Methods and compositions for inducing hematopoietic cell differentiation

Abstract

In various aspects, the present invention provides culture platforms, cell media, and methods for differentiating pluripotent cells into hematopoietic cells. In certain aspects, the present invention further provides pluripotent stem cell-derived hematopoietic cells produced using the culture media and methods disclosed herein. Secondary HE cells derived from pluripotent stem cells produced by the methods provided herein can be differentiated into hematopoietic lineage cells, including NK cell precursors, NK cells, NKT cells, or B cells, as well as T cell precursors and T cells.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 375,680, filed September 14, 2022, the disclosure of which is incorporated herein by reference in its entirety.

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

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

[0004] The in vitro derivation of HSCs with lymphocyte-generating potential is complicated by the existence of at least two temporally and spatially distinct waves of blood cell formation during embryonic development: primary and definitive hematopoiesis. Primary hematopoiesis begins in the extraembryonic yolk sac and generates a transient, limited hematopoietic repertoire containing primary erythroid and myeloid lineage cells. Nascent HSCs with lymphocyte-generating potential emerge only late in the secondary wave from specialized endothelial progenitor cells within the arterial vasculature, termed secondary hemogenic endothelium (HE). Secondary HE then undergoes an endothelial-to-hematopoietic transition to give rise to HSCs, which then ultimately migrate to the bone marrow to sustain multilineage hematopoiesis, including T, B, NKT, and NK lymphoid cells, throughout adulthood. Therefore, the generation of HSCs and subsequent lymphoid effector cells from pluripotent stem cells relies on the ability to accurately recapitulate the complex stages of early embryonic hematopoietic development toward the secondary program via well-designed and validated methods and compositions.

[0005] Only a limited number of studies have described the directed differentiation of iPSCs into secondary hiPSCs in vitro. A major obstacle to utilizing hiPSCs for therapeutic purposes has been the need to initially coculture such cells with mouse- or human-derived stromal cells in the presence of an undefined, serum-containing medium to maintain pluripotency and induce differentiation. Furthermore, existing protocols also employ strategies that involve culturing iPSCs to form embryoid bodies (EBs), which are heterogeneous aggregates of cells containing various differentiated cell types, including ectodermal, mesodermal, and endodermal cells. These procedures require the aggregation of pluripotent cells, for example, by spinning to form clumps, allowing cells to settle in wells, or allowing them to passively aggregate in suspension culture to form clumps. The formed EBs are maintained for a specific period, typically 7–10 days, in a differentiation-inducing culture system to allow appropriate differentiation. EBs are then transferred to adherent culture for further maturation or dissociated into single cells for cell type selection for 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 by treating pluripotent cells with collagenase and trypsin to scrape the cells and form small aggregates, which are then cultured to form EBs. While EB formation has been shown to promote the differentiation of pluripotent stem cells, the requirement to form aggregates and subsequent EBs is labor-intensive, cell number expansion is minimal in this process, and the cellular contents in the three-dimensional EB aggregates are inconsistently and non-uniformly exposed to media factors, resulting in heterogeneous cell products at various stages of differentiation, posing significant obstacles to the scalability and reproducibility of manufacturing processes that need to be efficient and streamlined. Summary of the Invention

[0006] In view of the above, there is a need for methods and compositions for efficiently and reliably differentiating stem cells into definitive hematopoiesis without co-culture with feeders or reliance on serum-containing media, and without the need for the formation of embryoid body aggregates as an intermediate. The compositions and methods disclosed herein address this need and provide other advantages.

[0007] In various aspects, the present invention relates generally to cell culture conditions, media, culture platforms, methods for culturing and differentiating stem cells toward a hematopoietic cell fate, and cell populations produced therefrom.

[0008] In some embodiments, the present invention provides methods and compositions, including novel cell surface markers that identify secondary hemogenic endothelial (HE) cells, for generating hematopoietic cell lineages from pluripotent stem cells, including iPSCs, under serum / feeder-free conditions and in an expandable monolayer culture platform that does not require EB formation. Cells that can be differentiated according to the methods disclosed herein range from pluripotent stem cells to committed progenitor cells to specific terminally differentiated and transdifferentiated cells, and to cells of various lineages that transition directly to a hematopoietic fate without passing through a pluripotent intermediate. Similarly, cells produced by stem cell differentiation range from multipotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0009] In one aspect, the present invention provides a method for detecting a compound comprising: (i) CD82 + , (ii) CD34 + CD82 + and / or (iii) CD34 + CD43 - CD82 +The present invention provides a cell population comprising cells having a phenotype of: wherein the cells comprise secondary hemogenic endothelial (HE) cells, and wherein the cells are derived from in vitro differentiation of iPSCs. In some embodiments of the cell population, the secondary HE cells are (i) enriched and / or (ii) capable of differentiating into hematopoietic lineage cells, including NK cell precursors, NK cells, NKT cells, or B cells, as well as T cell precursors and T cells. In some embodiments of the cell population, the iPSCs are clonal iPSCs, single-cell dissociated iPSCs, iPSC cell line cells, or iPSC master cell bank (MCB) cells. In some embodiments, the iPSCs are naive iPSCs. 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 non-pluripotent cells into iPSCs, the genetic imprints comprising (i) one or more genetic modification modalities introduced by genomic insertion, deletion, or substitution in the genome of the iPSCs, or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor, disease, or therapeutic response specific, and the iPSCs were reprogrammed from source-specific immune cells, and the cells comprise the same one or more genetic imprints.

[0010] In various embodiments of the cell population, differentiating the iPSCs to obtain HE cells comprises (i) differentiating the iPSCs to obtain hemogenic endothelial (HE) cells; and (ii) differentiating the iPSCs to obtain CD82 + 2. Sorting HE cells for cells that are CD82 (e.g., by using an anti-CD82 antibody) + and obtaining secondary HE cells that express a cell marker comprising:

[0011] In some embodiments of the cell population, the cell marker is CD34 + , CD43 -, RUNX1 + or any combination thereof (e.g., obtained by sorting with an anti-CD34 antibody and / or an anti-CD43 antibody), and the resulting secondary HE cells further comprise CD34 + CD82 + , CD34 + CD43 - CD82 + , CD34 + CD82 + RUNX1 + , or CD34 + CD43 - CD82 + RUNX1 + In various embodiments of the cell population, differentiation of the iPSCs comprises contacting the iPSCs 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 the 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, CD82 + At least 0.5%, at least 1%, or at least 2% of the cells that are secondary HE cells. In some embodiments, the cell population is characterized by its phenotype (e.g., CD82 + , CD34 + CD82 + , CD34 + CD43 - CD82 + , CD34 + CD82 + RUNX1 + , or CD34 + CD43 - CD82 + RUNX1 + ) is a substantially pure population of cells having

[0012] In another aspect, the invention provides a composition comprising a cell population described herein. In some embodiments, the composition further comprises a cryopreservation medium.

[0013] In another aspect, the present invention provides a method for producing iPSC-derived secondary HE, the method comprising differentiating iPSCs to obtain iPSC-derived hemogenic endothelial (HE) cells; and + sorting the HE cells for cells that are CD82 (e.g., using antibodies, including anti-CD82 antibodies); + and obtaining secondary HE cells expressing cell markers including CD34, wherein the secondary HE cells are capable of differentiating into hematopoietic lineage cells, including NK progenitor cells, NK cells, NKT cells, or B cells, as well as T cell progenitor cells and T cells. In various embodiments of the method, the sorting step comprises obtaining secondary HE cells expressing cell markers including CD34 + , CD43 - , RUNX1 + or any combination thereof (e.g., obtained by sorting with an anti-CD34 antibody and / or an anti-CD43 antibody), wherein the resulting secondary HE cells are + 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 iPSCs with a medium comprising a BMP activator and bFGF, thereby differentiating the iPSCs 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 optionally a p38 MAPK inhibitor, thereby differentiating the mesodermal progenitor cells to obtain HE cells. In various embodiments of the method, contacting with the p38 MAPK inhibitor improves maintenance of CD82 expression in the HE cells compared to contacting without the p38 MAPK inhibitor, and 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.

[0014] In some embodiments of the methods for producing secondary HE cells derived from iPSCs, the iPSCs include naive iPSCs and / or are derived from iPSCs containing one or more genetic imprints. In some embodiments, the one or more genetic imprints contained in the iPSCs are retained in the secondary HE cells derived from the iPSCs. In various embodiments, the method further includes cryopreserving the secondary HE cells.

[0015] In another aspect, the present invention provides a composition for generating secondary HE (hemogenic endothelial) cells from iPSCs, the composition comprising a BMP activator, bFGF, VEGF, a Wnt pathway activator, and optionally a p38 MAPK inhibitor. In various embodiments of the composition, (i) the composition does not comprise a TGFβ receptor / ALK inhibitor, (ii) the generated iPSC-derived secondary HE comprises increased RUNX1-expressing cells compared to differentiation without the BMP activator, and / or (iii) the generated iPSC-derived secondary HE comprises increased CD82-expressing cells compared to differentiation without the p38 MAPK inhibitor. 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, CAS 219138-24-6, 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 secondary HE cells.

[0016] In another aspect, the present invention provides a method for producing secondary HE from iPSCs, the method comprising: (i) differentiating iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain HE cells; and (iii) differentiating iPSCs from CD82 + 2. Sorting HE cells for cells that are CD82 (e.g., by using an anti-CD82 antibody) + and obtaining secondary hemogenic endothelial (HE) cells that express cell markers including CD34, wherein the secondary HE cells are capable of differentiating into hematopoietic lineage cells, including NK cell precursors, NK cells, NKT cells, or B cells, as well as T cell precursors and T cells. In various embodiments of the method, the sorting step comprises obtaining secondary hemogenic endothelial (HE) cells that express cell markers including CD34 + , CD43 - , RUNX1 +or any combination thereof (e.g., obtained by sorting with an anti-CD34 antibody and / or an anti-CD43 antibody), wherein the resulting secondary HE cells are + CD82 + , CD34 + CD43 - CD82 + , CD34 + CD82 + RUNX1 + , or CD34 + CD43 - CD82 + RUNX1 + In some embodiments of the method, 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 RUNX1-expressing HE 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 the 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 resulting secondary HE cells.

[0017] In another aspect, the present invention provides methods for producing iPSC-derived hematopoietic lineage cells by differentiating the secondary HE cells described herein, the method comprising contacting the secondary HE cells with a media 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 precursors and T cells in addition to NK cell precursors, NK cells, NKT cells, or B cells. In various embodiments of the method, the iPSC-derived hematopoietic lineage cells comprise NK cell precursors and / or NK cells, and (1) the media composition further comprises IL15, and / or (2) the secondary HE cells comprise a genetic 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. In some embodiments of the method, the media composition does not comprise OP9 stromal cells. In some embodiments, the differentiating step is carried out in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4 (human DLL4 Fc chimeric recombinant protein).

[0018] In another aspect, the present invention provides a method for producing iPSC-derived hematopoietic lineage cells, the method comprising differentiating iPSCs to express CD82 +and differentiating the secondary HE cells to obtain iPSC-derived hematopoietic lineage cells, wherein the iPSC-derived hematopoietic lineage cells include 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 iPSCs comprise one or more genetic imprints introduced into the iPSCs by genome editing during or after reprogramming of non-pluripotent cells into iPSCs, wherein the one or more genetic imprints comprise (i) one or more genetic modification modalities introduced by genomic insertion, deletion, or substitution in the genome of the iPSC, or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor-, disease-, or treatment-response-specific, wherein the iPSCs are reprogrammed from source-specific immune cells, and the one or more genetic imprints are retained in the iPSC-derived hematopoietic lineage cells. In some embodiments of the method, differentiating the iPSCs to obtain secondary hematopoietic endothelial (HE) cells comprises (i) differentiating the genetically engineered iPSCs to obtain mesodermal progenitor cells, (ii) differentiating the mesodermal progenitor cells to obtain HE cells, and (iii) differentiating the genetically engineered iPSCs to obtain secondary hematopoietic endothelial (HE) cells. + sorting the HE cells (e.g., by using an anti-CD82 antibody) for cells that are CD82 + and obtaining secondary HE cells that express cell markers including CD34. In some embodiments, the sorting step comprises: + , CD43 - , RUNX1 + or any combination thereof (e.g., obtained by sorting with an anti-CD34 antibody and / or an anti-CD43 antibody), and the resulting secondary HE cells are + CD82 + , CD34 + CD43 - CD82 + , CD34 + CD82 + RUNX1 + , or CD34 + CD43 - CD82 + RUNX1+ Includes the expression:

[0019] In various embodiments of the method, differentiating mesodermal progenitor cells into HE cells comprises contacting the mesodermal progenitor cells with (i) a cytokine that results in a higher percentage of RUNX1-expressing HE 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 the 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 the secondary HE cells, wherein the cryopreserved secondary HE cells are thawed prior to differentiation. In some embodiments, differentiating the secondary HE cells does not include OP9 stromal cells. In some embodiments, differentiating the secondary HE cells is performed in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4.

