Exemplary ROCK inhibitors for use in cell culture media according to embodiments of the present invention include thiazolylvin, Y27632, pyrintegrin, Blebbistatin, and functional variants or derivatives thereof. In certain embodiments, the ROCK inhibitor is thiazolylvin.

[0136] ERK / MEK inhibitors

[0137] Exemplary inhibitors of the ERK / MEK pathway suitable for use in the compositions contemplated herein include, but are not limited to, antibodies to MEK or ERK, dominant negative MEK or ERK variants, and siRNA and antisense nucleic acids that inhibit expression of MEK and / or ERK. Other exemplary ERK / MEK inhibitors (also referred to as "MEK inhibitors" or "MEKi") include, but are not limited to, PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, Vandetanib, pazopanib, Axitinib, GSK1120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEA119, AZD6244, FR180204, PTK787, and functional variants or fragments thereof.

[0138] Additional illustrative examples of MEK / ERK inhibitors include the following compounds: 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide; 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo- 2-Chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide oxy)-amide, 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(2,4-dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide oxy)-amide, hereinafter referred to as MEK inhibitor 1; 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide; hereinafter referred to as MEK inhibitor 2; and 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide or a pharmaceutically acceptable salt thereof.

[0139] In some embodiments, the MEK / ERK inhibitor is PD0325901.

[0140] Wnt activators

[0141] As used herein, the term "Wnt signaling promoter", "Wnt pathway activator", "Wnt activator" or "Wnt pathway agonist" refers to an agonist of the Wnt signaling pathway, including but not limited to agonists of one or more of Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15 or Wnt16. Wnt pathway agonists also include but are not limited to one or more of the following polypeptides or fragments thereof: Dkk polypeptide, crescent polypeptide, cerberus polypeptide, Axin polypeptide, Frzb polypeptide, T cell factor polypeptide or dominant negative promiscuous polypeptide.

[0142] Non-limiting examples of Wnt pathway agonists also include one or more of the following: a nucleic acid comprising a nucleotide sequence encoding a Wnt polypeptide; a polypeptide comprising an amino acid sequence of a Wnt polypeptide; a nucleic acid comprising a nucleotide sequence encoding an activated Wnt receptor; a polypeptide comprising an amino acid sequence of an activated Wnt receptor; an organic small molecule that promotes Wnt / β-catenin signaling; an organic small molecule that inhibits the expression or activity of a Wnt antagonist; an antisense oligonucleotide that inhibits the expression of a Wnt antagonist; a ribozyme that inhibits the expression of a Wnt antagonist; an RNAi construct, siRNA or shRNA that inhibits the expression of a Wnt antagonist; an antibody that binds to a Wnt antagonist and inhibits its activity; a nucleic acid comprising a nucleotide sequence encoding a β-catenin polypeptide; a polypeptide comprising an amino acid sequence of a β-catenin polypeptide; a nucleic acid comprising a nucleotide sequence encoding a Lef-1 polypeptide; a polypeptide comprising an amino acid sequence of a Lef-1 polypeptide; and functional variants or fragments thereof.

[0143] GSK-3β inhibitors

[0144] GSK-3β inhibitors (also referred to as "GSK3 inhibitors" or "GSK3i") are specific exemplary Wnt pathway agonists suitable for use in the compositions contemplated herein, and may include, but are not limited to, antibodies that bind to GSK-3β, dominant negative GSK-3β variants, and siRNA and antisense nucleic acids targeting GSK-3β. Other exemplary GSK-3β inhibitors include, but are not limited to, Kenpaullone, 1-azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT20026, SB216763, AR-A014418, lithium, SB 415286, TDZD-8, BIO, BIO-acetone oxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopentadienylruthenium complex, TDZD-8, 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-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazine-2-yl-pyrrole-2,5-dione; TWS1 19 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-l-(4,5-dibromo-phenylthio-2-yl)-ethanone, SB216763, SB415286 and functional variants or fragments thereof. Exemplary GSK3 inhibitors for use in cell culture media according to embodiments of the present invention include CHIR99021, BIO and kemparotone. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0145] TGFβ receptor / ALK5 inhibitors

[0146] TGFβ receptor (e.g., ALK5) inhibitors may include antibodies against TGFβ receptor (e.g., ALK5), dominant negative variants thereof, and antisense nucleic acids that inhibit their expression. Exemplary TGFβ receptor / ALK5 inhibitors (also referred to as "ALK5i") include, but are not limited to, SB431542, A-83-01, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO, BMP4, GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16, IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide), GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) and pyrimidine derivatives. In addition, although "ALK5 inhibitor" is not intended to cover non-specific kinase inhibitors, "ALK5 inhibitor" should be understood to cover inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as SB-431542. Without intending to limit the scope of the invention, it is believed that ALK5 inhibitors affect the process of mesenchymal to epithelial transformation / transition (MET). The TGFβ / activin pathway is a driver of epithelial to mesenchymal transition (EMT). Therefore, inhibition of the TGFβ / activin pathway can promote the MET (ie, reprogramming) process.

[0147] It has been shown that inhibition of the TGFβ / activin pathway has a similar effect of inhibiting ALK5. Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or in place of the ALK5 inhibitors as described in the various paragraphs herein. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, SMAD 2 / 3 phosphorylation inhibitors, SMAD 2 / 3 and SMAD 4 interaction inhibitors, and activators / agonists of SMAD 6 and SMAD 7. In addition, the classification described below is for organizational purposes only and those skilled in the art will know that compounds can affect one or more points within the pathway, and therefore compounds can act in more than one defined category.

[0148] Specific examples of TGFβ receptor inhibitors include, but are not limited to, SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550 410; LY580276; LY364947; LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (morin); activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumor cells targeting TGFβ receptor.

[0149] Inhibitors of SMAD 2 / 3 phosphorylation may include antibodies, dominant negative variants, and antisense nucleic acids targeting SMAD2 or SMAD3. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (mulberry pigment). Inhibitors of the interaction of SMAD 2 / 3 and SMAD4 may include antibodies, dominant negative variants, and antisense nucleic acids targeting SMAD2, SMAD3, and / or SMAD4. Specific examples of inhibitors of SMAD 2 / 3 and SMAD4 interactions include, but are not limited to, Trx-SARA, Trx-xFoxH1b, and Trx-Lef1. Activators / agonists of SMAD 6 and SMAD 7 include, but are not limited to, antibodies, dominant negative variants, and antisense nucleic acids targeting SMAD 6 or SMAD 7.

[0150] p38MAPK inhibitors

[0151] The p38 mitogen-activated protein kinase (MAPK) pathway plays a key role in the release of proinflammatory cytokines such as IL-6 and is stimulated by the inflammatory cytokine tumor necrosis factor-α (TNF-α) as well as other stressors. Four different subgroups within MAPK have been identified, including the extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK / SAPK), ERK / BigMAP kinase 1 (BMK1), and p38MAPK protein kinase group. The p38 MAPK family includes p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12), and p38δ (MAPK13). Although the four p38 MAPK family members have different tissue expression patterns, studies have shown that p38 MAPK plays a key role in cell response, proliferation, survival, cell cycle, and migration in cancer, and therefore, the use of p38 MAPK inhibitors in chemotherapy has attracted much attention.

[0152] The various p38 inhibitors are structurally distinct small molecules whose common mechanism of action involves competitive inhibition of the adenosine binding pocket (ATP binding site) of p38. These inhibitors were selected because they occupy less conserved hydrophobic regions around the p38 binding site that induce conformational reorganizations to block or reduce ATP binding to the p38 protein. Exemplary small molecule p38 MAPK inhibitors suitable for differentiation of iPSCs into definitive hemogenic endothelial (HE) cells and thus various derivative cells include, but are not limited to, cyclopropyl-{4-[4-(4-fluorophenyl)-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl}amine (referred to as "DBM1285") VX745, VX-702, RO-4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RV568, CAS219138-24-6, SB203580, and SB242235. DBM1285 has previously been shown to inhibit TNF-α production. However, the present application discloses the use of p38 MAPK inhibitors for CD82 expression and maintenance of CD82 expressing cell populations, which subsequently increases the efficiency of obtaining permanent HE cells.

[0153] AhR inhibitors

[0154] The aryl hydrocarbon receptor (AhR) is a member of the Pern-Arnt-Sim (PAS) superfamily of transcription factors that are involved in sensing environmental cues such as circadian rhythms (BMAL1 and BMAL2) and changes in oxygen tension or redox potential (HIF-1α, HIF-2α, HIF-3α). AhR is expressed in hematopoietic stem and progenitor cells (HSPCs) and plays an important physiological role in hematopoiesis. Inhibition of AhR is used to expand human umbilical cord blood-derived HSPCs. However, as demonstrated herein, AhR inhibition can be used to modulate PSC-derived effector cell activation and function. Exemplary AhR inhibitors suitable for the disclosed uses in the methods and compositions described herein include, but are not limited to, CH-223191 (CAS 301326-227), UM729 (a pyrimidine indole derivative), UM171, and SR1 (StemRegenin 1).

[0155] HDAC inhibitors

[0156] Exemplary HDAC (histone deacetylase) inhibitors may include antibodies binding to HDAC, dominant negative variants of HDAC, and siRNA and antisense nucleic acids targeting HDAC. Histone acetylation participates in histone and DNA methylation regulation. In general, at the overall level, pluripotent cells have more histone acetylation, and differentiated cells have less histone acetylation. HDAC inhibitors promote the activation of silent pluripotency genes. Exemplary HDAC inhibitors suitable for compositions contemplated herein include but are not limited to TSA (trichostatin A), VPA (valproic acid), sodium butyrate (NaB), SAHA (suberoylanilide hydroxamic acid or vorinostat (vorinostat)), sodium phenylbutyrate, depsipeptide (FR901228, FK228), trapoxin (trapoxin) (TPX), peptide 1 (CHAP1) containing cyclic hydroxamic acid, MS-275, LBH589 and PXD101.

[0157] Cytokines and growth factors

[0158] In some embodiments, the compositions and / or cell culture media provided herein are substantially free of cytokines and / or growth factors. In certain embodiments, the cell culture media contains one or more supplements, including but not limited to serum, extracts, growth factors, hormones, cytokines, etc., which can be added in a stage-specific manner to improve the quality and efficiency of the reprogramming, maintenance and / or differentiation process.

[0159] Various growth factors and their use in culture media are known, including, for example, ECM proteins, laminin 1, fibronectin, collagen IV isoforms, proteases, protease inhibitors, cell surface adhesion proteins, cell signaling proteins, cadherins, chloride intracellular channel 1, transmembrane receptor PTK7, insulin-like growth factor, inhibin βA, inducers of the TGFβ / activin / nodal signaling pathway, and activin A. The cytokines used in the culture medium may include, for example, one or more of the following: growth factors such as epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), 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), leukemia inhibitory factor (LIF), vascular endothelial cell growth factor (VEGF), transferrin, various interleukins (such as IL-1 to IL-18), various colony stimulating factors (such as granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (such as IFN-γ) and other cytokines that act on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0160] In certain embodiments, the composition and / or culture medium may include a protein of the TGFβ family as a cytokine / growth factor component of the composition. Examples of TGFβ family proteins include, but are not limited to, activin A, TGFβ, nodal, and functional variants or fragments thereof. These cytokines / growth factors are commercially available and may be natural or recombinant. Other cytokines, if used, may be added at concentrations determined empirically or guided by established cytokine technology.