[0020] In another aspect, the present invention provides a method for producing NK cells in a feeder-free environment, the method comprising: (a) differentiating iPSCs or secondary 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 (i) the culture medium comprises IL15, and / or (ii) the secondary HE cells further comprise genetic 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; and (b) expanding and activating the NK lineage cells to obtain NK cells having cytotoxicity against a target. In some embodiments, the culture medium further comprises one or more of a ROCK inhibitor, TPO, and IL3. In some embodiments, the secondary HE cells are (i) CD82+ , (ii) CD34 + CD82 + , (iii) CD34 + CD43 - CD82 + , and / or (iv) CD34 + and CD43 - , CD93 - , CXCR4 - , CD73 - , and RUNX1 + and a phenotype comprising at least one of:

[0021] In some embodiments of the method of generating NK cells, the step of differentiating the iPSCs comprises (i) differentiating the iPSCs to obtain mesodermal progenitor cells, (ii) differentiating the mesodermal progenitor cells to obtain hemogenic endothelial (HE) cells, and (iii) differentiating the iPSCs to obtain hemogenic endothelial (HE) cells. + sorting the HE cells (e.g., by using an anti-CD82 antibody) for cells that are CD82 + and obtaining secondary HE cells that express cell markers including CD34. In some embodiments of the method, the sorting step comprises obtaining secondary HE cells that express cell markers including CD34. + , CD43 - , RUNX1 + or any combination thereof (e.g., obtained by sorting with an anti-CD34 antibody and / or an anti-CD43 antibody), wherein the resulting secondary HE cells are + 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 RUNX1-expressing HE 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 the 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.

[0022] In some embodiments of the method, the differentiating step (a) further comprises contacting the secondary HE cells with an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4, and / or the expanding step (b) further comprises contacting the NK lineage cells with a proliferation composition comprising nicotinamide. In some embodiments, the expanding step (b) 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 explanation of the drawings]

[0023] [Figure 1] Flow cytometry analysis of D10 cells differentiated under control conditions and with cytokines that promote the specification of RUNX1+ HE (cytokine induction) is shown. [Figure 2] A summary of candidate HE surface markers identified by BioLegend LEGENDScreen is shown. [Figure 3A] UMAP visualization of the expression of curated genes used to identify cell clusters. [Figure 3B]Figure 1 shows the transcriptome profile of a D10 cell population visualized by UMAP. Each dot represents one cell. [Figure 3C] Violin plots of CD82 expression within each cell cluster from Figure 3B are shown. [Figure 4A] Flow cytometry analysis of cytokine-induced D10 cells comparing expression of HE candidate markers with RUNX1 and CD82. [Figure 4B] Flow cytometry analysis of cytokine-induced D10 cells comparing expression of HE candidate markers with RUNX1 and CD82. [Figure 5] Flow cytometry analysis of cytokine-induced D10 cells demonstrating enrichment of CD82+ cells within the CD73-CD93-CXCR4- endothelial population. Cells are pre-gated on single / live events. [Figure 6] The frequency (prevalence) of HE within cytokine-induced D10 populations subjected to fluorescence-activated cell sorting (FACs) based on the indicated markers on the x-axis is shown (mean±SD). [Figure 7] Flow cytometry analysis of D35 iT cells derived from cytokine-induced D10 populations FAC-sorted based on the markers indicated on the flow plots. [Figure 8] Flow cytometry analysis of D30 iNK cells derived from cytokine-induced D10 populations FAC-sorted based on the markers indicated on the flow plot. [Figure 9A] A comparison of fold expansion and iNK specification between cells differentiated on Retro / DLL4, irOP9-DLL4 and commercial kits is shown. [Figure 9B] A comparison of fold expansion and iNK specification between cells differentiated on Retro / DLL4, irOP9-DLL4 and commercial kits is shown. [Figure 9C] A comparison of fold expansion and iNK specification between cells differentiated on Retro / DLL4, irOP9-DLL4 and commercial kits is shown. [Figure 9D]A comparison of fold expansion and iNK specification between cells differentiated on Retro / DLL4, irOP9-DLL4 and commercial kits is shown. [Figure 10A] A comparison of the fold expansion of precursor iNK cells obtained using each of the three differentiation strategies when co-cultured with genetically engineered feeder cells to obtain activated NK cells is shown. [Figure 10B] A comparison of the fold expansion of precursor iNK cells obtained using each of the three differentiation strategies when co-cultured with genetically engineered feeder cells to obtain activated NK cells is shown. [Figure 10C] A comparison of the fold expansion of precursor iNK cells obtained using each of the three differentiation strategies when co-cultured with genetically engineered feeder cells to obtain activated NK cells is shown. [Figure 11A] Figure 1 shows that antigen-dependent caspase 3 / 7 activity is comparable between mature iNK cells differentiated with Retro / DLL4 or irOP9-DLL4. [Figure 11B] Figure 1 shows that antigen-dependent caspase 3 / 7 activity is comparable between mature iNK cells differentiated with Retro / DLL4 or irOP9-DLL4. [Figure 12A] Figure 1 shows that the release of IFNγ and TNFα cytokines is comparable between mature iNK cells differentiated with Retro / DLL4 or irOP9-DLL4. [Figure 12B] Figure 1 shows that the release of IFNγ and TNFα cytokines is comparable between mature iNK cells differentiated with Retro / DLL4 or irOP9-DLL4. [Figure 13] We show that Retro / DLL4-differentiated iNK cells exhibit antigen-dependent sequential killing capacity similar to that of irOP9-DLL4-differentiated cells. [Figure 14] We show that the addition of an AhR (aryl hydrocarbon receptor) inhibitor during the cell expansion stage of iPSC differentiation results in greater expansion fold and yield of differentiated NK cells. [Figure 15]1 shows that cells treated with AhR inhibitors before cryopreservation showed enhanced antitumor efficacy after thawing over time. DETAILED DESCRIPTION OF THE INVENTION

[0024] In various aspects, the present invention generally relates to methods and compositions for differentiating stem cells to a definitive hematopoietic cell fate. In certain aspects, the present invention provides a multi-stage differentiation platform that can induce iPSCs or iPSC-derived cells at various stages of development to definitive hematopoietic phenotypes ranging from secondary hemogenic endothelium to fully differentiated hematopoietic cells, including T cells, B cells, NKT cells, and NK cells. Definitive hematopoietic fate, e.g., CD34 + Methods and compositions are provided for making cells more susceptible to becoming secondary hematopoietic stem cells. In some embodiments, the methods and compositions of the present invention generate secondary hemogenic endothelium (HE) from naive iPSCs in a scalable manner by avoiding the formation of EBs or aggregates.

[0025] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0026] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. 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.

[0027] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0028] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0029] The term "and / or" should be understood to mean either one or both of the alternatives.

[0030] Throughout this specification, unless the context requires otherwise, "comprise," "comprises," and "comprising" will be understood to mean the inclusion of a stated step, element, or group of steps or elements, but not the exclusion of any other step, element, or group of steps or elements. In certain embodiments, the terms "include," "having," "contain," and "comprise" are used interchangeably.

[0031] "Consisting of" means including and limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0032] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the listed elements.

[0033] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0034] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0035] As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" or "essentially the same" refers to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about identical to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0036] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of a particular substance or source thereof, e.g., 95% free, 96% free, 97% free, 98% free, 99% free, etc., of a particular substance or source thereof, or is undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition but at such a low concentration that it is functionally inactive. A similar meaning may be applied to the term "absent," which refers to the absence of a particular substance or source thereof from a composition.

[0037] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of the body, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues removed from a living organism and cultured in a laboratory setting, usually under sterile conditions, typically for several hours or up to about 24 hours (although up to 48 hours or 72 hours or more, depending on the circumstances). In certain embodiments, such tissues or cells may be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissues are typically considered "in vitro," although in certain embodiments, the term may be used interchangeably with ex vivo.

[0038] The term "in vivo" generally refers to activities that occur inside a living organism.

[0039] The term "effector cell" generally applies to specific cells in the immune system that carry out a specific activity in response to stimulation and / or activation, or cells that provide a specific function upon activation. As used herein, the term "effector cell" includes, and in some contexts is interchangeable with, immune cells, "differentiated immune cells," and primary or differentiated cells that have been edited and / or modified to carry out a specific activity in response to stimulation and / or activation. Non-limiting examples of effector cells include primary- or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0040] As used herein, the terms "B lymphocyte" or "B cell" are used interchangeably and refer to a subset of lymphocytes defined by the expression of a B cell receptor composed of immunoglobulin heavy and light chains (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 or progenitor cells by directed differentiation. B cells include any subtype of B cells and can be at any stage of development, including, but not limited to, pro-B cells, pre-B cells, naive B cells, B-1 B cells, B-2 B cells, marginal zone B cells, follicular B cells, memory B cells, plasmablasts, plasma cells, and regulatory B cells.

[0041] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that has completed maturation in the thymus and has various 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. T cells can be any T cell, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. T cells are CD3 + T cells can be CD4 + / CD8 + Double positive T cells, CD4 +Helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells can be of any type and at any developmental stage, including, but not limited to, T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional 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 modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also be differentiated from stem or progenitor cells ("derived T cells" or "derived T cell-like effector cells," or collectively "derived T lineage cells"). Derived T cell-like effector cells may possess T cell lineage in some respects, but at the same time possess one or more functional attributes not present in primary T cells. In this 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."

[0042] "CD4 +"CD4 cells" refers to a subset of T cells that express CD4 on their surface and are associated with cellular immune responses. They are characterized by their secretory profile after stimulation, which may include secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. The "CD4" molecule is a 55 kD glycoprotein originally defined as a differentiation antigen for 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 involved as the relevant recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines helper / inducer subsets.

[0043] "CD8 + "CD8 cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the relevant recognition element in major histocompatibility complex class I-restricted interactions.

[0044] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). NK cells can be any NK cell, e.g., cultured NK cells (e.g., primary NK cells), or NK cells derived from cultured or expanded NK cells, or cell line NK cells (e.g., NK-92), or NK cells obtained from a mammal that is healthy or has a disease state. As used herein, the terms "adaptive NK cells" and "memory NK cells" are used interchangeably and are phenotypically CD3+. - and CD56 +and refers to a subset of NK cells that express at least one of NKG2C and CD57, and optionally CD16, but lack expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, CD56 + The isolated subpopulation of NK cells includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + may be dim or bright expression. NK cells or NK cell-like effector cells can be differentiated from stem or progenitor cells ("derived NK cells" or "derived NK cell-like effector cells," or collectively "derived NK lineage cells"). Derived NK cell-like effector cells may have NK cell lineage in some respects, but at the same time possess one or more functional attributes not present in primary NK cells. In this application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells, or derived NK lineage cells are collectively referred to as "NK lineage cells."

[0045] As used herein, the terms "NKT cells" or "natural killer T cells" or "NKT lineage cells" refer to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the nonclassical MHC molecule, CD1d. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., a canonical α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, termed nonclassical or non-invariant type II NKT cells, displays more heterogeneous TCRαβ usage. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0046] As used herein, the terms "secondary hemogenic endothelium" (HE) or "pluripotent stem cell-derived secondary hemogenic endothelium" (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem cells and progenitor cells in a process called endothelial-hematopoietic conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm through hemangioblasts to secondary hemogenic endothelium and hematopoietic precursors. In some embodiments, populations of iHE cells can be maintained, stored, and / or cryopreserved in multiple containers to reliably serve as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment.

[0047] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including pluripotent hematopoietic stem cells (blood cells), myeloid progenitors, megakaryocyte progenitors, erythroid progenitors, and lymphoid progenitors. 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 (T cells, B cells, NK cells) lineages. As used herein, the term "secondary hematopoietic stem cells" refers to cells that express the CD34 receptor and can give rise to both mature myeloid and lymphoid cell types, including T-lineage cells, NK-lineage cells, and B-lineage cells. + Refers to hematopoietic cells, which also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0048] As used herein, the term "embryonic stem cells" refers to naturally occurring pluripotent stem cells of the inner cell mass of the blastocyst. Embryonic stem cells are pluripotent and give rise to derivatives of all three primary germ layers, i.e., ectoderm, endoderm, and mesoderm, during development. They do not contribute to the extraembryonic membranes or placenta and are not totipotent.

[0049] As used herein, the term "pluripotent stem cells" refers to cells that have the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, pluripotent cells can also be 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" indicates that a cell can form many types of cells of a given lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Therefore, the term "multipotency" refers to a state of a cell that has a degree of developmental potential that is lower than totipotency and pluripotency.

[0050] As used herein, the term "pluripotency" refers to the ability of a cell (i.e., the embryo itself) to form all lineages of an organism or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0051] As used herein, the term "induced pluripotent stem cells" or "iPSCs" means stem cells generated from differentiated adult, neonatal, or fetal cells that have been induced or modified (i.e., reprogrammed) into cells that can differentiate into all tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm.

[0052] Pluripotency can be determined, in part, by assessing the pluripotency characteristics of cells, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the capacity for unlimited self-renewal, (iii) the 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.

[0053] Two types of pluripotency have been described: a "primed" or "metastable" state resembling the epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" state resembling the inner cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single-cell culture, (iii) globally reduced DNA methylation, (iv) reduced deposition of the H3K27me3 repressive chromatin mark on developmentally regulated gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard methodologies for cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or deleted from the resulting pluripotent cells, generally appear to have characteristics of a primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the primed state and characteristics of the ground state are observed unless exogenous transgene expression is maintained.

[0054] Pluripotency exists as a continuum, and induced pluripotent stem cells (iPSCs) appear to exist in both a "primed" and a "naive" state, with naive cells sometimes possessing greater differentiation potential. Induced pluripotent stem cells generated in conventional culture media exist in a primed state, more closely resembling cells derived from postimplantation blastocysts, while naive iPSCs exhibit pluripotent characteristics more closely resembling cells derived from mouse embryonic stem cells or preimplantation blastocysts. Primed and naive cell states can be defined by various differences, including differences in colony morphology, cellular response to inhibition or activation of key signaling pathways, gene expression signatures, and the ability to reactivate genes associated with extraembryonic cells. For example, conventional iPSCs, representing the primed pluripotent state, exhibit flat colony morphology, while naive iPSCs exhibit a compact, domed colony morphology similar to mouse embryonic stem cells. As used herein, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round, compact shape with a high nucleus-to-cytoplasm ratio, prominent nucleoli, and typical intercellular spacing.

[0055] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, a blood cell or a muscle cell. A differentiated or differentiation-induced cell is a cell that assumes a more specialized ("committed") position within the lineage of a cell. The term "committed," when applied to the process of differentiation, refers to a cell that has progressed along a differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a particular cell type or subset of cell types and, under normal circumstances, is unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0056] Differentiation of pluripotent stem cells requires changes in the culture system, including the addition of stimuli in the culture medium and changes in the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a common and critical intermediate for initiating lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development by generating multiple lineages within a three-dimensional space. Throughout the differentiation process, which typically takes hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, at which point they are manipulated to further differentiate, typically over days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity in a three-dimensional multilayered cluster of cells. Typically, this is achieved by one of several methods, including settling pluripotent cells in droplets, settling cells in "U"-bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB growth. Therefore, pluripotent stem cell aggregates must be transferred to a differentiation medium that provides the cues required to induce the desired lineage. EB-based culture of pluripotent stem cells typically generates differentiated cell populations (i.e., ectoderm, mesoderm, and endoderm germ layers) with moderate proliferation within the EB cell clusters. While EBs have been proven to promote cell differentiation, inconsistent exposure of the three-dimensional structure to differentiation cues in the environment can result in heterogeneous cells with various differentiation states. Additionally, EBs are laborious to create and maintain. Furthermore, cell differentiation through EB formation is accompanied by moderate cell proliferation, leading to reduced differentiation efficiency.

[0057] In contrast, "aggregate formation," unlike "EB formation," can be used to expand a population of pluripotent stem cell-derived cells. For example, during aggregate-based pluripotent stem cell expansion, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates to form larger aggregates, which can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell growth and increase cell number in culture. Unlike EB culture, cells cultured within aggregates in maintenance culture medium maintain markers of pluripotency. Pluripotent stem cell aggregates require additional differentiation cues to induce differentiation.

[0058] As used herein, "monolayer differentiation" is a term that refers to differentiation across three-dimensional, multi-layered clusters of cells, a differentiation method distinct from "embryoid bodies," "EBs," or "EB formation." Monolayer differentiation avoids the need for EB formation to initiate differentiation, among other advantages disclosed herein. Because monolayer culture does not mimic embryonic development as in the case of EB formation, differentiation into specific lineages is considered minimal compared to differentiation into all three germ layers with EB formation.

[0059] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case singular "medium"), "supplement," and "medium supplement" refer to a nutritional composition in which a cell culture is cultivated.

[0060] As used herein, "feeder cells" or "feeders" refers to one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate, since the feeder cells provide stimuli, growth factors, nutrients, and support the second cell type. Feeder cells may optionally be derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the proliferation and maturation of natural killer cells. When co-cultured with other cells, feeder cells are typically inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from outgrowing the supporting cells. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type may be a human feeder, such as a human dermal fibroblast. Another feeder cell type may be a mouse embryonic fibroblast (MEF). Generally, various feeder cells may be used in part to maintain pluripotency, direct differentiation to specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types, such as effector cells.

[0061] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is essentially free of feeder or stromal cells and / or has not been preconditioned by the culture of feeder cells. A "preconditioned" medium refers to a medium that has been harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. Preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain either feeder cells or stromal cells, nor has it been preconditioned by the culture of feeder cells. Feeder cells include, but are not limited to, stromal cells, mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells.

[0062] "Culturing" or "maintaining" refers to sustaining, propagating (growing), and / or differentiating cells outside of a tissue or body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" can utilize culture medium as a source of nutrients, hormones, and / or other factors that aid in the growth and / or maintenance of the cells.

[0063] As used herein, "passage" or "passaging" refers to the act of dividing cultured cells by subdividing and plating the cells onto multiple cell culture surfaces or vessels when the cells have proliferated to a desired extent. In some embodiments, "passage" or "passaging" refers to subdividing, diluting, and plating the cells. As cells are passed from a primary culture surface or vessel to a series of subsequent surfaces or vessels, subsequent cultures may be referred to herein as "secondary cultures" or "first passages," etc. Each act of subdividing and plating onto a new culture vessel is considered a passage. In some embodiments, cultured cells are passaged every 1, 2, 3, 4, 5, 6, 7, or more days. In some embodiments, iPSCs initially selected after reprogramming are passaged once every 3-7 days.

[0064] As used herein, "dissociated cells" or "single dissociated cells" refer to cells that have been substantially separated or purified away from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be enzymatically or mechanically dissociated from one another, e.g., by dissociation 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 involves disrupting the extracellular matrix (ECM) and cellular interactions with a substrate (e.g., a culture surface) or disrupting the ECM between cells.

[0065] As used herein, terms such as "isolated" refer to a cell or population of cells separated from its original environment; i.e., the environment of an isolated cell is substantially free of at least one component found in the environment of a "non-isolated" reference cell. This term includes cells that are removed from some or all components found in their naturally occurring environment, e.g., when isolated from a tissue or biopsy sample. This term also includes cells that are removed from at least one, some, or all components because they are found in a non-native environment, e.g., an environment isolated from a cell culture or cell suspension. Thus, an "isolated cell" is partially or completely separated from at least one component, including other substances, cells, or cell populations, as found in nature or when grown, stored, or persisted in a non-native environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a non-naturally occurring medium. Isolated cells can be obtained by separating a desired cell or population thereof from other substances or cells in the environment or by removing one or more other cell populations or subpopulations from the environment.

[0066] As used herein, terms such as "purify" refer to increasing 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%.

[0067] As used herein, "master cell bank" or "MCB" refers to a clonal master engineered iPSC line that is a clonal population of iPSCs that have been engineered to contain one or more therapeutic properties, characterized, tested, qualified, expanded, and shown to reliably serve as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic mutations and / or potential contamination by reducing and / or eliminating the total number of times the iPS cell line is passaged, thawed, or handled during the manufacturing process.

[0068] As used herein, the terms "reprogramming" or "dedifferentiation" or "increased cell potential" or "increased developmental potential" refer to a method of increasing the potential of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an unreprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in an unreprogrammed state. In contrast to "reprogrammed cells," "reprogrammed cells" refer to non-pluripotent cells that have undergone reprogramming / dedifferentiation to a pluripotent state and exhibit transitional morphology (i.e., a change in morphology) but do not possess the characteristics of pluripotent cells, including pluripotent stem cell morphology or the expression of stable endogenous pluripotency genes such as OCT4, NANOG, SOX2, SSEA4, TRA181, CD30, and / or CD50. The transitional morphology of "reprogrammed cells" distinguishes them from the starting non-pluripotent cells prior to the induction of reprogramming and from reprogrammed cells that have the characteristic morphology of embryonic stem cells. For example, when reprogramming fibroblasts, morphological changes in reprogrammed cells include MET (mesenchymal to epithelial transition). Those skilled in the art readily understand and identify such transitional morphologies for various types of somatic cells induced to reprogram. In some embodiments, reprogrammed cells are intermediate cells induced to reprogram for at least 1, 2, 3, 4, 5, 6, 7, 8, or more days, but not more than 21, 22, 24, 26, 28, 30, 32, 35, 40 days, or any number of days in between, where the cells have not entered a self-sustaining or self-sustaining pluripotent state. Non-pluripotent cells are induced to reprogram when one or more reprogramming factors are introduced into the cells. Reprogrammed cells induced to reprogram for 1, 2, 3, or 4 days are cells 1, 2, 3, or 4 days after transduction of reprogramming factors (the day of transduction is day 0).Unlike somatic cells prior to exposure to exogenous expression of reprogramming factors, "reprogrammed cells" can progress through the reprogramming process to reach a stable pluripotent state and, given a sufficient period of time, become "reprogrammed cells" even in the absence of exogenously expressed reprogramming factors.

[0069] "Pluripotency factor" or "reprogramming factor" refers to a factor or combination of factors used to induce or increase the developmental potential of a cell. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors OCT4 and SOX2, and small molecule reprogramming agents, such as TGFβ inhibitors, GSK3 inhibitors, MEK inhibitors, and ROCK inhibitors.

[0070] As used herein, "genetic modification" refers to gene editing, including (1) those naturally resulting from rearrangements, mutations, genetic imprinting, and / or epigenetic modifications occurring within a cell or during cell development, or (2) those obtained through genome engineering by cellular manipulation, including, but not limited to, insertions, deletions, or substitutions in the genome of a cell. As used herein, genetic modification also includes one or more retainable therapeutic properties of source-specific immune cells that are donor-, disease-, or therapeutic response-specific. Genetically modified cells are cells that contain a genetic modification (e.g., a gene edit) compared to a corresponding wild-type cell that does not have such a genetic modification.