[0161] Cell reprogramming and iPSC maintenance

[0162] Reprogramming factors known in the art for stem cell reprogramming can be used together with this reprogramming method. In one embodiment, reprogramming factors include but are not limited to OCT4, SOX2, NANOG, KLF, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, YAP1 and large T antigen (LTag), and any combination thereof, as disclosed in International Publication No. WO 2015 / 134652 and WO 2017 / 066634, the disclosure of which is incorporated herein by reference. Reprogramming factors can also be in the form of polynucleotides encoding reprogramming factors, and therefore can be introduced into non-pluripotent cells by carriers (such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids and mini-rings). In some embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by lentiviral vectors. In some embodiments, one or more polynucleotides are introduced by episomal vectors. In various other embodiments, one or more polynucleotides are introduced by Sendai virus vectors. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids. See, for example, International Publication No. WO 2019 / 075057A1, the disclosure of which is incorporated herein by reference.

[0163] The polynucleotides encoding these reprogramming factors may be included in a polycistronic construct (i.e., multiple coding sequences controlled by one promoter) or a non-polycistronic construct (having multiple coding sequences controlled by one promoter and some controlled by different promoters). The promoter may be, for example, CMV, EF1α, PGK, CAG, UBC, and other suitable promoters of constitutive, inducible, endogenous regulation, or time-specific, tissue-specific, or cell-type specific. In one embodiment, the promoter is CAG. In another embodiment, the promoter is EF1α. In some embodiments, a polycistronic construct may provide a single open reading frame (e.g., multiple coding sequences may be operably connected by self-cleaving peptide coding sequences such as 2A) or multiple open reading frames (e.g., multiple coding sequences connected by internal ribosome entry sites or IRES).

[0164] An alternative approach to obtaining iPSCs is to use plasmid systems that mediate short-term transient and temporary transgene expression (see, e.g., the “STTR system” in U.S. Appl. Pub. No. 20200270581 and the “STTR2 system” described in International Pub. No. WO 2022 / 072883, the relevant disclosures of each of which are incorporated herein by reference).

[0165] In some embodiments, reprogramming of non-pluripotent cells is initiated in the presence of a combination of small molecule compounds comprising a ROCK inhibitor, a MEK inhibitor, a WNT activator, an HDAC inhibitor and / or a TGFβ inhibitor, and iPSCs are generated after a sufficient period of time (see, e.g., International Publication No. WO 2022 / 072883, the relevant disclosure of which is incorporated herein by reference).

[0166] Cells suitable for reprogramming generally include any non-pluripotent cells. Non-pluripotent cells include, but are not limited to, terminally differentiated cells; or pluripotent cells or progenitor cells that cannot produce all three types of germ layer lineage cells. In some embodiments, the non-pluripotent cells used for reprogramming are primary cells, i.e., cells directly isolated from human or animal tissues. In some embodiments, the non-pluripotent cells used for reprogramming are source-specific cells, such as donors, diseases, or treatment responses specific. In some embodiments, the non-pluripotent cells used for reprogramming are primary immune cells. In some embodiments, the non-pluripotent cells used for reprogramming are themselves derived from pluripotent cells, including embryonic stem cells and / or induced pluripotent stem cells. In some embodiments, the non-pluripotent cells used for reprogramming are derived immune effector cells, for example, non-natural or synthetic T-like or NK-like cells derived from iPSC.

[0167] In some other embodiments, the non-pluripotent cell for reprogramming is a primary cell or a derived cell of genome modification.The genetic modification included in the non-pluripotent cell may include insertion, deletion or replacement in the genome, which causes knock-in, knock-out or knock-down of gene expression.The modified expression in the non-pluripotent cell for reprogramming may be constitutive or inducible (for example, developmental stage specificity, tissue specificity, cell specificity or inducer specificity).In some embodiments, insertion or replacement is locus specific targeted integration.In some embodiments, the selected locus for integration is a safe harbor locus or an endogenous locus of concern.

[0168] In one embodiment, the genetically modified non-pluripotent cells are reprogrammed to obtain genome-engineered iPSCs comprising the same genetic modifications. In some other embodiments, one or more such genome edits may be introduced into iPSCs after reprogramming to obtain genome-engineered iPSCs. In some embodiments, the genome-engineered iPSCs include polynucleotides encoding a cytokine signaling complex, which includes exogenous cytokines expressed on the cell surface and / or part or all of its receptor peptides, so that when subsequently differentiated into hematopoietic lineage cells by permanent hemogenic endothelial cells (HE), the culture medium does not need or need not include cytokines. See, for example, International Application No. PCT / US2022 / 073396 and International Publication Nos. WO 2022 / 098914 and WO 2022 / 098925, the entire disclosures of each of which are incorporated herein by reference. In one embodiment, the permanent HE cell comprises a gene insertion of a polynucleotide encoding a cytokine signaling complex for IL15 signaling (IL15 signaling complex), including but not limited to IL15, IL15RF (an IL15 / IL15R receptor fusion protein) or IL15Δ (an IL15 / IL15Rα fusion protein without an intracellular domain), as described in International Publication Nos. WO 2019 / 191495 and WO 2019 / 126748, the entire disclosures of each of which are incorporated herein by reference. In various embodiments, the iPSC used for genome editing is a clonal line or a clonal iPS cell population.

[0169] iPSC differentiation

[0170] (1) iPSC to iHE

[0171] One aspect of the present invention provides methods and compositions for obtaining permanent hemogenic endothelial cells (HE) using pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). As used herein, permanent hemogenic endothelial cells are a population of hemogenic cells that are committed to permanent hematopoiesis and have the ability to generate all hematopoietic cells (including but not limited to pre-T cell progenitors, T cell progenitors, T cells, and pre-NK cell progenitors, NK cell progenitors, NK cells, NKT cells, B cells, and other hematopoietic cells). In some aspects, the present invention provides compositions and methods for obtaining hematopoietic lineage cells from permanent HE cells or permanent hemogenic endothelial cells differentiated from iPSCs.

[0172] Typically, the technology for differentiating iPSCs or permanent HE cells derived therefrom involves directly or indirectly regulating specific cell pathways using polynucleotide-based, polypeptide-based and / or small molecule-based methods. For example, the developmental capacity of cells can be regulated by contacting cells with one or more regulators. As used herein, "contact" may involve culturing cells in the presence of one or more factors (such as, for example, small molecules, proteins, peptides, etc.). In some embodiments, cells are contacted with one or more agents to induce cell differentiation. Such contact can occur, for example, by introducing one or more agents into cells during in vitro culture. Thus, contact can occur by introducing one or more agents into cells in a cell culture medium. Cells can be maintained in a culture medium comprising one or more agents for a time sufficient for the cells to achieve the desired differentiation phenotype. In some other embodiments, "contact" occurs when one or more factors are introduced into cells via a vector, as discussed below. In some embodiments, one or more vectors are introduced via retrovirus, Sendai virus, adenovirus, episome, mini-circle, vector system with expression cassette, or mRNA.

[0173] In various embodiments, the culture platform for generating hematopoietic cell lineages from iPSC or permanent hemogenic endothelial cells derived therefrom as provided herein does not contain or is substantially free of inhibitors of TGFβ / activin signaling pathways, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In some embodiments, the cell culture medium for differentiating permanent hemogenic endothelial cells does not contain stromal cells, such as OP9 stromal cells. In one embodiment, the culture platform includes an inoculation medium for maintaining the original iPSC. It is also important to achieve the basal state or original state multipotency of iPSC for obtaining permanent HE cells and hematopoietic lineage cells by differentiating iPSC without forming EB intermediates. In addition, the use of monolayer culture without forming EB and its aggregates will also greatly affect the efficiency of the original iPSC differentiation into permanent HE. In some embodiments, the inoculation medium includes a ROCK inhibitor and does not contain or is substantially free of TGFβR / ALK5 inhibitors. In some other embodiments, the inoculation medium includes a GSK3 inhibitor, but does not contain a TGFβR / ALK5 inhibitor. In yet other embodiments, the inoculation medium comprises a GSK3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor.

[0174] One aspect of the present invention provides a culture medium for obtaining permanent hemogenic endothelial (HE) cells differentiated from pluripotent stem cells including iPSC. In one embodiment, the culture medium comprises one or more of a BMP activator, bFGF and optional VEGF, a Wnt pathway activator, a p38 MAPK inhibitor or any combination thereof. In one embodiment, the culture medium comprises a BMP activator, bFGF, VEGF and a Wnt pathway activator. In some other embodiments, the culture medium comprises a BMP activator, bFGF, VEGF, a Wnt pathway activator and a p38 MAPK inhibitor. In one embodiment, after the differentiated cells from iPSC obtain specialized mesoderm progenitor cells, VEGF, a Wnt pathway activator and / or a p38 MAPK inhibitor are added to a culture medium comprising a BMP activator and bFGF. In one embodiment, the above culture medium does not contain a TGFβ receptor / ALK inhibitor. In one embodiment, the Wnt pathway activator is a GSK3β inhibitor. Without being limited by theory, a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor contributes to improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor; whereas a BMP activator results in a higher percentage of RUNX1 expressing cells in the HE population compared to differentiation without cytokines. In some embodiments, the BMP activator comprises BMP4. In some embodiments, the p38 MAPK inhibitor comprises at least one of DBM1285, VX-745, VX-702, RO-4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RV568, CAS219138-24-6, SB203580, and SB242235. In one embodiment, the p38 MAPK inhibitor comprises DBM1285.

[0175] The pluripotent stem cells, optionally seeded and expanded as described above, are then differentiated into mesodermal progenitor cells expanded at this stage. The expanded mesodermal progenitor cell population is then differentiated into a mesodermal population with permanent hemogenic endothelial cell potential, and then differentiated into a permanent hemogenic endothelial (HE) cell population. Alternatively, the method for generating iPSC-derived permanent hemogenic endothelial cells (iHE) can start from pluripotent stem cell-derived mesodermal progenitor cells, wherein the method includes differentiating mesodermal progenitor cells into permanent hemogenic endothelial (iHE) cells. In some embodiments of the above methods, iHE is suitable for cryopreservation. In some embodiments, the pluripotent stem cell is iPSC. In some embodiments, iPSC is an original iPSC.

[0176] In some embodiments of the above-described method for obtaining iPSC-derived hemogenic endothelial cells, the method comprises (i) differentiating pluripotent stem cells to obtain mesodermal progenitor cells or a population thereof by contacting the iPSCs with a medium comprising a BMP activator and bFGF in addition to a basal medium; and (ii) differentiating the mesodermal progenitor cells to obtain HE cells by contacting the mesodermal progenitor cells with a medium comprising a BMP activator, bFGF, VEGF, a Wnt pathway activator, and optionally a p38 MAPK inhibitor, wherein the obtained HE cells comprise definitive HE cells.