[0071] "Functional" 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, refers to (1) successful transgenic or controlled gene expression, such as inducible or transient expression, at a desired stage of cellular development, achieved by knock-in, knock-out, knock-down gene expression, direct genome editing or modification, or by "passaging" through differentiation from or reprogramming of an initially genomically engineered starting cell, or (2) at the cellular level, (i) the successful editing or modification of a gene in a cell through direct genome editing. (ii) gene expression modifications that are maintained in a cell through "passage" via differentiation or reprogramming of the original genomically engineered starting cell; (iii) downstream gene regulation in a cell as a result of gene expression modifications that are only apparent in an earlier developmental stage of the cell or only apparent in the starting cell that gave rise to the cell via differentiation or reprogramming; or (iv) enhanced or newly achieved cellular function or property exhibited in a mature cell product originally derived from genome editing or modifications performed on an iPSC, progenitor cell, or dedifferentiated cell source.

[0072] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to preferential and / or enhanced therapeutic properties of source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can be further differentiated into specific cell types, including any hematopoietic lineage cell. Source cell-derived iPSCs and cells differentiated therefrom may be collectively referred to as "derived" or "derived" cells, depending on the context. For example, as used throughout this application, derived effector cells, or derived NK cells or derived T lineage cells are cells differentiated from iPSCs, compared to their primary counterparts obtained from natural / original sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, genetic imprints that confer preferential and / or enhanced therapeutic properties are incorporated into iPSCs by reprogramming selected source cells that are specific to the donor, disease, or therapeutic response, or by using genome editing to introduce genetic modification modalities into the iPSCs. In embodiments of source cells obtained from specifically selected donors, diseases, or therapeutic situations, genetic imprints that contribute to preferential therapeutic properties can include situation-specific genetic or epigenetic modifications that represent a retainable phenotype, i.e., preferential therapeutic property, that are passed on to iPSC-derived cells of the selected source cells, regardless of whether the underlying molecular events have been identified.Source cells specific to a donor, disease, or therapeutic response may contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-positioned monospecific TCRs, e.g., from virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, e.g., homozygosity for a point mutation encoding the high-affinity CD16 receptor in the selected donor; and selected HLA-matched donor cells that exhibit predetermined HLA requirements, i.e., haplotypes, in an expanded population. As used herein, preferential and / or enhanced therapeutic properties include improved engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and modulation, survival, and cytotoxicity of derived cells. Preferential therapeutic properties are also manifested by antigen-targeting receptor expression, HLA presentation or lack thereof, tolerance to the tumor microenvironment, induction of bystander immune cells and immune modulation, improved on-target specificity with reduced extratumoral effects, and resistance to treatments such as chemotherapy. When derivative cells with one or more therapeutic properties are obtained from differentiating iPSCs that incorporate genetic imprints that confer preferential therapeutic properties, such derivative cells are also referred to as "synthetic cells." Generally, synthetic cells have one or more non-native cellular functions when compared to their closest corresponding primary cells, regardless of whether the synthetic cells are differentiated from engineered pluripotent cells or obtained by manipulating primary cells from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissue. For example, synthetic effector cells, or synthetic NK cells or synthetic T cells, as used throughout this application, are cells differentiated from genomically modified iPSCs when compared to their primary counterparts obtained from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissue. In some embodiments, synthetic cells have one or more non-native cellular functions when compared to their closest corresponding primary cells.

[0073] As used herein, the term "exogenous" is intended to mean that a referenced molecule or activity is introduced into a host cell or is non-native to the host cell. An exogenous molecule can be introduced, for example, by integrating into a host chromosome or by introducing an encoding nucleic acid into the host's genetic material as non-chromosomal genetic material, such as a plasmid. Thus, the term used with respect to expression of an encoding nucleic acid refers to introducing the encoding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity that is present in a host cell. Similarly, when used with respect to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0074] A "construct" refers to a macromolecule or complex of molecules containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell and that can replicate and / or express the material in the target cell. Thus, the term "vector" includes the delivered construct. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. Major types of vectors include, but are not limited to, plasmids, episomal 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, etc.

[0075] "Integration" means that one or more nucleotides of a construct are stably inserted into a cell's genome, i.e., covalently linked to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that nucleotides of a construct are inserted into the chromosome or mitochondrial DNA of a cell at a preselected site or "integration site." As used herein, the term "integration" also refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of the endogenous sequence or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" can further include replacement of the deleted nucleotide with the endogenous sequence or one or more inserted nucleotides.

[0076] As used herein, the term "encoding" refers to the inherent property of a particular 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 biological processes that have a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually set forth in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to "encode" the protein or other product of that gene or cDNA.

[0077] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. A gene of interest or polynucleotide may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may encode an miRNA, an shRNA, a naturally occurring polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a variant of a naturally occurring polypeptide having less than 100% sequence identity with the naturally occurring polypeptide) or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selection marker, etc.

[0078] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases adenine (A), cytosine (C), guanine (G), and thymine (T); if the polynucleotide is RNA, thymine is uracil (U). Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. "Polynucleotide" also refers to both double-stranded and single-stranded molecules.

[0079] "Operably-linked" or "operatively linked" are interchangeable with "operably connected" or "operatively connected" and refer to the association of nucleic acid sequences (or amino acids in a polypeptide having multiple domains) on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it is capable of affecting the expression of that 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 a further example, a receptor binding domain can be operably connected to an intracellular signaling domain such that binding of the receptor to a ligand transduces a signal in response to that binding.

[0080] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also generally referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as polypeptides or proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include naturally occurring polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0081] A "fusion protein" or "chimeric protein," as used herein, is a protein created by genetic engineering that joins two or more partial or complete polynucleotide sequences encoding separate proteins, such that expression of these joined polynucleotides results in a single peptide or multiple polypeptides that possess functional properties derived from each of the original proteins or fragments thereof. A linker (or spacer) peptide can be added between the two adjacent polypeptides of different sources in a fusion protein.

[0082] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell (e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil) and a tumor cell and activate the immune cell. Examples of engagers include, but are not limited to, a bi-specific T cell engager (BiTE), a bi-specific killer cell engager (BiKE), a tri-specific killer cell engager (TriKE), or a multispecific killer cell engager, or a universal engager that is compatible with multiple immune cell types.

[0083] As used herein, the term "surface triggering receptor" refers to a receptor that can induce or initiate an immune response, e.g., a cytotoxic response. Surface triggering receptors can be engineered and expressed on effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, the surface triggering receptor promotes bispecific or multispecific antibody binding between effector cells and specific target cells (e.g., tumor cells) regardless of the effector cell's native receptor and cell type. Using this approach, iPSCs containing universal surface triggering receptors can be generated, and such iPSCs can be differentiated into populations of various effector cell types that express the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed and activated on any effector cell, regardless of cell type; all effector cells that express the universal receptor can bind or be linked to an engager that can be recognized by the surface triggering receptor, regardless of the engager's tumor-binding specificity. In some embodiments, engagers with the same tumor-targeting specificity are used to bind to the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to a universal surface triggering receptor. Thus, one or more effector cell types may engage to kill one specific type of tumor cell or two or more types of tumors. The surface triggering receptor generally contains a costimulatory domain for effector cell activation and an anti-epitope specific for the engager epitope. Bispecific engagers are specific for the anti-epitope of a surface triggering receptor on one end and a tumor antigen on the other end.

[0084] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. In some instances, expression of the safety switch protein is conditionally controlled to address safety concerns for transplanted, engineered cells that have permanently integrated a gene encoding the safety switch protein into their genome. This conditional regulation can be variable and may include post-translational activation via small molecules and tissue-specific and / or transient transcriptional regulation. Safety switch proteins may mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some instances, safety switch proteins are activated by an exogenous molecule, e.g., a prodrug, and upon activation, trigger apoptosis and / or cell death of therapeutic cells. 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.

[0085] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative, therapeutic, or palliative properties for disease and can be administered to ameliorate, relieve, alleviate, reverse, or lessen the severity of the disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the onset of disease or to reduce the severity of such disease or pathological condition once it appears. "Pharmaceutically active protein" includes whole 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 multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0086] As used herein, the term "signaling molecule" refers to any molecule that modifies, participates in, inhibits, activates, reduces, or enhances cell signaling. "Signaling" refers to the transmission of a molecular signal in the form of a chemical modification through the recruitment of protein complexes along a pathway that ultimately leads to a biochemical event within the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Tollgate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0087] The term "ligand" refers to a substance that forms a complex with a target molecule and generates a signal by binding to a site on the target. A ligand may be a natural or artificial substance that can specifically bind to a target. A ligand may 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 to which a ligand binds may be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and alters its function, triggering a signaling response, is called "agonistic" or "agonist." A ligand that binds to a target and blocks or reduces the signaling response is "antagonistic" or "antagonist."

[0088] As used herein, the terms "specific" or "specificity" may be used to refer to the ability of a molecule, e.g., a receptor, antibody, or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0089] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, that are genetically incorporated into cells to promote antigen and / or epitope specificity, including, but not limited to: i) antigen specificity when associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when associated with a monoclonal antibody or bispecific engager; iii) targeting of transformed cells; iv) targeting of cancer stem cells; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0090] "HLA deficiency," including HLA class I deficiency, HLA class II deficiency, or both, refers to cells that lack or no longer maintain or have reduced levels of surface expression of complete MHC complexes containing HLA class I protein heterodimers and / or HLA class II heterodimers, where the reduced or reduced levels are lower than those naturally detectable by other cells or synthetic methods. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21) or by deletion or reduced expression levels of HLA class I-associated genes, including, but not limited to, the beta-2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 gene, and tapasin. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-associated genes, including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. It was previously unclear whether HLA complex-deficient or modified iPSCs have the ability to enter development, mature, and generate functional differentiated cells while retaining regulated activity. Additionally, it was previously unclear whether HLA complex-deficient differentiated cells could be reprogrammed into iPSCs and maintained as pluripotent stem cells while retaining HLA complex deficiency. Unexpected failures during cell reprogramming, maintenance of pluripotency, and differentiation may be related to aspects including, but not limited to, developmental stage-specific gene expression or lack thereof, the requirement for HLA complex presentation, protein shedding of introduced surface expression modalities, the need for proper and efficient clonal reprogramming, and the need for reconfiguration of differentiation protocols.

[0091] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that have been further modified by introducing genes expressing proteins related to improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as, but not limited to, 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 HLA-deficient" cells also include cells other than iPSCs.

[0092] The term "antibody" is used in the broadest sense herein and generally refers to an immune response-generating molecule that contains at least one binding site that specifically binds to a target, which may be an antigen or a receptor capable of interacting with a particular antibody. For example, NK cells can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptor (FcγR), thereby triggering ADCC (antibody-dependent cellular cytotoxicity)-mediated effector cell activation. The specific fragment or portion of an antigen or receptor, or generally the target, that an antibody binds is known as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, natural antibodies and variants thereof, fragments of natural antibodies and variants thereof, peptibodies and variants thereof, and antibody mimetics that mimic the structure and / or function of antibodies or specific fragments or portions thereof, including single-chain antibodies and fragments thereof. The antibody may be a murine antibody, a human antibody, a humanized antibody, a camelid IgG, a single variable new antigen receptor (VNAR), a shark heavy chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single domain antibody (dAb), an anti-idiotypic antibody, a bispecific, multispecific, or multimeric antibody, or a fragment thereof. An anti-idiotypic antibody is specific for binding to the idiotope of another antibody, an idiotope being an antigenic determinant of an antibody. A bispecific antibody may be a BiTE (bispecific T cell engager) or BiKE (bispecific killer cell engager), and a multispecific antibody may be a TriKE (trispecific killer cell engager).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, diabodies, triabodies, tetrabodies, single domain antigen binding fragments (sdAb), camelid heavy chain IgG and Nanobody® fragments, recombinant heavy chain antibodies (VHH), and other antibody fragments that maintain the binding specificity of the antibody.

[0093] "Fc receptors," abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind to the most common class of antibody, IgG, are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). Classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγR) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different antibody affinities due to their different molecular structures.