[0177] In some embodiments of the above method comprising (i) and (ii), the method further comprises (iii) sorting the obtained HE cells using antibodies that recognize cell surface markers including CD34, CD82, CD43, CD73, CXCR4 and / or CD93, thereby generating an enriched permanent HE cell subpopulation. In some embodiments, the sorting uses an anti-CD34 antibody, and the enriched iHE cell subpopulation is CD34 + In some embodiments, the sorting uses an anti-CD82 antibody and the enriched permanent HE cell subpopulation is CD82 + In some embodiments, the sorting uses both anti-CD34 and anti-CD82 antibodies, and the enriched subpopulation of permanent HE cells is CD34 + CD82 + In some embodiments, the sorting uses anti-CD34, anti-CD82, and anti-CD43 antibodies, and the enriched permanent HE cell subpopulation is CD34 + CD82 + CD43 - In some embodiments, sorting uses CD34 + 、CD82 + 、CD43 - and CD73 - In some other embodiments, the sorting uses CD34 + 、CD82 + 、CD43 - 、CD73 - and CXCR4 - In some embodiments, sorting uses CD34 + 、CD82 + and CD93 - In some embodiments, sorting uses CD34 + 、CD82 + 、CD43 - and CD93 - In some embodiments, sorting uses CD34 +、CD43 - and CD73 - In some other embodiments, the sorting uses CD34 + 、CD43 - 、CD73 - and CXCR4 - .

[0178] (2) iPSC or iHE to T lineage cells

[0179] Provided herein is a culture platform for generating T cell progenitors or T cells from permanent hemogenic endothelial cells. In one embodiment, the culture medium comprises SCF, Flt3L, IL7 and optional ROCK inhibitors, TPO and IL3, wherein the culture medium does not contain one or more of VEGF, bFGF and BMP activators. In some embodiments, the culture medium for differentiating permanent HE into T cell progenitors comprises ROCK inhibitors, SCF, Flt3L, TPO and IL7; and does not contain BMP activators. In some embodiments, the culture medium for differentiating T cell progenitors into T cells comprises SCF, Flt3L and IL7; and does not contain TPO, IL3, BMP activators or ROCK inhibitors. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, Notch factors are used in a culture platform for generating T cell progenitors or T cells from iPSC or permanent HE cells. In some embodiments, Notch factors (including Jag1, Jag2, DLL-1, DLL-3 and DLL-4) can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to a culture surface, peptides contained in an extracellular matrix coated on a culture surface, or peptides presented by stromal cells.

[0180] A method for generating pluripotent stem cell derived T cell progenitors or T cells using a multi-stage process is also provided. Typically, the method begins with inoculation and optionally amplification of pluripotent stem cells, which differentiate into mesodermal progenitors and then differentiate into HE cells, and HE cells can be optionally sorted to obtain enriched permanent HE for subsequent T lineage cell differentiation. Alternatively, HE can be used as a starting cell for T lineage cell differentiation. The method and various embodiments (including for methods) for differentiating iPSC into HE cells are as described above, and a method for differentiating HE cells into T lineage cells after or independently of iPSC differentiation into HE cells is provided herein.

[0181] In some embodiments of the method for differentiating HE cells into T cell progenitors or T cells, the method comprises contacting HE cells with a culture medium comprising one or more growth factors and cytokines (including SCF, Flt3L and IL7) and optionally one or more factors (including TPO, IL3 and ROCK inhibitors); wherein the culture medium does not contain or substantially does not contain one or more of VEGF, bFGF and BMP activators. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments of the above methods, differentiating iHE cells into pre-iproTs comprises contacting iHE cells with a culture medium comprising a ROCK inhibitor, SCF, Flt3L, TPO and IL7. In other embodiments, differentiating pre-iproTs into ipro-Ts or iTs comprises contacting pre-iproT cells with a culture medium comprising SCF, Flt3L and IL7, wherein the culture medium does not contain or substantially does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors. In some embodiments of the above methods, Notch factors are used in the culture medium for generating T cell progenitors or T cells. In some embodiments, Notch factors (including Jag1, Jag2, DLL-1, DLL-3, and DLL-4) can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, peptides contained in an extracellular matrix coated on a cell culture surface, or peptides presented by cells. In some embodiments, differentiation of iHE cells into T lineage cells occurs in the absence of OP9 stromal cells (OP9-free hematopoietic cell differentiation). In some embodiments, OP9-free differentiation of iHE is performed in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4. In some embodiments, recombinant human fibronectin is

[0182] (3) iPSC or iHE to NK lineage cells

[0183] NK lineage cells can be differentiated from iPSCs to iPSC-derived permanent hemogenic endothelial (HE) cells, or directly from permanent HE cells.

[0184] Compositions and methods for differentiating iPSCs into permanent HE cells (including HE cell sorting) and various embodiments thereof are described in the previous section. Additional culture platforms and methods for differentiating permanent hemogenic endothelial cells to obtain NK lineage cells (including NK progenitors and NK cells) are also provided herein.

[0185] In some embodiments of the culture platform for obtaining NK lineage cells from HE cells, the culture platform comprises at least a culture medium comprising SCF, Flt3L, IL7, IL3, and optionally IL15, IL21, TPO, ROCK inhibitor, AhR antagonist, 4-1BB agonist, and nicotinamide, wherein the culture medium does not contain VEGF, bFGF, BMP activator, OP9 stromal cells, and feeder cells. In some embodiments of the culture medium for generating NK progenitor cells or NK cells, the culture medium does not contain K562 feeder cells or engineered variants thereof. In some embodiments, the ROCK inhibitor comprises Thiazole Vivin or Y27632.

[0186] In some embodiments of the culture medium for generating NK progenitor cells or NK cells, the culture medium comprises an AhR antagonist. In some embodiments, the AhR antagonist is a small molecule AhR inhibitor. Without being limited by theory, in some embodiments, the AhR inhibitor in the culture medium can be used for NK cell progenitor cells or NK cell expansion and / or activation. In some embodiments, the AhR inhibitor comprises at least one of CH-223191, UM729, UM171 and SR1. In some embodiments, the culture medium for generating NK progenitor cells or NK cells comprises CH-223191. In some embodiments, in addition to one or more of the AhR antagonists, NK expansion, maturation and / or activation are also carried out in the presence of nicotinamide (NAM) contained in the culture medium for generating NK progenitor cells or NK cells.

[0187] A method for generating NK progenitor cells or NK cells derived from pluripotent stem cells using a multi-stage process is also provided. Typically, the method begins with inoculation and optionally amplification of pluripotent stem cells, which differentiate into mesodermal progenitor cells and then differentiate into HE cells, and HE cells can be optionally sorted to obtain enriched permanent HE for subsequent NK lineage cell differentiation. Alternatively, HE can be used as a starting cell for NK lineage cell differentiation. The method and various embodiments (including for methods) for differentiating iPSC into HE cells are as described above, and a method for differentiating HE cells into NK lineage cells after or independently of iPSC differentiation into HE cells is provided herein.

[0188] In some embodiments, the method of differentiating HE cells into NK lineage cells comprises contacting iHE cells with a composition comprising SCF, Flt3L, IL3 and IL7; and optionally one or more of a ROCK inhibitor, TPO, IL15, immobilized IL21, a 4-1BB agonist, an AhR antagonist, and nicotinamide, thereby obtaining NK lineage cells, including NK progenitor cells and NK cells. In some embodiments of the method, IL15 is contained in the culture medium. In some other embodiments of the method, IL15 is in the form of an IL15 signaling complex encoded by an exogenous polynucleotide, which is introduced into permanent HE cells by gene insertion, wherein the IL15 signaling complex comprises a portion or all of a peptide of an exogenous IL15 and / or its receptor expressed on the cell surface. In some embodiments, differentiation of iHE cells into NK progenitor cells or NK cells occurs in the absence of OP9 stromal cells. In some embodiments, differentiation of iHE cells into NK progenitor cells or NK cells occurs in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4, such that OP9 stromal cells are not required. In some embodiments, recombinant human fibronectin is In some embodiments, the method of differentiating iHE cells into iNK cells comprises contacting the iHE cells with a composition comprising SCF, Flt3L, IL3, IL7, a ROCK inhibitor, immobilized IL21, a 4-1BB agonist, and optionally one or both of TPO and IL15, thereby obtaining iPSC-derived NK progenitor cells or NK cells.

[0189] In some embodiments, the method of differentiating iHE cells into iNK cells further comprises contacting iPSC-derived NK progenitors or NK cells with one or both of an AhR antagonist and nicotinamide, thereby expanding and / or activating iPSC-derived NK cells. In some embodiments, NK activation occurs in the presence of a small molecule AhR inhibitor, thereby regulating NK lineage cell maturation.

[0190] B. Cell Populations and Cell Lines Generated by the Methods and Compositions Provided Herein

[0191] According to the above, one of the advantages provided by the method described herein is: in the case of not forming EB, the viability and survival rate of culturing, passage and dissociating single pluripotent cells are enhanced for pluripotent stem cell differentiation. Cell dissociation into single cells can be achieved by enzymatic or mechanical means, such as dissociating into single cell suspensions. Any enzyme agent known in the art that allows cells to be dissociated into single cells can be used in the embodiments of the present method. In one embodiment, the dissociating agent is selected from trypsin / EDTA, TrypLE-Select, collagenase IV and dispase. Chelating agents (such as EDTA, Accutase or AccuMax) can also be used alone or in combination with enzyme agents to dissociate cells according to the method envisioned herein. The dissociating agent can be dissolved in PBS without calcium and magnesium to promote dissociation into single cells. In order to enhance the survival rate of cells during and after dissociation, in some embodiments, substances that promote survival are added, for example, one or more growth factors, inhibitors or conditioned medium of cell pathways involved in cell death and apoptosis. In one embodiment, the substance that promotes survival is a ROCK inhibitor, including but not limited to Thiazole Weiwen.

[0192] In some embodiments, pluripotent stem cells are iPSC. In some embodiments, iPSC is original iPSC. In some embodiments, iPSC is reprogrammed by immune cells of specific donors or patients. In some embodiments, the cells cultured after reprogramming have been induced to differentiate for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 15 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days, 35 days, 40 days, 42 days or 45 days, or any number of days therebetween. In some embodiments, the cells cultured after reprogramming have been induced to differentiate for about 1 day to 42 days, 2 days to 40 days, 2 days to 35 days, 2 days to 20 days, 2 days to 10 days, 4 days to 30 days, about 4 days to 24 days, about 6 days to 22 days or about 8 days to about 12 days.

[0193] In some embodiments, iPSC is genome engineered. In some embodiments, the iPSC for differentiation comprises one or more genetic imprints. In some embodiments, the genetic imprints in pluripotent stem cells include (i) one or more gene modification patterns obtained by genome insertion, deletion or substitution in the pluripotent cell genome during or after reprogramming non-pluripotent cells to iPSCs; or (ii) one or more of the source-specific immune cells specific to the donor, disease or treatment response can retain therapeutic properties, and wherein the pluripotent cells are reprogrammed by source-specific immune cells, wherein the iPSC retains the source therapeutic properties, and the iPSC-derived hematopoietic lineage cells also include the source therapeutic properties. In some embodiments, the gene modification pattern includes one or more of the following: safety switch proteins, targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote transplantation, transportation, homing, vitality, self-renewal, retention, immune response regulation and regulation and / or survival of iPSC or its derivative cells. In some other embodiments, the gene modification pattern comprises one or more of the following: (i) deletion, disruption or reduced expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CITTA, RFX5 or RFXAP; or (ii) deletion, disruption or reduced expression of HLA-E, HLA-G, hnCD16, 4-1BBL, CD3, CD4, CD8, CD47, CD137, CD80, PDL1, A 2A R, CAR, TCR or bispecific or multispecific adapter surface triggering receptor introduction or increased expression. In some embodiments, the surface triggering receptor is universal, that is, compatible with any effector cell type, and the effector cell expressing the universal surface triggering receptor can be coupled with the same bispecific or multispecific adapter, regardless of its cell type. In some other embodiments, the hematopoietic lineage cell comprises the therapeutic properties of the source-specific immune cell associated with one or more of the following: (i) antigen targeting receptor expression; (ii) HLA presentation or its lack; (iii) resistance to the tumor microenvironment; (iv) induction and immunomodulation of bystander immune cells; (v) as the tumor off-effect decreases, the on-target specificity is improved; (vi) resistance to treatments such as chemotherapy; and (vii) improved homing, retention and cytotoxicity.