[0094] Two isoforms of the FcγR receptor CD16 have been identified: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and promoting antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high-affinity CD16," "non-cleavable CD16," or "high-affinity non-cleavable CD16" (abbreviated as hnCD16) refer to naturally occurring or non-naturally occurring variants of CD16. Wild-type CD16 has low affinity, and upon NK cell activation, it undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary high-affinity CD16 polymorphic variants. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) has been altered or eliminated do not undergo shedding. The cleavage site and membrane proximal region are described in detail in International Publication No. WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high affinity and non-cleavable. Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 containing an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0095] The term "adoptive cell therapy," as used herein, refers to cell-based immunotherapy involving the transfusion of autologous or allogeneic lymphocytes, such as genetically modified or unmodified CD34 cells, hemogenic endothelial cells, hematopoietic stem or progenitor cells, hematopoietic multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, B cells, or immunoregulatory cells, which have been expanded ex vivo prior to transfusion.

[0096] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domestic animal.

[0097] As used herein, the terms "treat," "treatment," and the like, when used in reference to a subject in need of therapeutic treatment, refer to obtaining a desired pharmacological and / or physiological effect, including, but not limited to, achieving an improvement or elimination of symptoms of a disease. The effect may be preventative, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it achieves an improvement or elimination of symptoms, or it may be therapeutic, in that it provides a partial or complete cure of the disease and / or side effects caused by the disease. The term "treatment" includes any treatment of a disease in a mammal, particularly a human, including (a) preventing a disease from occurring in a subject who may be susceptible to, but has not yet been diagnosed with, the disease; (b) inhibiting or halting the development of, the disease; (c) relieving the disease or causing regression of, the disease or completely or partially eliminating symptoms of the disease; and / or (d) restoring an individual to a pre-disease state, e.g., reconstituting the hematopoietic system.

[0098] As used herein, a "therapeutically sufficient amount" includes within its meaning a non-toxic but sufficient and / or effective amount of the particular therapeutic agent and / or pharmaceutical composition being referred to to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, 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, alleviate, and / or improve at least one symptom associated with the disease or condition being treated in the subject.

[0099] A. Compositions and Methods for the Production and Differentiation of Induced Pluripotent Stem Cells In some embodiments, the present invention generally provides a method for identifying naive pluripotent cells by isolating them from mesodermal progenitor cells, mesodermal cells, secondary hematopoietic endothelial cells, secondary hematopoietic stem or progenitor cells, CD34 + The present invention relates to a multi-step process for differentiating non-pluripotent or partially differentiated cells, including cells, multipotent progenitor cells (MPPs) (which can differentiate into myeloid cells, including neutrophil progenitors), T cell progenitors, NK cell progenitors, or fully differentiated terminal hematopoietic cells, such as T cells, B cells, NKT cells, or NK cells. In various embodiments, such naive pluripotent cells can be obtained by reprogramming source non-pluripotent cells into induced pluripotent stem cells (iPSCs), which retain one or more therapeutic properties of the source cells. In some aspects, the invention relates to compositions used in the disclosed methods, and to cell populations, cell lines, clonal cells, or master cell banks produced using the disclosed methods.

[0100] Existing methods for culturing pluripotent cells, such as iPSCs, rely heavily on feeder cells or media pretreated with feeder cells and containing fetal bovine serum, but such environments may not be suitable for producing cells for clinical and therapeutic use. For example, cells cultured in such heterogeneous and contaminated environments are generally considered unsuitable for human cell transplantation because exposure to animal components can pose a serious risk of immune rejection and transmission of unidentified pathogens to treated patients, and can potentially reactivate animal retroviruses. The culture systems contemplated herein using animal-component-free and feeder-free culture media facilitate the production of clinical-grade cell lines, particularly T, B, NKT, or NK cell lines derived from ESCs, iPSCs, and pluripotent stem cells.

[0101] In some embodiments, the feeder-free environment is essentially free of human feeder cells and is not pre-treated 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 further does not contain stromal cells, such as OP9 stromal cells. The feeder-free cell culture medium is suitable for use in culturing pluripotent cells, single cell culture, dissociation, and passaging of pluripotent cells, cell sorting of pluripotent cells, generating ground-state pluripotent cells, maintaining ground-state pluripotency, inducing differentiation of pluripotent cells, and maturation of effector cells from differentiation of pluripotent cells.

[0102] In contrast to methods used in the art, various aspects of the present invention avoid EB formation during differentiation. As provided in various embodiments, hematopoietic lineage cells derived from iPSCs are obtained by using a stepwise strategy of seeding clonal iPSC cells in a TGFβ-free culture medium to maintain the pluripotency of their basal or naive state, differentiating the clonal iPSCs in a monolayer without EB formation, and applying appropriate combinations of small molecules, growth factors, and / or cytokines in the early and middle stages of differentiation. Thus, aspects of the present invention allow expanded clonal iPSCs to be directly transferred to adherent culture in a monolayer and immediately differentiated without the need for EB formation from the iPSCs.

[0103] The compositions provided herein are useful, in part, for the production of industrial or clinical-grade pluripotent cells that exhibit reduced spontaneous differentiation compared to cells generated or cultured in the absence of the composition. In one embodiment, non-pluripotent cells are induced to become pluripotent and cultured to maintain pluripotency for an extended period of time. In another embodiment, non-pluripotent cells are induced to become pluripotent and cultured to achieve and / or maintain reduced spontaneous differentiation compared to cells cultured in the absence of the composition. In another embodiment, non-pluripotent cells are induced to become pluripotent and cultured to achieve and / or maintain ground-state pluripotency (see, e.g., the compositions in Tables 1 and 2).

[0104] In various embodiments, the compositions provided herein maintain ground 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 number of passages in between. In other embodiments, the compositions provided herein (see, e.g., Table 2) maintain a decrease in 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, 60, 70, 80, 90, 100 or more passages, including any intervening passage numbers.

[0105] In various embodiments, the culture media provided herein may comprise any defined basal medium suitable for supporting the maintenance and / or proliferation of stem cells, such as conventional human embryonic stem cell medium. Examples of defined basal media that may be used in accordance with embodiments of the present invention include, but are not limited to, Dulbecco's Modified Eagle Medium ("DMEM"), Basal Media Eagle (BME), DMEM / F-12 (1:1 DMEM and F-12 vol vol), Medium 199, F-12 (Ham) Nutrient Mix, F-10 (Ham) Nutrient Mix, Minimum Essential Medium (MEM), StemPro®-34, William's Medium E, and RPMI 1640, all of which are available from, among others, Gibco-BRL / Life Technologies, Inc., Gaithersburg, Md. Many of these media are available in several versions, including, but not limited to, DMEM 11966, DMEM 10314, MEM 11095, Williams' Medium E 12251, Ham's F12 11059, MEM-α 12561, 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.

[0106] Small molecules and classes thereof for use in cell culture media according to embodiments of the present invention are described more fully below. In one embodiment, the composition comprises a cell culture medium and one or more of a TGFβ family protein, a Rho kinase inhibitor (ROCKi), a MEK inhibitor (MEKi), and a WNT activator. In various embodiments, the composition does not comprise a TGFβ inhibitor (TGFβi). In various embodiments, one or more of a TGFβ family protein, a ROCKi, a MEKi, and a WNT activator may be added at one or more specific stages during the generation, maintenance, and / or differentiation of iPSCs over a predetermined period of time. Such specific stages during iPSC generation (reprogramming) include, but are not limited to, somatic cell transfection (day 0), exogenous gene expression, increased heterochromatin, loss of somatic identity, and iPSC colony formation. Specific steps in maintaining iPSCs include, but are not limited to, single cell dissociation of iPSC colonies, single cell sorting of dissociated iPSCs, clonal expansion of iPSC single cells, cryopreservation of master cell banks (MCBs) of clonal iPSCs, thawing of iPSC MCBs, and optionally further cryopreservation-thaw cycles of iPSC MCBs.

[0107] [Table 1]

[0108] [Table 2]

[0109] Suitable nutrients / extracts may include, for example, knockout serum replacement (KOSR), L-glutamic acid (L-glutamic acid), and non-essential amino acids (NEAA). 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 beta (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, various interleukins (e.g., IL-1 through IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFNγ), and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines are commercially available, for example, from R&D Systems (Minneapolis, MN), and may be either natural or recombinant. In some other embodiments, the culture media of the invention comprise one or more of bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-I), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factors (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (Flt3L), and one or more cytokines, for example, one or more of leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, and IL15. In some embodiments, the growth factors / mitogens and cytokines are stage- and / or cell type-specific, at concentrations empirically determined or guided by established cytokine techniques.In some embodiments, cytokines are added at specific stages of differentiation.

[0110] Any suitable vessel or cell culture container can be used as a support for cell culture in basal media and / or cell culture supplements. However, in some embodiments, coating the surface of the culture vessel with an adhesion-promoting matrix / substrate (e.g., collagen, fibronectin, RGD-containing polypeptides, gelatin, etc.) can promote cell attachment and, in some embodiments, enhance the effectiveness of the cell culture media and supplements disclosed herein and / or provide feeder-free differentiation. Suitable substrates for cell culture and passaging are known in the art and include, but are not limited to, mixtures of naturally occurring cell line-produced matrices such as vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombospondin, Matrigel™, and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminated monolayers. In some embodiments, providing feeder-free conditions involves culturing cells on a matrix-coated surface / 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 Retronectin®. In other embodiments, the matrix comprises Matrigel™ or vitronectin.

[0111] ROCK inhibitors Rho-associated kinase (ROCK) is a serine / threonine kinase that acts as a downstream effector of Rho kinase (there are three isoforms: RhoA, RhoB, and RhoC). ROCK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ROCK inhibitors contemplated herein (also referred to as "ROCKi") can reduce the expression and / or activity of ROCK. Exemplary ROCK inhibitors include, but are not limited to, antibodies against ROCK, dominant-negative ROCK variants, and siRNAs and antisense nucleic acids that suppress ROCK expression. Other exemplary ROCK inhibitors include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.

[0112] Exemplary ROCK inhibitors for use in cell culture media according to embodiments of the present invention include thiazovivin, Y27632, pirintegrin, blebbistatin, and functional variants or derivatives thereof. In certain embodiments, the ROCK inhibitor is thiazovivin.

[0113] ERK / MEK inhibitors 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 siRNAs and antisense nucleic acids that suppress 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, GSKl 120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEAl 19, AZD6244, FR180204, PTK787, and functional variants or fragments thereof.

[0114] Further illustrative examples of MEK / ERK inhibitors include the following compounds: 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo-2-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-methyl-3H-benzimidazol-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide ethoxy)-amide, 6-(4-bromo-2-chloro-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, Examples of MEK inhibitors include 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 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.

[0115] In some embodiments, the MEK / ERK inhibitor is PD0325901.

[0116] Wnt activator As used herein, the terms "Wnt signal promoter," "Wnt pathway activator," "Wnt activator," or "Wnt pathway agonist" refer to agonists of the Wnt signaling pathway, including, but not limited to, agonists of one or more of Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, or Wnt16. Wnt pathway agonists further include, but are not limited to, one or more of the following polypeptides or fragments thereof: Dkk polypeptide, Crescent polypeptide, Cerberus polypeptide, Axin polypeptide, Frzb polypeptide, T-cell factor polypeptide, or dominant negative disheveled polypeptide.

[0117] Non-limiting examples of Wnt pathway agonists further include one or more of a nucleic acid comprising a nucleotide sequence encoding a Wnt polypeptide, a nucleotide sequence encoding a Wnt polypeptide, a polypeptide comprising the amino acid sequence of a Wnt polypeptide, a nucleic acid comprising a nucleotide sequence encoding an activated Wnt receptor, a polypeptide comprising the amino acid sequence of an activated Wnt receptor, a small organic molecule that promotes Wnt / β-catenin signaling, a small organic molecule that inhibits the expression or activity of a Wnt antagonist, an antisense oligonucleotide that inhibits the expression of a Wnt antagonist, a ribozyme, an RNAi construct, siRNA, or shRNA that inhibits the expression of a Wnt antagonist, an antibody that binds to and inhibits the activity of a Wnt antagonist, a nucleic acid comprising a nucleotide sequence encoding a β-catenin polypeptide, a polypeptide comprising the amino acid sequence of a β-catenin polypeptide, a nucleic acid comprising a nucleotide sequence encoding a Lef-1 polypeptide, a polypeptide comprising the amino acid sequence of a Lef-1 polypeptide, and functional variants or fragments thereof.

[0118] GSK-3β inhibitors GSK-3β inhibitors (also referred to as "GSK3 inhibitors" or "GSK3i") are particular 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 that target GSK-3β. Other exemplary GSK-3β inhibitors include kenpaullone, 1-azakempaullone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, lithium, SB 415286, TDZD-8, BIO, BIO-acetoxime, (5-methyl 1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopenadienyl ruthenium complex, TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, α-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione, TWSI 19 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-1(4,5-dibromo-thiophen-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 invention include CHIR99021, BIO, and Kenpaullone. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0119] TGFβ receptor / ALK5 inhibitor TGFβ receptor (e.g., ALK5) inhibitors include antibodies to TGFβ receptors (e.g., ALK5), dominant negative variants thereof, and antisense nucleic acids that suppress their expression. Exemplary TGFβ receptor / ALK5 inhibitors (also referred to as "ALK5i") include 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. Furthermore, "ALK5 inhibitors" is not intended to encompass non-specific kinase inhibitors, but should be understood to encompass inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as, for example, SB-431542. Although not intending to limit the scope of the present invention, it is believed that ALK5 inhibitors affect the mesenchymal to epithelial conversion / transition (MET) process. The TGFβ / activin pathway is a driving factor for epithelial to mesenchymal transition (EMT). Therefore, inhibiting the TGFβ / activin pathway can promote the MET (i.e., reprogramming) process.

[0120] Inhibition of the TGFβ / activin pathway has been shown to have a similar effect to inhibiting ALK5. Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or instead of the ALK5 inhibitors described in each paragraph of this specification. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, inhibitors of SMAD 2 / 3 phosphorylation, inhibitors of the interaction between SMAD 2 / 3 and SMAD 4, and activators / agonists of SMAD 6 and SMAD 7. Furthermore, the classifications described below are merely for organizational purposes, and those skilled in the art will know that a compound may affect more than one point in the pathway and therefore may function in more than one of the defined classifications.

[0121] Specific examples of TGFβ receptor inhibitors include SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124), lerdelimumab (CAT-152), meterimumab (CAT-192), GC-1008, ID11, AP-12009, AP-11014, LY550410, LY580276, LY364947, These include, but are not limited to, LY2109761, SB-505124, SB431542, SD-208, SM16, NPC-30345, Ki26894, SB-203580, SD-093, Gleevec, 3,5,7,2',4'-pentahydroxyflavone (morin), activin-M108A, P144, soluble TBR2-Fc, and antisense transfected tumor cells targeting the TGFβ receptor.

[0122] Inhibitors of SMAD2 / 3 phosphorylation include dominant-negative variants of nucleic acids targeting SMAD2 or SMAD3 and antibodies against antisense nucleic acids. Specific examples of inhibitors include PD169316, SB203580, SB-431542, LY364947, A77-01, and 3,5,7,2',4'-pentahydroxyflavone (Morin). Inhibitors of the interaction between SMAD2 / 3 and SMAD4 include dominant-negative variants of antisense nucleic acids targeting SMAD2, SMAD3, and / or SMAD4 and antibodies against antisense nucleic acids. Specific examples of inhibitors of the interaction between SMAD2 / 3 and SMAD4 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 that target SMAD 6 or SMAD 7.

[0123] p38 MAPK inhibitors 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 proinflammatory cytokine tumor necrosis factor-α (TNF-α), among other stressors. Four distinct subgroups within MAPKs have been identified, including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK / SAPK), ERK / Big MAP kinase 1 (BMK1), and the p38 MAPK group of protein kinases. The p38 MAPK family includes p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12), and p38δ (MAPK13). Although the four p38 MAPK family members have distinct tissue expression patterns, p38 MAPKs have been shown to play important roles in cellular responses, proliferation, survival, cell cycle, and migration in cancer, and p38 MAPK inhibitors for use in chemotherapy have attracted attention.

[0124] Various p38 inhibitors are structurally diverse small molecules that share a common mechanism of action involving competitive inhibition of the adenosine-binding pocket (ATP-binding site) of p38. These inhibitors were selected to occupy the less conserved hydrophobic region surrounding the p38 binding site, inducing a conformational rearrangement that blocks or reduces ATP binding to the p38 protein. Exemplary small molecule p38 MAPK inhibitors suitable for differentiation of iPSCs into secondary hematopoietic endothelial (HE) cells and, subsequently, 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 (known as "DBM1285"), VX745, VX-702, RO-4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RV568, CAS 219138-24-6, SB203580, and SB242235. DBM1285 has previously been shown to inhibit TNF-α production. However, the present application discloses the use of p38 MAPK inhibitors to maintain CD82 expression and CD82-expressing cell populations, which subsequently improves the efficiency of obtaining secondary HE cells.

[0125] AhR inhibitors The aryl hydrocarbon receptor (AhR) is a member of the Pern-Arnt-Sim (PAS) superfamily of transcription factors (HIF-1α, HIF-2α, HIF-3α) that is involved in, among other things, circadian rhythms (BMAL1 and BMAL2) and sensing environmental signals such as changes in oxygen tension gradient or redox potential. AhR is expressed in hematopoietic stem and progenitor cells (HSPCs) and plays an important physiological role in hematopoiesis. Inhibition of AhR has been used to expand human umbilical cord blood-derived HSPCs. However, as shown herein, AhR inhibition is useful for regulating the activation and function of PSC-derived effector cells. Exemplary AhR inhibitors suitable for use in the methods and compositions described herein include, but are not limited to, CH-223191 (CAS 301326-227), UM729 (a pyrimidoindole derivative), UM171, and SR1 (StemRegenin 1).

[0126] HDAC inhibitors Exemplary HDAC (histone deacetylase) inhibitors include antibodies that bind to dominant negative variants of siRNA and antisense nucleic acids targeting HDAC. Histone acetylation is involved in histone and DNA methylation regulation. Generally, at a global level, pluripotent cells have more histone acetylation, while differentiated cells have less histone acetylation. HDAC inhibitors promote the activation of silenced pluripotency genes. Exemplary HDAC inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, TSA (trichostatin A), VPA (valproic acid), sodium butyrate (NaB), SAHA (suberoylanilide hydroxamic acid or vorinostat), sodium phenylbutyrate, depsipeptide (FR901228, FK228), trapoxin (TPX), cyclic hydroxamic acid-containing peptide 1 (CHAP I), MS-275, LBH589, and PXDIOI.

[0127] Cytokines and Growth Factors 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 contain 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 processes.

[0128] Various growth factors and their use in culture media are known, including, for example, ECM proteins, laminin 1, fibronectin, collagen type IV isotype, proteases, protease inhibitors, cell surface adhesion proteins, cell signaling proteins, cadherins, chloride intracellular channel 1, transmembrane receptor PTK7, insulin-like growth factors, inhibin beta A, inducers of the TGFβ / activin / nodal signaling pathway, and activin A. Cytokines used in the culture medium can include one or more of the following growth factors, for example, 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 beta (TGF-β), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), transferrin, various interleukins (such as IL-1 through IL-18), various colony-stimulating factors (such as granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (such as IFN-γ), and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo).

[0129] In certain embodiments, the composition and / or culture medium may include a protein of the TGFβ family as the 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 may be commercially available and may be either natural or recombinant. Other cytokines, if used, may be added at concentrations determined empirically or as guided by established cytokine techniques.

[0130] Cellular reprogramming and iPSC maintenance Any reprogramming factor known in the art for stem cell reprogramming can be used in the present reprogramming method. In one embodiment, the reprogramming factor includes, but is 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), as well as any combination thereof, as disclosed in International Publication Nos. 2015 / 134652 and 2017 / 066634, the disclosures of which are incorporated herein by reference. Reprogramming factors can also be in the form of polynucleotides encoding the reprogramming factors and thus can be introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. 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 an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by a plasmid combination. See, for example, International Publication No. 2019 / 075057(A1), the disclosure of which is incorporated herein by reference.

[0131] Polynucleotides encoding these reprogramming factors may be included in polycistronic constructs (i.e., multiple coding sequences controlled by one promoter) or non-polycistronic constructs (multiple coding sequences, some controlled by one promoter and some controlled by different promoters). Promoters may be, for example, CMV, EF1α, PGK, CAG, UBC, and other suitable promoters that are constitutive, inducible, endogenously regulated, or temporally, tissue, or cell type specific. In one embodiment, the exogenous promoter is CAG. In another embodiment, the promoter is EF1α. In some embodiments, polycistronic constructs may provide a single open reading frame (e.g., multiple coding sequences operably linked by a self-cleaving peptide coding sequence such as 2A) or multiple open reading frames (e.g., multiple coding sequences linked by an internal ribosome entry site, or IRES).

[0132] An alternative method of obtaining iPSCs is to use a plasmid system that mediates short-lived, transient and temporary transgene expression (see, e.g., the "STTR system" in U.S. Patent Application Publication No. 20200270581 and the "STTR2 system" described in WO 2022 / 072883, the relevant disclosures of each of which are incorporated herein by reference).

[0133] In some embodiments, reprogramming of non-pluripotent cells is initiated in the presence of a combination of small molecule compounds including 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., WO 2022 / 072883, the relevant disclosures of which are incorporated herein by reference).

[0134] Cells suitable for reprogramming generally include any non-pluripotent cell. Non-pluripotent cells include, but are not limited to, terminally differentiated cells, or multipotent or progenitor cells, which are unable to give rise to all three types of germ layer lineage cells. In some embodiments, the non-pluripotent cells for reprogramming are primary cells, i.e., cells isolated directly from human or animal tissue. In some embodiments, the non-pluripotent cells for reprogramming are source-specific stem cells, e.g., donor-, disease-, or therapeutic response-specific. In some embodiments, the non-pluripotent cells for reprogramming are primary immune cells. In some embodiments, the non-pluripotent cells 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 for reprogramming are induced immune effector cells, e.g., non-natural or artificial T- or NK-like cells derived from iPSCs.

[0135] In some other embodiments, the non-pluripotent cells for reprogramming are genomically modified primary or derived cells. The genetic modifications included in the non-pluripotent cells may include insertions, deletions, or substitutions in the genome that result in knock-in, knock-out, or knock-down of gene expression. The modified expression in the non-pluripotent cells for reprogramming may be constitutive or inducible (e.g., developmental stage-, tissue-, cell-, or inducer-specific). In some embodiments, the insertion or replacement is a locus-specific targeted integration. In some embodiments, the locus selected for integration is a safe harbor locus or an endogenous locus of interest.

[0136] In one embodiment, genetically modified non-pluripotent cells are reprogrammed to obtain genetically engineered iPSCs containing the same genetic modifications. In some other embodiments, one or more such genome edits can be introduced into iPSCs after reprogramming to obtain genetically engineered iPSCs. In some embodiments, the genetically engineered iPSCs contain polynucleotides encoding cytokine signaling complexes containing partial or complete peptides of exogenous cytokines and / or their receptors expressed on the cell surface, thereby eliminating or requiring the inclusion of cytokines in the culture medium during subsequent differentiation into hematopoietic lineages via secondary hemogenic endothelium (HE). See, e.g., International Application No. PCT / US2022 / 073396 and International Publication Nos. 2022 / 098914 and 2022 / 098925, the entire disclosures of each of which are incorporated herein by reference. In one embodiment, the secondary HE cells comprise a genetic 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 the intracellular domain), as described in WO 2019 / 191495 and WO 2019 / 126748, the entire disclosures of each of which are incorporated herein by reference. In various embodiments, iPSCs for genome editing are clonal lines or populations of clonal iPS cells.

[0137] iPSC differentiation (1) From iPSC to iHE In one aspect of the present invention, methods and compositions are provided for obtaining secondary hemogenic endothelium (HE) using pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). As used herein, secondary hemogenic endothelium refers to a population of hematopoietic cells committed to definitive hematopoiesis, capable of giving rise to all hematopoietic cells, including, but not limited to, pre-NK cell precursors, NK cell precursors, NK cells, NKT cells, B cells, and other hematopoietic cells, as well as pre-T cell precursors, T cell precursors, and T cells. In some aspects, the present invention provides compositions and methods for obtaining hematopoietic lineage cells from secondary HE cells or from iPSCs via secondary hemogenic endothelium differentiated from the iPSCs.