[0194] In some embodiments, the adaptor is cell type specific, i.e., the adaptor binds and / or activates a specific immune cell type. In certain embodiments, the adaptor is cell type independent, i.e., the adaptor binds and / or activates a variety of immune cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils.

[0195] In some embodiments, iPSCs include one or more targeted edits at one or more desired sites, wherein the one or more targeted edits remain intact and functional at the corresponding selected edit sites in the expanded iPSCs or iPSC-derived non-pluripotent cells. Targeted editing introduces insertions, deletions, and / or substitutions into the genome of iPSCs and their derived cells (i.e., introduces targeted integration and / or insertions / deletions at selected sites). In some embodiments, iPSCs and their derived hematopoietic cells comprise B2M negative, HLA-E / G, PDL1, A 2A One or more of R, CD47, LAG3 negative, TIM3 negative, TAP1 negative, TAP2 negative, Tapasin negative, NLRC5 negative, PD1 negative, RFKANK negative, CITTA negative, RFX5 negative and RFXAP negative. These cells with modified HLA class I and / or class II have increased resistance to immune detection and therefore exhibit improved in vivo retention. In addition, such cells can avoid the need for HLA matching in adoptive cell therapy and therefore provide a source of universal, ready-made treatment options.

[0196] In some embodiments, iPSCs and derived hematopoietic cells thereof comprise one or more of hnCD16, 4-1BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells have improved immune effector capabilities.

[0197] In some embodiments, iPSC and its derived hematopoietic cells include surface triggering receptors for coupling with bispecific or multispecific adapters. Such cells have improved tumor targeting specificity. In some embodiments, iPSC and its derived hematopoietic cells are antigen-specific. In some embodiments, iPSC and its derived hematopoietic cells include polynucleotides encoding cytokine signaling complexes, which include exogenous cytokines expressed on the cell surface and / or part or all of their receptors, so that cytokines are not required or necessary to be included in the culture medium.

[0198] As described above, these methods include strategies for enriching cell colonies with specific characteristics at different stages of the method. In one embodiment, the method for enriching pluripotent stem cells from a cell colony includes preparing a single cell suspension by dissociating the cells in the colony and resuspending the cells. The dissociated cells can be resuspended in any suitable solution or culture medium to maintain the cells or perform cell sorting. In various embodiments, enrichment provides a method for deriving cloned iPSC colonies in a relatively short period of time, thereby improving the efficiency of iPSC production. Enrichment may include sorting cell colonies by identifying and obtaining cells expressing pluripotency markers, thereby obtaining enriched pluripotent cell colonies. Additional enrichment methods include exhausting cells expressing differentiation markers, non-reprogrammed or non-pluripotent cells. In some embodiments, the cells for sorting are pluripotent cells. In some embodiments, the cells for sorting are reprogrammed cells. In some embodiments, the cells used for sorting have been induced to reprogram for at least 1, 2, 3, 4, 5, 6, 7, 8 days or more, but no more than 25, 26, 28, 30, 32, 35, 40 days, or any days in between.

[0199] Cells can be sorted by any suitable cell sorting method, such as by magnetic bead sorting or flow cytometry (FACS) sorting. iPSC can be sorted based on one or more pluripotency markers, including but not limited to SSEA3 / 4, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD105, OCT4, NANOG, SOX2, KLF4, SSEA1 (mouse), CD30, SSEA5, CD90 and / or CD50 expression. In various embodiments, iPSC is sorted based on at least two, at least three or at least four pluripotency markers. In certain embodiments, iPSC is sorted based on the expression of SSEA4, and in some embodiments, iPSC is sorted based on the expression of SSEA4 in combination with TRA1-81 and / or TRA1-60. In certain embodiments, iPSC is sorted based on the expression of SSEA4, TRA1-81 or TRA1-60 and / or CD30. In one embodiment, iPSC is sorted based on SSEA4, TRA1-81 and CD30. In another embodiment, iPSC is sorted based on SSEA4, TRA1-60 and CD30. In some embodiments, non-reprogrammed cells in cells are initially depleted using one or more surface markers of differentiated cells (including but not limited to CD13, CD26, CD34, CD45, CD31, CD46 and CD7), and then pluripotent markers are enriched, such as SSEA4, TRA1-81 and / or CD30.

[0200] In one embodiment, enrichment provides a method for obtaining cloned pluripotent stem cell-derived differentiated cell colonies in a relatively short period of time, thereby improving the efficiency of generating pluripotent stem cell-derived differentiated cells at different stages. In one embodiment, enrichment provides a method for deriving a HE cell population expressing CD34, an HSC cell population expressing CD34, an iHE cell population, a T or NK progenitor cell population, and / or a T or NK cell population, thereby improving the efficiency of generating each cell population. Enrichment may include sorting the cell population to identify and obtain cells expressing specific characteristic markers indicating the differentiation stage / cell type. As described above, one or more antibodies that recognize one or more cell surface markers (including CD34, CD82, CD43, CD73, CXCR4 and / or CD93) can be used to generate enriched iHE cell subpopulations. In some embodiments, the antibody used for cell sorting comprises an anti-CD34 antibody, and the enriched iHE cell subpopulation is CD34. +In some embodiments, the antibody used for cell sorting comprises an anti-CD82 antibody, and the enriched iHE cell subpopulation is CD82 + In some embodiments, the antibodies used for sorting comprise both anti-CD34 antibodies and anti-CD82 antibodies, and the enriched iHE cell subpopulation is CD34 + CD82 + In some embodiments, the antibodies used for cell sorting comprise anti-CD34, anti-CD82, and anti-CD43 antibodies, and the enriched iHE cell subpopulation is CD34 + CD82 + CD43 - Additional enrichment methods include depleting cells expressing markers representative of undesirable cell types to obtain an enriched population of the desired cell type.

[0201] Therefore, another aspect of the present invention provides a composition comprising one or more cell populations, cell lines or cloned cells of: (i) a pluripotent stem cell-derived CD34 + HE cells (iCD34), wherein the iCD34 cells have the ability to differentiate into multipotent progenitor cells, and wherein the iCD34 cells are CD34 + CD43 - (ii) pluripotent stem cell-derived permanent hemogenic endothelial cells (iHE), wherein the iHE cell line or clone is CD82 + or CD82 + and CD34 + 、CD93 - CXCR4 - 、CD73 - and CXCR4 - CD73 - (iii) pluripotent stem cell-derived multipotent progenitor cells (iMPP), wherein the iMPP cells are CD34 + CD45 + (iv) pluripotent stem cell-derived T cell progenitors (ipro-T), wherein the T cell progenitors are CD34 + CD45 + CD7 + (v) pluripotent stem cell-derived T cells (iT), wherein the T cells are CD45 + CD4 + CD3 + or CD45 + CD8 + CD3 + (vi) pluripotent stem cell-derived NK progenitor cells (ipro-NK), wherein the NK progenitor cells are CD45+ CD56 + CD7 + CD3 - ; and (vii) pluripotent stem cell-derived NK cells (iNK), wherein the NK cells are CD45 + CD56 + NKp46 + In some embodiments, the above compositions, cell populations, cell lines, or cloned cells are suitable for cryopreservation. In some embodiments, these compositions, cell populations, cell lines, or cloned cells are suitable for ambient storage conditions for more than 12 hours, 24 hours, 36 hours, 48 ​​hours, but not more than 3 days, 4 days, 5 days, 6 days, or a week.

[0202] C. Therapeutic Uses of iPSC-Derived Immune Cells

[0203] In one aspect, the present invention also provides a composition comprising an immune cell colony or subpopulation derived from the separation of iPSC using the disclosed methods and compositions, wherein these immune cells are suitable for cell-based adoptive therapy. In one embodiment, the immune cell colony or subpopulation of separation comprises HSC cells derived from iPSC. In one embodiment, the immune cell colony or subpopulation of separation comprises T cells derived from iPSC. In one embodiment, the immune cell colony or subpopulation of separation comprises NK cells derived from iPSC. In some embodiments, the immune cell colony or subpopulation derived from the separation of iPSC comprises the original T cells, stem cell memory T cells and / or central memory T cells of the increased number or ratio. In one embodiment, the immune cell colony or subpopulation derived from the separation of iPSC comprises the type I NKT cells of the increased number or ratio. In another embodiment, the immune cell colony or subpopulation derived from the separation of iPSC comprises the adaptive NK cells of the increased number or ratio. In some embodiments, the HSC cells, T cells or NK cell colonies or subpopulations derived from the separation of iPSC are allogeneic. In some other embodiments, the isolated population or subpopulation of HSC cells, T cells, or NK cells derived from iPSCs is autologous.

[0204] A variety of diseases can be improved by introducing immune cells according to various aspects of the present invention to a subject suitable for adoptive cell therapy. Examples of diseases including a variety of autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome ( syndrome), systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease); hematological malignancies, including but not limited to acute and chronic leukemias, lymphomas, multiple myeloma, and myelodysplastic syndrome; solid tumors, including but not limited to brain, prostate, breast, lung, colon, uterine, skin, liver, bone, pancreatic, ovarian, testicular, bladder, kidney, head, neck, stomach, cervix, rectal, laryngeal, or esophageal tumors; and infections, including but not limited to HIV (human immunodeficiency virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), adenovirus, and BK polyomavirus-associated conditions.

[0205] Specific embodiments of the present invention relate to methods of treating a subject by administering to a subject in need thereof a composition comprising any of the cells described herein. In some embodiments, the composition comprising any of the cells described herein may be administered in combination with a therapeutic agent. The therapeutic agent and / or composition may be administered before, during, or after the onset of the disease or injury. The treatment of developing diseases is of particular interest, wherein the treatment stabilizes or reduces the patient's undesirable clinical symptoms.

[0206] In some embodiments, the subject suffers from a disease, condition and / or injury that can be treated, improved and / or improved by cell therapy. Some embodiments envision that the subject in need of cell therapy is a subject suffering from damage, disease or condition, whereby cell therapy (e.g., wherein the cell material is applied to the subject's therapy) can treat, improve, improve and / or reduce the severity of at least one symptom associated with damage, disease or condition. Some embodiments envision that the subject in need of cell therapy includes but is not limited to bone marrow or stem cell transplant candidates, subjects who have received chemotherapy or irradiation therapy, subjects suffering from or at risk of producing hyperproliferative disorders or cancer (e.g., hyperproliferative disorders or hematopoietic cancer), subjects suffering from tumors (e.g., solid tumors) or at risk of suffering from tumors, subjects suffering from viral infection or diseases related to viral infection or subjects at risk of suffering from viral infection or diseases related to viral infection.

[0207] Therefore, aspects of the present invention also provide pharmaceutical compositions, which include hematopoietic lineage cells derived from pluripotent cells prepared by the methods and compositions disclosed herein, wherein the pharmaceutical compositions also include a pharmaceutically acceptable medium. In one embodiment, the pharmaceutical compositions include T cells derived from pluripotent cells prepared by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical compositions include NK cells derived from pluripotent cells prepared by the methods and compositions disclosed herein.

[0208] In addition, various aspects of the present invention provide therapeutic uses of the above-mentioned pharmaceutical compositions by introducing / administering the compositions to a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disorder; a hematological malignancy; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0209] As will be understood by those of ordinary skill in the art, both autologous and allogeneic immune cells (including hematopoietic lineage cells) derived from iPSC based on the methods and compositions provided herein can be used in cell therapy. Autologous cell therapy can have reduced infection, low GvHD probability and rapid immune reconstruction. Allogeneic cell therapy can have immune-mediated graft-versus-host disease (GVM) effect and low relapse rate. For autologous transplantation, in some embodiments, the separated derived hematopoietic lineage cell colony is completely or partially HLA matched relative to the patient. In other embodiments, derived hematopoietic lineage cells are not matched with subject HLA, wherein derived hematopoietic lineage cells are NK cells or T cells lacking HLA-I and / or HLA-II. Based on the specific conditions of patients or subjects requiring cell therapy, those of ordinary skill in the art will be able to determine which specific type of therapy is administered.

[0210] In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is at least 0.1×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 5×10 6 cells, at least 1×10 7 cells, at least 5×10 7 cells, at least 1×10 8 cells, at least 5×10 8 cells, at least 1×10 9 cells or at least 5×10 9 In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1×10 5 cells to about 1×10 6 cells; approximately 0.5×10 per dose 6 cells to about 1×10 7 cells; approximately 0.5×10 per dose 7 cells to about 1×10 8 cells; approximately 0.5×10 per dose 8 cells to about 1×10 9 cells; approximately 1×10 per dose 9 cells to about 5×10 9 cells; approximately 0.5×10 per dose 9 cells to about 8×10 9 cells; approximately 3×10 per dose 9 cells to about 3×10 10 cells, or any range in between. Generally, for a 60 kg patient, 1×10 8 cells / dose is equivalent to about 1.67×10 6 cells / kg.

[0211] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a portion or a single umbilical cord blood, or 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 0.75×10 6 cells / kg body weight, at least 1.25×106 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, at least 30×10 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight or 1×10 9 cells / kg body weight.

[0212] In one embodiment, a dose of derived hematopoietic lineage cells is delivered to a subject. In an exemplary embodiment, an effective amount of cells provided to a subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg or at least 10×10 6 cells / kg or more, including all intermediate cell doses.

[0213] In some embodiments, the therapeutic use of derived hematopoietic lineage cells is single-dose treatment. In some embodiments, the therapeutic use of derived hematopoietic lineage cells is multi-dose treatment. In some embodiments, multi-dose treatment is a dose per day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days or every 50 days or any number of days during. In some embodiments, multi-dose treatment includes three times, four times or five times a week, a dose. In some embodiments, including three times, four times or five times a week, a dose of multi-dose treatment also includes an observation period for determining whether additional single doses or multiple doses are needed.

[0214] Some variation in dosage, frequency and regimen will necessarily occur, depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage, frequency and regimen for the individual subject in each case.

[0215] Compositions comprising derived hematopoietic lineage cell groups of embodiments of the present invention may be sterile, and may be suitable for administration and ready for administration (i.e., administration without any further treatment) to human patients / subjects. Cell-based compositions prepared for administration mean that the composition does not require any additional processing or manipulation before transplantation or administration to a subject. In other embodiments, the present invention provides a separate population of derived hematopoietic lineage cells that are expanded and / or regulated before administration with one or more agents including small chemical molecules. Compositions and methods for regulating immune cells (including iPSC-derived effector cells) are described in more detail in, for example, International Publication No. WO 2017 / 127755, and its related disclosures are incorporated herein by reference. For derived hematopoietic lineage cells that are genetically engineered to express recombinant TCR or CAR, activation and amplification cells may be used as described in, for example, U.S. Patent No. 6,352,694. In certain embodiments, different schemes may be used to provide primary stimulation signals and costimulatory signals to derived hematopoietic lineage cells. For example, the reagent providing each signal may be present in a solution or coupled to a surface. When coupled to the surface, these agents can be coupled to the same surface (i.e., "cis" form) or individual surfaces (i.e., "trans" form). Alternatively, one agent can be coupled to the surface and another agent is present in solution. In one embodiment, the reagent providing the co-stimulatory signal can be bound to the cell surface and the reagent providing the main activation signal is present in solution or coupled to the surface. In certain embodiments, both agents can be present in solution. In another embodiment, the agent can be in soluble form and then cross-linked to the surface, such as cells or antibodies or other adhesives expressing Fc receptors, which will be combined with agents such as those disclosed in U.S. Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPC), which are contemplated for activation and amplification of T lymphocytes according to the present disclosure.

[0216] A sterile, therapeutically acceptable composition suitable for administration to a patient may include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable culture media, such as cell culture media) or other pharmaceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents are determined in part by the particular composition being administered and the particular method for administering the therapeutic composition. Thus, there are a variety of suitable formulations for the therapeutic compositions of the present disclosure (see, e.g., Remington's Pharmaceutical Sciences, 17th edition, 1985, the disclosure of which is hereby incorporated by reference in its entirety).

[0217] These pharmaceutically acceptable carriers and / or diluents may be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer may account for up to about 5% (weight / weight) of the total composition. Electrolytes may also be included in the therapeutic composition, such as, but not limited to, sodium chloride and potassium chloride. In one aspect, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, in the range of about 6 to about 9, or in the range of about 6.5 to about 8. In another embodiment, the therapeutic composition includes a buffer having a pH in the pH range. In another embodiment, the pH of the therapeutic composition is about 7. Alternatively, the pH of the therapeutic composition is in the range of about 6.8 to about 7.4. In yet another embodiment, the pH of the therapeutic composition is about 7.4.

[0218] In some embodiments, the present invention also provides the use of a pharmaceutically acceptable cell culture medium in a specific composition and / or culture disclosed herein. Such compositions are suitable for administration to human subjects. In general, any culture medium that supports the maintenance, growth and / or health of iPSC-derived effector cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In a specific embodiment, a pharmaceutically acceptable cell culture medium is serum-free and / or feeder-free culture medium. In various embodiments, serum-free culture medium is animal-free and may optionally be protein-free. Optionally, the culture medium may contain biopharmaceutically acceptable recombinant proteins. "Animal-free" culture medium refers to a culture medium in which components are derived from non-animal sources. Recombinant proteins replace protozoan proteins in animal-free culture mediums and nutrition is obtained from synthetic, plant or microbial sources. In contrast, "protein-free" culture medium is defined as being substantially free of protein. Those skilled in the art will appreciate that the above culture medium examples are illustrative and in no way limit the culture medium formulations applicable to the present invention, and there are many known and suitable culture media available for use by those skilled in the art.

[0219] Example

[0220] The following examples are offered by way of illustration and not by way of limitation.

[0221] Example 1 - Materials and Methods

[0222] Maintenance of hiPSCs in small molecule cultures: Once the culture reaches 75%-90% confluency, hiPSCs are routinely passaged as single cells. For single cell dissociation, hiPSCs are washed once with PBS (Mediatech) and treated with Accutase (Millipore) at 37°C for 3-5 minutes, followed by pipetting to ensure single cell dissociation. The single cell suspension is then mixed with an equal volume of traditional culture medium, centrifuged at 225×g for 4 minutes, resuspended in FMM and plated on a Matrigel-coated surface. The number of passages is typically 1:6–1:8, transferred to tissue culture plates pre-coated with Matrigel at 37°C for 2 to 4 hours, and fed with FMM every 2 to 3 days. Cell cultures are maintained in a humidified incubator set at 37°C and 5% CO2.

[0223] Human iPSC engineering with ZFN, CRISPR for targeted editing of the mode of interest: Using ROSA26 targeted insertion as an example, for ZFN-mediated genome editing, 2 million iPSCs were transfected with a mixture of 2.5 μg ZFN-L (FTV893), 2.5 μg ZFN-R (FTV894), and 5 pg donor constructs for AAVS1 targeted insertion. For CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 μg ROSA26-gRNA / Cas9 (FTV922) and 5 μg donor constructs for ROSA26 targeted insertion. Transfection was performed using the Neon transfection system (Life Technologies) using the following parameters: 1500 V, 10 ms, 3 pulses. On the 2nd or 3rd day after transfection, transfection efficiency was measured using flow cytometry to assess whether the plasmid contained an artificial promoter-driven factor GFP and / or RFP expression cassette. On day 4 after transfection, puromycin was added to the culture medium at a concentration of 0.1 μg / ml for the first 7 days and 0.2 μg / ml for the next 7 days to select target cells. During the puromycin selection, cells were passaged onto fresh wells coated with Matrigel on day 10. On day 16 or later of puromycin selection, surviving cells were analyzed by flow cytometry for GFP + Percentage of iPS cells.

[0224] Bulk sorting and clonal sorting of iPSCs: After 20 days of puromycin selection, iPSCs with or without targeted genome editing were stained with GFP. + SSEA4 + TRA181 +Bulk sorting and clonal sorting of iPSCs. Single cell dissociated targeted iPSC pools were resuspended in staining buffer containing Hank's balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1× penicillin / streptomycin (Mediatech), and 10 mM Hepes (Mediatech). Conjugated primary antibodies, including SSEA4-PE, TRA181-Alexa, and Fluor-647 (BD Biosciences), were added to the cell solution. All antibodies were used at 7 μL / 100 μL staining buffer per million cells. The solution was washed in staining buffer, centrifuged and resuspended in staining buffer containing 10 μM thiazole for flow cytometric sorting. Flow cytometric sorting was performed on a FACS Aria II (BD Biosciences). For bulk sorting, GFP + SSEA4 + TRA181 + Cells are gated and sorted into FMM. For clone sorting, the sorted cells are directly ejected into 96-well plates, each well is coated with 5 × matrigel, and pre-filled with 200 μL FMM supplemented with 5 μg / mL fibronectin and 1 × penicillin / streptomycin (Mediatech). After sorting, before incubation, the 96-well plates are centrifuged at 225g for 1 minute to 2 minutes. The culture plate remains undisturbed for seven days. On the seventh day, 150 μL culture medium is removed from each well and replaced with 100 μL FMM. On the 10th day after sorting, 100 μL FMM additionally fed into the well is re-fed. As early as the 2nd day, colony formation was detected and most of the colonies were expanded between the 7th day and the 10th day after sorting. In the first passage, each well was washed with PBS and dissociated with 30 μL aku enzymes for about 10 minutes at 37°C. After cell dissociation was observed, the dissociated colonies were transferred to another well of a 96-well culture plate previously coated with 5× Matrigel and then centrifuged at 225 g for 2 minutes before incubation. Subsequent passages were routinely treated with acridine for 3-5 minutes and expanded at 1:4-1:8 after reaching 75%-90% confluency in FMM in larger wells pre-coated with 1× Matrigel. Each clonal cell line was analyzed for GFP fluorescence levels and TRA1-81 expression levels. Cells with approximately 100% GFP were selected. + and TRA1-81 + The cloned lines were used for further PCR screening and analysis and cryopreserved as master cell banks.