[0138] Generally, techniques for differentiating iPSCs or secondary HE cells derived therefrom involve modulating specific cellular pathways, either directly or indirectly, using polynucleotide-, polypeptide-, and / or small molecule-based approaches. Cell developmental potential may be modulated, for example, by contacting the cells with one or more regulatory factors. "Contacting," as used herein, may involve culturing the cells in the presence of one or more factors (e.g., small molecules, proteins, peptides, etc.). In some embodiments, cells are contacted with one or more factors to induce cell differentiation. Such contacting may be performed, for example, by introducing one or more factors into the cells during in vitro culture. Thus, contacting may be performed by introducing one or more factors into the cells in cell culture medium. The cells may be maintained in culture medium containing one or more factors for a sufficient time for the cells to acquire the desired differentiation phenotype. In some other embodiments, "contacting" is performed when one or more factors are introduced into the cells via a vector, as discussed below. In some embodiments, the one or more vectors are introduced by retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system with an expression cassette, or mRNA.

[0139] In various embodiments, the culture platform provided herein for generating hematopoietic cell lineages from iPSCs or secondary hemogenic endothelium derived therefrom does not contain or is essentially free of inhibitors of the TGFβ / activin signaling pathway, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In some embodiments, the cell culture medium for differentiating secondary hemogenic endothelium does not contain stromal cells, such as OP9 stromal cells. In one embodiment, the culture platform includes a seeding medium for maintaining naive iPSCs. Obtaining the pluripotency of the ground state or naive state of iPSCs is also important for obtaining secondary HE cells and hematopoietic lineage cells by differentiating iPSCs without forming EB intermediates. Additionally, the efficiency of differentiation of naive iPSCs into secondary HE cells is also significantly affected by the use of monolayer cultures that do not form EBs and their aggregates. In some embodiments, the seeding medium includes a ROCK inhibitor but does not contain or is essentially free of a TGFβR / ALK5 inhibitor. In some other embodiments, the seeding medium comprises a GSK3 inhibitor but does not comprise a TGFβR / ALK5 inhibitor, hi yet some other embodiments, the seeding medium comprises a GSK3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor.

[0140] One aspect of the present invention provides a culture medium for obtaining secondary hemogenic endothelial (HE) cells differentiated from pluripotent stem cells, including iPSCs. In one embodiment, the culture medium comprises a BMP activator, bFGF, and optionally one or more of 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, VEGF, a Wnt pathway activator, and / or a p38 MAPK inhibitor are added to the medium containing a BMP activator and bFGF after differentiated cells from iPSCs have been specified into mesodermal progenitor cells. In one embodiment, the culture medium does not comprise a TGFβ receptor / ALK inhibitor. In one embodiment, the Wnt pathway activator is a GSK3β inhibitor. Without being bound by theory, small molecule p38 MAPK (mitogen-activated protein kinase) inhibitors contribute to improved maintenance of CD82 expression in HE cells compared to differentiation without the inhibitor, while BMP activators result 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, CAS 219138-24-6, SB203580, and SB242235. In one embodiment, the p38 MAPK inhibitor comprises DBM1285.

[0141] Pluripotent stem cells are optionally seeded and expanded as described above, and then differentiated into mesodermal progenitor cells, which are expanded at this stage. The expanded population of mesodermal progenitor cells is then differentiated into a mesodermal population with secondary hemogenic endothelial potential, which is then differentiated into a population of secondary hemogenic endothelial (HE) cells. Alternatively, a method for producing iPSC-derived secondary hemogenic endothelium (iHE) may begin with mesodermal progenitor cells derived from pluripotent stem cells, which method includes differentiating the mesodermal progenitor cells into secondary hemogenic endothelial (iHE) cells. In some embodiments of the above method, the iHE is suitable for cryopreservation. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

[0142] In some embodiments of the above-described methods for obtaining iPSC-derived hemogenic endothelial cells, the method comprises: (i) differentiating the pluripotent stem cells to obtain mesodermal progenitor cells or a population thereof by contacting the iPSCs with a medium comprising a basal medium in addition to a BMP activator and bFGF; 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 secondary HE cells.

[0143] In some embodiments of the above methods, including (i) and (ii), the method further comprises (iii) sorting the resulting HE cells using an antibody that recognizes a cell surface marker including CD34, CD82, CD43, CD73, CXCR4, and / or CD93, thereby producing an enriched subpopulation of secondary HE cells. In some embodiments, the sorting uses an anti-CD34 antibody, and the enriched subpopulation of iHE cells is CD34 + In some embodiments, an anti-CD82 antibody is used for sorting, and the enriched subpopulation of secondary HE cells is CD82 + In some embodiments, both anti-CD34 and anti-CD82 antibodies are used for sorting, and the enriched subpopulation of secondary HE cells is CD34 + CD82 +In some embodiments, anti-CD34, anti-CD82, and anti-CD43 antibodies are used for sorting, and the enriched subpopulation of secondary HE cells is CD34 + CD82 + CD43 - In some embodiments, the selection is performed using CD34 + , CD82 + , CD43 - , and CD73 - In some other embodiments, the selection is performed using CD34 + , CD82 + , CD43 - , CD73 - , and CXCR4 - In some embodiments, the selection is performed using CD34 + , CD82 + , and CD93 - In some embodiments, the selection is performed using CD34 + , CD82 + , CD43 - , and CD93 - In some embodiments, the selection is performed using CD34 + , CD43 - , and CD73 - In some other embodiments, the selection is performed using CD34 + , CD43 - , CD73 - , and CXCR4 - is used.

[0144] (2) T-lineage cells from iPSCs or iHE Provided herein is a culture platform for generating T cell progenitors or T cells from secondary hemogenic endothelium. In one embodiment, the culture medium comprises SCF, Flt3L, IL7, and optionally a ROCK inhibitor, TPO, and IL3, and the medium does not comprise one or more of VEGF, bFGF, and a BMP activator. In some embodiments, the medium for differentiating secondary hemogenic endothelium into T cell progenitors comprises a ROCK inhibitor, SCF, Flt3L, TPO, and IL7, but does not comprise a BMP activator. In some embodiments, the medium for differentiating T cell progenitors into T cells comprises SCF, Flt3L, and IL7, but does not comprise TPO, IL3, a BMP activator, or a ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, Notch factors are used in culture platforms for generating T cell progenitors or T cells from iPSCs or secondary 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.

[0145] Also provided are methods for generating T cell precursors or T cells derived from pluripotent stem cells using a multi-step process. Generally, the method begins with seeding and optionally expanding pluripotent stem cells, differentiating the pluripotent stem cells into mesodermal progenitor cells and subsequently into HE cells, which may optionally be sorted to obtain enriched secondary HE cells for subsequent differentiation of T lineage cells. Alternatively, the HE may be used as starting cells for differentiation of T lineage cells. The methods and various embodiments for differentiating iPSCs into HE cells, including methods for sorting HE cells, are described above. Provided herein are methods for differentiating HE cells into T lineage cells, either after or independently of differentiation of iPSCs into HE cells.

[0146] In some embodiments of methods for differentiating HE cells into T cell precursors or T cells, the method includes contacting the HE cells with a medium comprising a growth factor and one or more cytokines including SCF, Flt3L, and IL7, and optionally one or more factors including TPO, IL3, and a ROCK inhibitor, wherein the medium does not include or is essentially free of 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-iProt cells includes contacting iHE cells with a medium comprising a ROCK inhibitor, SCF, Flt3L, TPO, and IL7. In other embodiments, differentiating pre-iProt cells into ipro-T or iT cells includes contacting pre-iProt cells with a medium comprising SCF, Flt3L, and IL7, wherein the medium does not include or is essentially free of one or more of VEGF, bFGF, BMP activators, and a ROCK inhibitor. In some embodiments of the above methods, Notch factors are used in the culture medium for generating T cell precursors or T cells. In some embodiments, Notch factors, including Jag1, Jag2, DLL-1, DLL-3, and DLL-4, can be introduced as soluble peptides, peptides conjugated to beads, peptides conjugated to surfaces, peptides contained in an extracellular matrix coated on a cell culture surface, or peptides presented by cells. In some embodiments, the step of differentiating iHE cells into T lineage cells is performed in the absence of OP9 stromal cells (OP9-free hematopoietic cell differentiation). In some embodiments, OP9-free differentiation of iHE cells is in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4. In some embodiments, the recombinant human fibronectin is Retronectin®.

[0147] (3) NK lineage cells from iPSCs or iHE NK lineage cells can be differentiated either from iPSCs via iPSC-derived secondary hemogenic endothelial (HE) cells or directly from secondary HE cells.

[0148] Compositions and methods for differentiating iPSCs into secondary HE cells, including sorting of HE cells, and various embodiments thereof, are described in the previous section. Further provided herein are additional culture platforms and methods for differentiating secondary hemogenic endothelial cells to obtain NK lineage cells, including NK progenitor cells and NK cells.

[0149] In some embodiments of a culture platform for obtaining NK-lineage cells from HE cells, the culture platform includes at least a medium containing SCF, Flt3L, IL7, IL3, and optionally one or more of IL15, IL21, TPO, a ROCK inhibitor, an AhR antagonist, a 4-1BB agonist, and nicotinamide, and the medium does not include one or more of VEGF, bFGF, a BMP activator, OP9 stromal cells, and feeder cells. In some embodiments of a culture medium for generating NK progenitor cells or NK cells, the medium does not include K562 feeder cells or engineered variants thereof. In some embodiments, the ROCK inhibitor includes thiazovivin or Y27632.

[0150] In some embodiments of the medium for generating NK progenitor cells or NK cells, the medium comprises an AhR antagonist. In some embodiments, the AhR antagonist is a small molecule AhR inhibitor. Without being bound by theory, in some embodiments, the AhR inhibitor in the medium is useful for the proliferation and / or activation of NK cell progenitor cells or NK cells. In some embodiments, the AhR inhibitor comprises at least one of CH-223191, UM729, UM171, and SR1. In some embodiments, the medium for generating NK progenitor cells or NK cells comprises CH-223191. In some embodiments, in addition to one or more AhR antagonists, NK proliferation, maturation, and / or activation is further in the presence of nicotinamide (NAM), which is included in the medium for generating NK progenitor cells or NK cells.

[0151] Also provided are methods for generating NK progenitor cells or NK cells from pluripotent stem cells using a multi-step process. Generally, the method begins with seeding and optionally expanding pluripotent stem cells, differentiating the pluripotent stem cells into mesodermal progenitor cells and subsequently differentiating them into HE cells, which may optionally be sorted to obtain enriched secondary HE cells for subsequent differentiation into NK-lineage cells. Alternatively, the HE cells may be used as starting cells for differentiation into NK-lineage cells. Methods and various embodiments for differentiating iPSCs into HE cells, including methods for sorting HE cells, are described above. Provided herein are methods for differentiating HE cells into NK-lineage cells, either after or independently of differentiation of iPSCs into HE cells.

[0152] In some embodiments, a method for differentiating iHE cells into NK-lineage cells includes 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 this method, IL15 is included in the culture medium. In some other embodiments of this method, IL15 is in the form of an IL15 signaling complex encoded by an exogenous polynucleotide introduced into the secondary HE cells by gene insertion, and the IL15 signaling complex includes a partial or complete peptide of exogenous IL15 and / or its receptor expressed on the cell surface. In some embodiments, differentiating iHE cells into NK progenitor cells or NK cells is performed in the absence of OP9 stromal cells. In some embodiments, differentiating iHE cells into NK progenitor cells or NK cells is performed 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, the recombinant human fibronectin is Retronectin®. In some embodiments, a method of differentiating iHE cells into iNK cells comprises contacting iHE cells with a composition comprising SCF, Flt3L, IL3, IL7, 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.

[0153] In some embodiments, the method of differentiating iHE cells into iNK cells further comprises contacting the iPSC-derived NK progenitor or NK cells with one or both of an AhR antagonist and nicotinamide, thereby expanding and / or activating the iPSC-derived NK cells. In some embodiments, NK activation is performed in the presence of a small molecule AhR inhibitor, thereby modulating the maturation of NK lineage cells.