[0225] Hematopoietic differentiation: To initiate differentiation toward the hematopoietic lineage, hiPSCs were seeded as a monolayer in maintenance medium at day 0 (D) and allowed to adhere and expand for about 24 hours. The monolayer cells were maintained until around D5-D6, at which time they were dissociated into single cells and seeded as a low-density monolayer until differentiation around D10. The D10 dissociated single cell population was sorted by FACS for further analysis and marker profiling for permanent HE characterization. Alternatively, or in addition, the D10 cell population was cell sorted using one or more of anti-CD82, anti-CD34, and anti-CD43 antibodies as disclosed herein to obtain cells containing CD82 + 、CD34 + CD82 + or CD34 + CD43 - CD82 + These cells can be optionally cryopreserved in cryopreservation medium or continue to undergo effector cell differentiation.

[0226] Differentiation of HE cells to T and NK lineages: Sorted D10 HE cells were further differentiated to iT and iNK lymphoid lineages. For iT cells, after sorting, HE cells were transferred to low attachment tissue culture plates for serum-free differentiation. After about 10 days in culture (after HE isolation), the generation of T cell progenitors in cell culture was assessed by co-expression of cell surface markers CD34 and CD7. After further differentiation for about 15 to 20 days, these CD34 + CD7 + T cell progenitors give rise to distinct populations of mature iT cells, as observed by the expression of CD4 and CD8.

[0227] For iNK cells, after sorting, HE cells are cultured in serum-free differentiation medium for about 10 to 15 days. The cell culture is evaluated for NK cell progenitor generation. After an additional 10 to 15 days of culture and expansion and optional conditioning as further disclosed herein, markers including CD56, CD122, NKp30, CD94, CD16, NKG2D, and KIR are used to identify the presence of activated / mature NK cells.

[0228] Example 2 - Screening of permanent hemogenic endothelial cell surface markers

[0229] Cell markers act as letter combinations that help identify and classify cells. Most markers are molecules or antigens within the plasma membrane of the cell. Many surface markers are classified by their clusters of differentiation (CD), which are recognized by specific antibodies. Typically, a specific combination of markers is unique to different cell types.

[0230] During in vitro differentiation of pluripotent stem cells through endothelial cells of mesodermal origin, heterogeneous endothelial cells (ECs) acquire arterial, venous or hemogenic fates and form corresponding subtypes of endothelial cells that are phenotypically and functionally specialized. These cell subtypes are formed in close space and time and are currently distinguished mainly by gene expression profiles. Hematopoietic cells are generated by endothelial-hematopoietic transformation (EHT) from a unique endothelial cell population called hemogenic endothelial cells (HE). EHT represents a continuous process in which cells with endothelial properties gradually acquire hemogenic morphology and phenotype, and endothelial progenitor cells that undergo this hemogenic transformation are called permanent hemogenic endothelial cells. Currently, permanent HE cells are identified as CD34 that do not express CD43, CD73, CD93 and CXCR4. + Cells. + CD43 in cells - CD73 - CD93 - CXCR4 - Compared with such negative screening of cell subsets, identification of one or more positive markers specific for the definitive HE cell subtype would greatly improve the efficiency and accuracy of isolating early cell populations of desired quality and purity for subsequent hematopoietic cell differentiation.

[0231] To efficiently screen for novel positive markers of HE, Applicants' proprietary hiPSC platform was used, which is capable of single-cell passaging and high-throughput flow cytometry sorting to allow the derivation of clonal hiPSCs. To identify additional markers for enriching for definitive HE cells with the ability to generate hematopoietic cells, hiPSCs were seeded as a monolayer and differentiated toward hematopoietic cells using the methods and compositions disclosed herein.

[0232] Flow cytometry analysis was performed to compare D10 cells differentiated under control conditions with cells differentiated under cytokine conditions including BMP4. + The specificity of HE is cytokine-driven, such as Figure 1 RUNX1 is a transcription factor expressed in cells and is thought to be primarily associated with HE features. Cells were gated for single / live events. CD34 + RUNX1 - Cells and CD34 + RUNX1 + Cells were stained with anti-human antibodies and analyzed by LEGEND Screen TM This screen subsequently identified many cells that were CD34 + RUNX1 +Candidate surface markers with higher expression percentages within subgroups ( Figure 2 ).like Figure 2 As shown, among the other candidate markers shown, CD34 - Compared with the CD34 + The expression of glioma in CD34 + Higher expression in the population was also associated with cytokine-driven RUNX1 + Among the above candidate markers, CD143 and CD82 are the most promising because their expression is different and closely correlated with the expression of RUNX1.

[0233] Example 3 - Validation of permanent hemogenic endothelial cell surface markers

[0234] Purification of cytokine-driven D10 cells using CD34 magnetic-activated cell sorting. Visualization using UMAP (Uniform Manifold Approximation and Projection) Figure 3A The scRNAseq analysis of the expression of the validation signature genes is presented in Figure 2, and the UMAP visualization is then used to identify cell clusters expressing the corresponding signature genes (CD34, RUNX1, SPN, or ITGA2B). Figure 3A As shown, each dot represents a cell, and the color density indicates the characteristic gene expression level.

[0235] Then use Figure 3A UMAP visualization of the validation signature genes to define Figure 3B Each cell cluster in . Figure 3B The cell clusters in the 24 samples included: (1) RUNX1 cells expressing the endothelial marker CD34 but not expressing RUNX1 or the hematopoietic markers SPN (CD43) or ITGA2B (CD41); - EC, which is non-hematopoietic CD34 + (2) RUNX1 cells expressing CD34 and RUNX1 but not SPN or ITGA2B + EC, which are the desired HE cells; (3) any cells expressing SPN or ITGA2B, which are (differentiated) hematopoietic cells.

[0236] Once in D10 CD34 + Three cell clusters were identified in the D10 cells, and violin plots were used to show the CD82 expression in each of the three subpopulations of the D10 cell population ( Figure 3C ).like Figure 3CAs shown, using the width of the violin plot representing the number of cells at each CD82 expression level, and the mean and median CD82 expression for each cell subset marker, the violin plots show that although hematopoietic cells are known to express both RUNX1 and CD82, RUNX1 + CD82 gene expression and RUNX1 in EC(HE) population - CD82 gene expression was significantly higher in the non-hematopoietic population, supporting the hypothesis that CD82 is a marker that can distinguish HE from non-HE.

[0237] Flow cytometric analysis of cytokine-driven D10 cells was then performed to compare the expression of other selected HE candidate markers with RUNX1 and CD82. Since RUNX1 is a transcription factor and not expressed on the cell surface, it cannot be used as a marker to enrich for intact HE cells by flow sorting. Figure 4A As shown, the first row of flow cytometry plots depict candidate HE markers compared to CD34 to determine if the candidate markers are restricted to the endothelial cell population (CD34 + ); all tested markers except CD44 were primarily restricted to the endothelial cell population. The second row ( Figure 4A ) shows a comparison of CD34 and CD43 to identify CD34 + CD43 - Group. The third and fourth rows ( Figure 4B ) depicts the expression of CD34 + CD43 - Comparison of population-gated candidate HE markers with RUNX1 (third row) and CD82 (fourth row). As shown, all candidate markers were expressed in RUNX1 + It was observed that CD61 and CD226 were not expressed by the entire RUNX1 + This suggests that they only mark the HE subset at D10 of differentiation. Since both CD82 and CD143 are restricted to the endothelial cell population and are expressed in RUNX1 + Therefore, the data support each of them as viable candidate surface markers for HE.

[0238] Further observations revealed that the addition of a p38 MAPK (mitogen-activated protein kinase) inhibitor to the composition used to derive permanent HE cells from iPSCs was beneficial in maintaining CD82 expression, thereby leading to CD34 + CD82 of HE cells + RUNX1 +Therefore, it is contemplated that, in addition to the BMP4 cytokine, the composition may optionally include a p38 MAPK inhibitor, such as DBM1285 (an exemplary small molecule inhibitor tested), to increase the efficiency of obtaining permanent HE cells differentiated from iPSCs, wherein the permanent HE cells have a phenotype that includes CD82 + and optionally CD34 + 、CD43 - 、CD93 - CXCR4 - and CD73 - One or more of .

[0239] Example 4 - Application of permanent hemogenic endothelial cell surface markers

[0240] Current methods for identifying permanent HE rely on the exclusion of endothelial cell populations (CD34 + CD43 - ), so that the selected HE usually has CD34 + CD43 - CD73 - CD93 - CXCR4 - However, endothelial progenitor cells that precede HE in cell development also share this phenotype. Since cells usually differentiate slowly and have high CD34 + CD43 - CD73 - CD93 - CXCR4 - A percentage of the population will be more skewed toward immature endothelial progenitor cell development rather than definitive HE, making negative marker screening an unreliable method for detecting definitive HE cells that have the potential to develop into cells of the T lineage in addition to the NK, NKT, and B cell lineages.

[0241] For the CD82 and CD143 markers from the above screen, CD82 was used as a demonstration to show their validity as permanent HE markers. Flow cytometric analysis of cytokine-driven D10 cells was performed to show CD82 + Cells in CD73 - CD93 - CXCR4 - Enrichment within the endothelial cell population, where cells were pre-gated for single / live events. Figure 5 As shown, CD82 expression is restricted to CD34 + CD43 - CD73 -CD93 - CXCR4 - The data therefore support that CD82 is a reliable marker that replaces triple-negative markers for identifying permanent HE.

[0242] In a separate experiment, the frequency of permanent HE within the cytokine-driven D10 population was calculated using limiting dilution analysis (ELDA) software. The cytokine-driven D10 population was subjected to fluorescence activated cell sorting (FAC) based on the markers indicated along the x-axis (mean ± SD) and Figure 6 The mean permanent HE frequency is indicated above each column in the table. + CD43 - CD82 + 1 in 38 cells in the population and CD82 + One in 44 cells in the population was definitive HE, further supporting the use of CD82 as a reliable positive marker for the identification of definitive HE. The data also showed that D10 cell sorting for definitive HE could be performed using a single marker, CD82. + , or use CD43 - CD82 + (Through CD43 - to eliminate differentiated hematopoietic cells), or using CD34 + CD43 - CD82 + To achieve a satisfactory permanent HE frequency, each relative to CD34 + Cell populations improved.

[0243] The definitive hematopoietic potential represented by the cell population obtained using various marker combinations was determined by iT differentiation. D10 CD34 + CD43 - CD82 + and CD34 + CD43 - CD82 - The cell fractions were plated in cultures as disclosed herein (iTC-A2 and iTC-B2). At about D35 of iT differentiation, the presence of iT cells in the culture was assessed by the expression of the pan-hematopoietic marker CD45 and the lymphoid markers CD5 and CD7. Since the iT cells in this example have a CD19-CAR knocked into the TRAC locus, thereby preventing the assembly of the T cell receptor on the cell surface, the detectable intracellular T cell co-receptor CD3 (icCD3) is used as a marker for differentiated T cells. Figure 7As shown in the figure, the populations were FAC-sorted by the markers indicated above the flow cytometry plots, and iT cells differentiated from each indicated D100 population primarily produced CD8 + Cytotoxic T cells, but not CD4 + Further, without being limited by theory, CD8aa (CD8a + CD8b - ) compared with CD8ab (CD8a + CD8b + ) indicated that the cells were more adaptable. + CD43 - CD82 + The successful differentiation of the fraction into T cells indicated the permanent nature of the HE cells identified by CD82.

[0244] For iNK differentiation, the presence of iNK cells in D30 cultures was assessed by the expression of the pan-hematopoietic marker CD45 and the NK marker CD56. Figure 8 The population was FAC sorted for the markers indicated above the flow cytometry plot. Although a relatively small myeloid cell population (CD11b / CD14 + ),but Figure 8 The robust expression of the lymphoid marker CD7 and the expression of the NK cell receptor NKG2A shown in Figure 3 indicate successful NK cell differentiation.

[0245] In conclusion, the data demonstrate that CD82 is a reliable cell surface marker for permanent HE detection during iPSC differentiation. + or CD34 + CD82 + The cells represent a permanent HE population in the early stages of directed differentiation of iPSCs into hematopoietic cells; and the expression of CD82 can be used as an alternative positive marker to replace negative markers (including CD73, CD93, CXCR4 or any combination thereof) for identifying the (HE, or more specifically, permanent HE) subpopulation.

[0246] Example 5 - OP9-free stroma differentiation of HE into mature and functional lymphocytes

[0247] To differentiate iPSCs in chemically defined, serum-free medium without murine or human stromal cells, sorted permanent HE cells (iCD34; CD82 + ;CD34 + CD82 + ; or CD34 + CD43 - CD82 +) are plated in a non-passaged culture medium for about 18 to 20 days, or up to 27 to 30 days, and then the differentiated CD56-expressing NK cells are prepared for collection, storage, or further maturation and expansion. Notably, this differentiation process as described herein is completely free of feeder cells, particularly OP9 stromal cells expressing DLL4 (OP9-DLL4) or their irradiated counterparts (irOP9-DLL4), thereby making the differentiated cell product free of exogenous undefined cellular components, which is desirable for regulatory compliance in manufacturing cell therapies.

[0248] A composition for replacing OP9-DLL4 for differentiation of hematopoietic cells (including NK cells) comprising human DLL4 Fc chimeric recombinant protein (Fc-rhDLL4) and (Takara Bio USA, Inc., San Jose, CA). RetroNectin in the Retro / DLL4 extracellular matrix is ​​a recombinant human fibronectin with three functional domains: a human fibronectin cell binding domain (C-domain), a heparin binding domain (H-domain), and a CS-1 sequence domain. The culture medium containing the Retro / DLL4 extracellular matrix also contains IL7, Flt3L, and SCF, and optionally one or more of a ROCK inhibitor, IL3, TPO, and VEGF. For example, after seeding iCD34 cells, a ROCK inhibitor may be present in the culture medium for the first 2 to 4 days, TPO and VEGF may be present in the culture medium for the first 4 to 8 days, and IL3 may be present in the culture medium for the first 7 to 11 days.

[0249] Stromal-free differentiation of permanent HE cells was compared with that using irOP9-DLL4 or a commercially available NK differentiation stem cell kit (Stemcell Technologies) (for differentiation of CD45 + CD56 + 、CD7 + and CD1lb + / CD14 + / CD15 + The frequency of cells depicting iNK expansion fold and iNK progenitor cell specialization / characteristics) were compared to those undergoing differentiation. 9A to 9D As shown in Figure 2, all three differentiation strategies met the minimum expansion fold (i.e., approximately 25-fold) and generated iNK progenitor cells with similar specialization (e.g., CD45 + CD56 + and CD7 +Although Retro / DLL4 differentiation may not have as robust cell expansion as observed with irOP9-DLL4 differentiation, it is beneficial in generating fewer myeloid cells (e.g., CD11b + / CD14 + / CD15 + frequency) is superior to the stem cell kit.

[0250] The collected iNK progenitor cells were combined with irradiated K562 cells (irK562-41BBL-IL21) engineered to express 41BBL and IL21 in a tissue culture container for two rounds of maturation and expansion during a 7-day expansion period. After the first 7-day maturation and expansion period (R1D7), the expansion fold of the cells was determined and restimulated with additional irK562-41BBL-IL21 to continue maturation and expansion. After the second 7-day maturation and expansion period (R2D7), the expansion fold of the cells was determined and cryopreserved for in vitro cytotoxicity assays. FIG. 10A to FIG. 10C As shown in , although iNK progenitors differentiated using the stem cell kit failed to expand after R1D7, those differentiated on irOP9-DLL4 and Retro / DLL4 showed similar expansion levels, far exceeding those differentiated on the stem cell kit. In addition, after R2D7, the Retro / DLL4 group surprisingly showed superior expansion to the irOP9-DLL4 and stem cell kit groups, resulting in a more desirable overall expansion fold ( Fig. 10C ). Furthermore, the mature and expanded CD45 + CD56 + iNK cells and those obtained using conventional irOP9-DLL4 differentiation displayed similar features depicted by various inhibitory, activating, and co-activating NK receptors, demonstrating that the matrix-free Retro / DLL4 system is an effective and efficient solution for differentiating HE cells toward cells of the NK lineage.

[0251] To demonstrate the functionality of mature iNK cells differentiated from permanent HE cells using a matrix-free approach, cryopreserved iNK cells from the irOP9-DLL4 and Retro / DLL4 groups were each thawed and co-cultured with Nalm6 target cells or Nalm6 cells engineered to express exemplary surface antigens (e.g., Nalm6-KLK2). After four hours of co-culture, apoptosis was measured using a caspase 3 / 7 activity assay. Fig.11A and Fig. 11BAs shown in , although iNK cells from the Retro / DLL4 group showed lower antigen-independent spontaneous killing of Nalm6 target cells than those from the irOP9-DLL4 group across a range of effector:target cell ratios, iNK cells from both groups had equally effective antigen-specific killing directed by CAR. The lower antigen-independent spontaneous killing of iNK cells differentiated from the matrix-free Retro / DLL4 approach may be advantageous for therapeutic effector cells as a desired safety attribute by reducing unspecified innate NK killing.

[0252] In separate experiments, thawed iNK cells from the irOP9-DLL4 and Retro / DLL4 groups were co-cultured with PC3 cells or PC3 cells engineered to express antigens on their surface (e.g., PC3-KLK2). After 16 hours, cytokine release in the supernatant of each group was measured by ELISA. Fig. 12A and Fig. 12B As shown, iNK cells from both groups displayed similar levels of maximal release of IFNγ and TNFα upon PMA / ionomycin stimulation, as well as similar levels of antigen-dependent cytokine release of IFNγ and TNFα, confirming that the matrix-free Retro / DLL4 differentiation system supports the functionality of differentiated effector cells.

[0253] To demonstrate the persistence of differentiated effector cells, mature and expanded iNK cells from the irOP9-DLL4 and Retro / DLL4 groups were further subjected to Continuous restimulation assay. In this assay, iNK cells differentiated from each group are combined with PC3 parental cells or PC3 cells expressing cell surface antigens (e.g., PC3-KLK2) at various E:T (effector: target cell) ratios. The number of live target cells is monitored by hourly fluorescence imaging over 48 hours. The number of live cells is quantified and normalized to the number of live cells remaining in the target cell only control group. After 48 hours, iNK cells from R1 are restimulated with fresh PC3 parental cells or PC3-KLK2 target cells to generate normalized live cell counts (R2 restimulation). After 48 hours, iNK cells from R2 are restimulated again to generate normalized live cell counts (R3 restimulation). As Fig.13 As shown in , iNK cells from the Retro / DLL4 group maintained similar capabilities as those from the irOP9-DLL4 group in antigen-dependent serial killing of target cells, further confirming that the matrix-free Retro / DLL4 differentiation system supports long-term effector cell functionality.

[0254] Example 6 - Altered expansion composition of mature lymphocytes

[0255] To regulate effector cell activation during the cell expansion phase of iPSC differentiation, an AhR (aryl hydrocarbon receptor) inhibitor was found to improve effector cell function, including anti-tumor efficacy. iPSCs were differentiated into permanent HE cells as previously described, and then differentiated into pre-expansion NK cells, at which point they were expanded by co-culturing with IL21 and 41BBL expressed by irK562 feeder cells for about 7 days. On day 2, the exemplary AhR inhibitor CH-223191 (and other inhibitors such as UM729, UM171, SRI) was added to the expansion medium at a final concentration of about 3 μM to enhance the amplification signal. Fig.14 showed that in the presence of CH-223191, a greater expansion fold and yield of differentiated NK cells were achieved compared to iNK cells expanded in the absence of the small molecule AhR inhibitor.

[0256] The cells treated with CH-223191 were cryopreserved in liquid nitrogen for a long time. In order to evaluate the recovery of cells from cryopreservation, the cells were thawed and cultured in B0 medium for 7 days, and cell counting was performed during the culture process. Cells treated with AhR inhibitors showed greater post-thaw recovery and persistence over time. Further, the in vitro and in vivo functions of cells treated with CH-223191 and cryopreserved in liquid nitrogen were further evaluated. Compared with untreated or control cells, cells treated with CH-223191 and cryopreserved in liquid nitrogen showed enhanced anti-tumor efficacy at the end point, which proves that AhR inhibitor treatment produces better anti-tumor efficacy over time ( Fig.15 ).

[0257] It will be readily appreciated by those skilled in the art that the methods, compositions and products described herein represent exemplary embodiments and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various substitutions and modifications may be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0258] All patents and publications mentioned in this specification are indicative of the skill levels of those skilled in the art to which the disclosure pertains. All patents and publications are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0259] The present disclosure illustratively described herein may be appropriately implemented in the absence of any component, limitation, not specifically disclosed herein. Thus, for example, in each case herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by any of the other two terms. The terms and expressions used are used as terms of description rather than limitation, and when such terms and expressions are used, it is not intended to exclude any equivalents of the features shown and described, or a portion thereof, but it should be recognized that various modifications may exist within the scope of the claimed disclosure. Therefore, it should be understood that although the present disclosure has been particularly disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are deemed to be within the scope of the invention as defined in the appended claims.

Claims

1. A cell population, comprising cells having the following phenotypes: (i)CD82 + ; (ii) CD34 + CD82 + ; and / or (iii)CD34 + CD43 - CD82 + , wherein the cells comprise definitive hemogenic endothelial (HE) cells, and wherein the cells are derived from in vitro iPSC differentiation.

2. The cell population according to claim 1, wherein the permanent HE cells (i) is enriched; and / or (ii) capable of differentiating into cells of the hematopoietic lineage including T cell progenitors and T cells in addition to NK progenitors, NK cells, NKT cells or B cells.

3. The cell population of claim 1, wherein the iPSCs are clonal iPSCs, single-cell dissociated iPSCs, iPSC line cells, or iPSC master cell bank (MCB) cells. The cell population of claim 3 , wherein the iPSCs are naive iPSCs.

5. The cell population of claim 3, wherein the iPSCs further comprise one or more genetic imprints introduced into the iPSCs by genome editing during or after reprogramming of non-pluripotent cells into the iPSCs, wherein the genetic imprints comprise: (i) one or more genetic modification patterns introduced by genomic insertion, deletion or substitution in the genome of the iPSC; or (ii) one or more of donor, disease or treatment response specific source-specific immune cells may retain therapeutic properties, and wherein the iPSCs are reprogrammed from the source-specific immune cells; And wherein the cells comprise the same one or more genetic imprints.

6. The cell population according to claim 1, wherein the differentiation of iPSCs to obtain the cell population comprises: (i) differentiating iPSCs to obtain hemogenic endothelial (HE) cells, and (ii) Targeting CD82 + HE cells were sorted to obtain cells expressing CD82 + Permanent HE cells, a cell marker The definitive HE cells are capable of differentiating into cells of the hematopoietic lineage.

7. The cell population according to claim 6, wherein differentiating iPSCs to obtain HE cells further comprises: (a) differentiating iPSCs to obtain mesodermal progenitor cells; as well as (b) differentiating the mesodermal progenitor cells to obtain HE cells.

8. The cell population according to claim 6, wherein the cell markers further comprise CD34 + 、CD43 - ,RUNX1 + or any combination thereof, and wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + phenotype.

9. The cell population of claim 6, wherein the iPSC differentiation comprises contacting the iPSC with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) A small molecule p38 MAPK (mitogen activated protein kinase) inhibitor, which resulted in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor.

10. The cell population of claim 9, wherein the cytokine comprises BMP4, and / or wherein the small molecule p38 MAPK inhibitor comprises DBM1285.

11. The cell population of claim 1, wherein (i) at least 0.5%, at least 1%, or at least 2% of the cells have CD82 + The cells are permanent HE cells; and / or (ii) the cell population is a substantially pure cell population having the phenotype.

12. A composition comprising the cell population according to any one of claims 1 to 11.

13. The composition of claim 12, further comprising a cryopreservation medium.

14. A method for generating iPSC-derived permanent HE, wherein the method comprises differentiating iPSCs to obtain iPSC-derived hemogenic endothelial (HE) cells and targeting CD82 + The HE cells were sorted to obtain cells expressing CD82 + Permanent HE cells, a cell marker The permanent HE cells are capable of differentiating into hematopoietic lineage cells including T cell progenitor cells and T cells in addition to NK progenitor cells, NK cells, NKT cells or B cells.

15. The method according to claim 14, wherein the sorting further comprises sorting for the following cells: CD34 + 、CD43 - ,RUNX1 + or any combination thereof; and wherein the permanent HE cells obtained comprise CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + phenotype.

16. The method according to claim 14, wherein the method further comprises: (i) contacting the iPSCs with a culture medium containing a BMP activator and bFGF, thereby differentiating the iPSCs to obtain mesodermal progenitor cells; as well as (ii) contacting the mesodermal progenitor cells with a culture medium comprising a BMP activator, bFGF, VEGF, a Wnt pathway activator, and optionally a p38 MAPK inhibitor, thereby differentiating the mesodermal progenitor cells to obtain HE cells.

17. The method of claim 16, wherein contact with the p38 MAPK inhibitor increases maintenance of CD82 expression in HE cells compared to the absence of the p38 MAPK inhibitor; wherein the BMP activator comprises BMP4; and / or wherein the Wnt pathway activator comprises a GSK3 inhibitor.

18. The method of claim 17, wherein the p38 MAPK inhibitor comprises DBM1285; and / or wherein the GSK3 inhibitor comprises CHIR99021.

19. The method of claim 16, wherein the iPSCs comprise naive iPSCs, and / or are derived from iPSCs comprising one or more genetic imprints.

20. The method of claim 19, wherein the one or more genetic imprints contained in the iPSCs are retained in the iPSC-derived definitive HE cells.

21. The method of claim 14, further comprising cryopreserving the permanent HE cells.

22. A composition for generating iPSC-derived definitive HE (hemogenic endothelial) cells, the composition comprising: a BMP activator, bFGF, VEGF, a Wnt pathway activator, and optionally a p38 MAPK inhibitor.

23. The composition of claim 22, wherein: (i) the composition does not contain a TGFβ receptor / ALK inhibitor; (ii) the iPSC-derived definitive HE comprises increased RUNX1-expressing cells compared to differentiation without the BMP activator; and / or (iii) The iPSC-derived permanent HE contained increased CD82-expressing cells compared to differentiation without the p38 MAPK inhibitor.

24. The composition of claim 22, wherein the BMP activator comprises BMP4; and / or wherein the p38 MAPK inhibitor comprises DBM1285, VX-745, VX- At least one of 702, RO-4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RV568, CAS219138-24-6, SB203580 and SB242235.

25. The composition of claim 22, wherein the p38 MAPK inhibitor comprises DBM1285.

26. The composition of claim 22, further comprising iPSCs, mesodermal cells or the definitive HE cells.

27. A method for generating iPSC-derived permanent HE, the method comprising: (i) differentiating iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain HE cells; as well as (iii) Targeting CD82 + The HE cells were sorted to obtain cells expressing CD82 + permanent hemogenic endothelial (HE) cells, The permanent HE cells are capable of differentiating into hematopoietic lineage cells including T cell progenitor cells and T cells in addition to NK progenitor cells, NK cells, NKT cells or B cells.

28. The method according to claim 27, wherein the sorting further comprises sorting for the following cells: CD34 + 、CD43 - ,RUNX1 + or any combination thereof, and wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + phenotype.

29. The method of claim 27, wherein step (ii) of differentiating the mesodermal progenitor cells into HE cells comprises contacting the mesodermal progenitor cells with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) A small molecule p38 MAPK (mitogen activated protein kinase) inhibitor, which resulted in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor.

30. The method of claim 29, wherein the cytokine comprises BMP4; and / or wherein the small molecule p38 MAPK inhibitor comprises DBM1285.

31. The method of claim 27, further comprising cryopreserving the obtained permanent HE cells.

32. A method for generating iPSC-derived hematopoietic lineage cells by differentiating the permanent HE cells according to any one of claims 1 to 11, wherein the method comprises contacting the permanent HE cells with a culture medium composition comprising SCF, Flt3L and IL7; and optionally one or more of a ROCK inhibitor, TPO and IL3, thereby obtaining the iPSC-derived hematopoietic lineage cells comprising T cell progenitors and T cells in addition to NK cell progenitors, NK cells, NKT cells or B cells.

33. The method of claim 32, wherein the iPSC-derived hematopoietic lineage cells comprise NK cell progenitors and / or NK cells, and wherein (1) the culture medium composition further comprises IL15; and / or (2) the definitive HE cells comprise a gene insertion of a polynucleotide encoding a cytokine signaling complex comprising exogenous IL15 and / or a partial or complete peptide of its receptor expressed on the cell surface.

34. The method of claim 33, wherein the culture medium composition is free of OP9 stromal cells.

35. The method of claim 34, wherein the differentiation occurs in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4.

36. A method for producing iPSC-derived hematopoietic lineage cells, the method comprising: Differentiate iPSCs to obtain permanent hemogenic endothelial (HE) cells, wherein the permanent HE cells express CD82 + cell markers; and differentiating the permanent HE cells to obtain iPSC-derived hematopoietic lineage cells; The iPSC-derived hematopoietic lineage cells further comprise T cell progenitors and T cells in addition to NK cell progenitors, NK cells, NKT cells or B cells.

37. The method of claim 36, wherein the iPSC comprises one or more genetic imprints introduced into the iPSC by genome editing during or after reprogramming of a non-pluripotent cell to the iPSC, wherein the one or more genetic imprints comprise: (i) one or more genetic modification patterns introduced by genomic insertion, deletion or substitution in the genome of the iPSC; or (ii) one or more of donor, disease or treatment response specific source-specific immune cells retain therapeutic properties, wherein the iPSCs are reprogrammed from the source-specific immune cells, and wherein the one or more genetic imprints are retained in the iPSC-derived hematopoietic lineage cells.

38. The method of claim 36, wherein differentiating the iPSCs to obtain definitive hemogenic endothelial (HE) cells comprises: (i) differentiating genetically engineered iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain HE cells; as well as (iii) Targeting CD82 + HE cells were sorted to obtain cells expressing CD82 + The permanent HE cells with cell markers.

39. The method according to claim 38, wherein the sorting further comprises sorting for the following cells: CD34 + 、CD43 - ,RUNX1 + or any combination thereof, and wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + phenotype.

40. The method of claim 38, wherein differentiating the mesodermal progenitor cells into HE cells comprises contacting the mesodermal progenitor cells with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) A small molecule p38 MAPK (mitogen activated protein kinase) inhibitor, which resulted in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor.

41. The method of claim 40, wherein the cytokine comprises BMP4; and / or wherein the small molecule p38 MAPK inhibitor comprises DBM1285.

42. The method of claim 36, further comprising cryopreserving the permanent HE cells, wherein the cryopreserved permanent HE cells are thawed prior to differentiation thereof.

43. The method of claim 36, wherein the definitive HE cells are differentiated in the absence of OP9 stromal cells.

44. The method of claim 43, wherein differentiating the permanent HE cells occurs in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4.

45. A method for generating NK cells in a feeder-free environment, the method comprising: (a) differentiating iPSCs or permanent HE cells derived therefrom into NK lineage cells in a medium comprising one or more growth factors and cytokines comprising SCF, Flt3L and IL7; wherein the medium does not contain OP9 stromal cells; and further, wherein: (i) the culture medium contains IL15, and / or (ii) the permanent HE cells comprise a gene insertion of a polynucleotide encoding a cytokine signaling complex, wherein the cytokine signaling complex comprises a portion or all of a peptide of exogenous IL15 and / or its receptor expressed on the cell surface; and (b) expanding and activating the NK lineage cells to obtain NK cells that are cytotoxic to the target.

46. ​​The method of claim 45, wherein the culture medium further comprises one or more of a ROCK inhibitor, TPO, and IL3.

47. The method of claim 45, wherein the permanent HE cell comprises a phenotype comprising: (i)CD82 + ; (ii) CD34 + CD82 + ; (iii) CD34 + CD43 - CD82 + ; and / or (iv) CD34 + , and CD43 - 、CD93 - CXCR4 - 、CD73 - and RUNX1 + At least one of .

48. The method of claim 45, wherein differentiating the iPSCs further comprises: (i) differentiating the iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain hemogenic endothelial (HE) cells; as well as (iii) Targeting CD82 + sorting HE cells to obtain the permanent HE cells, wherein the permanent HE cells express CD82 + cell markers.

49. The method of claim 48, wherein the sorting further comprises sorting for the following cells: CD34 + 、CD43 - ,RUNX1 + or any combination thereof, and wherein the obtained permanent HE cells contain CD34 + CD82 + 、CD34 + CD43 - CD82 + 、CD34 + CD82 + RUNX1 + or CD34 + CD43 - CD82 + RUNX1 + phenotype.

50. The method of claim 48, wherein differentiating the mesodermal progenitor cells into HE cells comprises contacting the mesodermal progenitor cells with: (i) a cytokine that results in a higher percentage of HE cells expressing RUNX1 compared to the absence of the cytokine; and / or (ii) A small molecule p38 MAPK (mitogen activated protein kinase) inhibitor, which resulted in improved maintenance of CD82 expression in HE cells compared to the absence of the inhibitor.

51. The method of claim 50, wherein the cytokine comprises BMP4; and / or wherein the small molecule p38 MAPK inhibitor comprises DBM1285.

52. The method of claim 45, wherein step (a) differentiating further comprises contacting the definitive HE cells with an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4; and / or wherein step (b) expanding further comprises contacting the NK lineage cells with an expansion composition comprising nicotinamide.

53. The method of claim 45, wherein step (b) expanding further comprises contacting the NK lineage cells with a small molecule AhR inhibitor, thereby modulating activation of the NK lineage cells.

54. The method of claim 53, wherein the small molecule AhR inhibitor comprises CHIR223191, UM729, UM171, or SR1.

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