[0154] B. Cell populations and cell lines generated from the methods and compositions provided herein In view of the above, one advantage provided by the methods described herein is improved viability and survival of single pluripotent cell culture, passaging, and dissociation without EB formation for differentiation of pluripotent stem cells. Dissociation of cells into single cells, such as a single-cell suspension, can be achieved by enzymatic or mechanical means. Any enzymatic agent known in the art capable of dissociating cells into single cells can be used in embodiments of the methods herein. In one embodiment, the dissociation 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 enzymatic agents in dissociating cells according to the methods contemplated herein. The dissociation agent is dissolved in calcium- and magnesium-free PBS to facilitate dissociation into single cells. To improve cell survival during and after dissociation, some embodiments add a survival-promoting substance, such as one or more growth factors, inhibitors of cellular pathways involved in cell death and apoptosis, or treated media. In one embodiment, the pro-survival agent is a ROCK inhibitor, including but not limited to thiazovivin.

[0155] In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iPSCs are reprogrammed from immune cells of a particular donor or patient. In some embodiments, the cells cultured after reprogramming are induced to differentiate for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 22, 24, 26, 28, 30, 32, 35, 40, 42, or 45 days, or any number of days therebetween. In some embodiments, the cells cultured after reprogramming are induced for about 1 to 42 days, 2 to 40 days, 2 to 35 days, 2 to 20 days, 2 to 10 days, 4 to 30 days, about 4 to 24 days, about 6 to 22 days, or about 8 to about 12 days.

[0156] In some embodiments, the iPSCs are genetically engineered. In some embodiments, the iPSCs for differentiation contain one or more genetic imprints. In some embodiments, the genetic imprints of the pluripotent stem cells include (i) one or more genetic modification modalities obtained by genomic insertion, deletion, or substitution in the genome of the pluripotent cells during or after reprogramming of non-pluripotent cells into iPSCs, or (ii) one or more retainable therapeutic properties of source-specific immune cells that are specific to the donor, disease, or therapeutic response, where the pluripotent cells are reprogrammed from source-specific immune cells and the iPSCs retain the source therapeutic property, which is also contained in the iPSC-derived hematopoietic lineage cells. In some embodiments, the genetic modification modality includes one or more of a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival of iPSCs or their derivatives. In some other embodiments, the genetic modification modality is (i) deletion, disruption, or reduced expression of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, 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 2AIn some embodiments, the surface triggering receptor is universal, i.e., compatible with any effector cell type, and effector cells expressing the universal surface triggering receptor can bind to the same bispecific or multispecific engager regardless of their cell type. In yet some other embodiments, the hematopoietic lineage cells comprise therapeutic properties of source-specific immune cells related to one or more of: (i) antigen-targeting receptor expression, (ii) HLA presentation or lack thereof, (iii) tolerance to the tumor microenvironment, (iv) induction of bystander immune cells and immunomodulation, (v) improved on-target specificity with reduced off-tumor effects, (vi) resistance to treatments such as chemotherapy, and (vii) improved homing, persistence, and cytotoxicity.

[0157] In some embodiments, the engager is cell type specific, i.e., the engager binds to and / or activates a particular immune cell type, hi certain embodiments, the engager is cell type independent, i.e., the engager binds to and / or activates multiple immune cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils.

[0158] In some embodiments, the iPSCs comprise one or more targeted edits at one or more desired sites, and the one or more targeted edits remain intact and functional at each selected edit site in the expanded iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions (i.e., targeted integrations and / or in / dels at selected sites) into the genome of the iPSCs and their derived cells. In some embodiments, the iPSCs and their derived hematopoietic cells are B2M-negative, HLA-E / G, PDL1, A 2AThese cells may be R, CD47, LAG3-negative, TIM3-negative, TAP1-negative, TAP2-negative, tapasin-negative, NLRC5-negative, PD1-negative, RFKANK-negative, CITTA-negative, RFX5-negative, and / or RFXAP-negative. These cells with modified HLA class I and / or II have increased resistance to immune detection and therefore improved in vivo persistence. Furthermore, such cells can circumvent the need for HLA matching in adoptive cell therapy, thus providing a source of universal, off-the-shelf therapeutic regimens.

[0159] In some embodiments, iPSCs and their derived hematopoietic cells comprise one or more of hnCD16, 4-1BBL, CD3, CD4, CD8, CAR, TCR, CD137, or CD80. Such cells have improved immune effector capabilities.

[0160] In some embodiments, iPSCs and their derived hematopoietic cells comprise surface triggering receptors for binding to bi- or multispecific engagers. Such cells have improved tumor-targeting specificity. In some embodiments, iPSCs and their derived hematopoietic cells are antigen-specific. In some embodiments, iPSCs and their derived hematopoietic cells comprise polynucleotides encoding cytokine signaling complexes comprising exogenous cytokines and / or partial or complete peptides of their receptors expressed on the cell surface, thereby not requiring or requiring the inclusion of cytokines in the culture medium.

[0161] As described above, the method includes strategies for enriching cell populations with specific characteristics at various stages of the method. In one embodiment, a method for enriching pluripotent stem cells from a cell population includes dissociating the cells in the population and resuspending the cells to create a single cell suspension. The dissociated cells can be resuspended in any suitable solution or medium for maintaining the cells or for cell sorting. In various embodiments, enrichment provides a method for inducing clonal iPSC colonies in a relatively short period of time, thereby improving the efficiency of iPSC generation. Enrichment can include sorting the population of cells by identifying and obtaining cells that express markers of pluripotency, thereby obtaining an enriched population of pluripotent cells. Further enrichment methodologies include depletion of cells expressing markers of differentiation, unreprogrammed cells, 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 for selection have been induced to reprogram for at least 1, 2, 3, 4, 5, 6, 7, 8 days or more, but not more than 25, 26, 28, 30, 32, 35, 40 days, or any number of days in between.

[0162] Cells can be sorted by any suitable method for sorting cells, such as by magnetic beads or flow cytometry. iPSCs can be sorted based on expression of one or more markers of pluripotency, including, but not limited to, expression of 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. In various embodiments, iPSCs are sorted based on at least two, at least three, or at least four markers of pluripotency. In certain embodiments, iPSCs are sorted based on expression of SSEA4, and in some embodiments, based on expression of SSEA4 in combination with TRA1-81 and / or TRA1-60. In certain embodiments, iPSCs are sorted based on SSEA4, TRA1-81, or TRA1-60, and / or CD30 expression. In one embodiment, iPSCs are sorted based on SSEA4, TRA1-81, and CD30. In another embodiment, iPSCs are sorted based on SSEA4, TRA1-60, and CD30. In some embodiments, cells are first depleted of non-reprogrammed cells using one or more surface markers of differentiated cells, including, but not limited to, CD13, CD26, CD34, CD45, CD31, CD46, and CD7, and then enriched for pluripotency markers such as SSEA4, TRA1-81, and / or CD30.

[0163] In one embodiment, enrichment provides a method for obtaining clonal pluripotent stem cell-derived differentiated cell colonies in a relatively short time, thereby improving the efficiency of generating differentiated cells from pluripotent stem cells at various stages. In one embodiment, enrichment provides a method for deriving a population of CD34-expressing HE cells, a population of CD34-expressing HSC cells, a population of iHE cells, a population of T cell progenitor or NK cell progenitor cells, and / or a population of T cells or NK cells, thereby improving the efficiency of generating each of the cell populations. Enrichment may also include sorting the cell population to identify and obtain cells expressing specific characteristic markers indicative of the differentiation stage / cell type. As discussed above, enriched iHE cell subpopulations can be generated using one or more antibodies that recognize one or more cell surface markers, including CD34, CD82, CD43, CD73, CXCR4, and / or CD93. In some embodiments, the antibody for cell sorting includes an anti-CD34 antibody, and the enriched subpopulation of iHE cells is characterized by CD34. + In some embodiments, the antibody for cell sorting comprises an anti-CD82 antibody, and the enriched subpopulation of iHE cells is CD82 + In some embodiments, the antibodies for selection include both anti-CD34 and anti-CD82 antibodies, and the enriched subpopulation of iHE cells is CD34 + CD82 + In some embodiments, the antibodies for cell sorting include anti-CD34, anti-CD82, and anti-CD43 antibodies, and the enriched subpopulation of iHE cells is CD34 + CD82 + CD43 - Further enrichment methodologies include depleting cells expressing markers indicative of undesired cell types to obtain a population enriched for the desired cell type.

[0164] Accordingly, another aspect of the present invention is a composition comprising: (i) CD34 derived from pluripotent stem cells; + HE cells (iCD34) have the ability to differentiate into multipotent progenitor cells and + CD43 -(ii) a secondary hematopoietic endothelium (iHE) derived from a pluripotent stem cell, wherein the iHE cell line or cloned cells are CD82 + , or CD82 + and CD34 + , CD93 - , CXCR4 - , CD73 - , and CXCR4 - CD73 - (iii) iHE, which is a pluripotent stem cell-derived multipotent progenitor cell (iMPP), which is a pluripotent stem cell-derived multipotent progenitor cell that expresses CD34 + CD45 + (iv) iMPP cells derived from pluripotent stem cells, which are T cell progenitor cells (ipro-T), and which are CD34 + CD45 + CD7 + (v) T cell precursors derived from pluripotent stem cells (iT), which are CD45 + CD4 + CD3 + or CD45 + CD8 + CD3 + (vi) NK progenitor cells (iproNK) derived from pluripotent stem cells, which are CD45 + CD56 + CD7 + CD3 - and (vii) NK progenitor cells derived from pluripotent stem cells (iNK), which are CD45 + CD56 + NKp46 + In some embodiments, the compositions, cell populations, cell lines, or clonal cells are suitable for cryopreservation. In some embodiments, the compositions, cell populations, cell lines, or clonal cells are suitable for storage under ambient conditions for more than 12 hours, 24 hours, 36 hours, or 48 hours, but not more than 3 days, 4 days, 5 days, 6 days, or 1 week.

[0165] C. Therapeutic Uses of iPSC-Derived Immune Cells In one aspect, the invention also provides a composition comprising an isolated population or subpopulation of immune cells derived from iPSCs using the disclosed methods and compositions, wherein the immune cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of immune cells comprises iPSC-derived T cells. In one embodiment, the isolated population or subpopulation of immune cells comprises iPSC-derived NK cells. In one embodiment, the isolated population or subpopulation of immune cells derived from iPSCs has an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of immune cells derived from iPSCs has an increased number or proportion of type I NKT cells. In another embodiment, the isolated population or subpopulation of immune cells derived from iPSCs has an increased number or proportion of adaptive NK cells. In some embodiments, the isolated population or subpopulation of iPSC-derived HSC cells, T cells, or NK cells is allogeneic. In some other embodiments, the isolated population or subpopulation of HSC cells, T cells, or NK cells derived from iPSCs is autologous.

[0166] Introducing immune cells according to embodiments of the present invention into a subject suitable for adoptive cell therapy can ameliorate a variety of diseases, including alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (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, pemphigoid / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease); acute and chronic leukemia, and lymphoma. hematological malignancies, including but not limited to, lymphoma, multiple myeloma, and myelodysplastic syndromes; solid tumors, including but not limited to, tumors of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testicle, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus; and various autoimmune diseases, including but not limited to, infectious diseases, including but not limited to, HIV- (human immunodeficiency virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), adenovirus, and BK polyomavirus-associated disorders.

[0167] Certain embodiments of the present invention relate to methods of treating a subject in need thereof by administering to the subject a composition comprising any of the cells described herein. In some embodiments, a 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 disease or injury. Treatment of ongoing disease, where treatment stabilizes or alleviates undesirable clinical symptoms in the patient, is particularly important.

[0168] In some embodiments, the subject has a disease, condition, and / or injury that can be treated, ameliorated, and / or improved by cell therapy. Some embodiments contemplate that the subject in need of cell therapy is a subject having an injury, disease, or condition whereby cell therapy, e.g., therapy in which cellular material is administered to the subject, can treat, ameliorate, improve, and / or reduce the severity of at least one symptom associated with the injury, disease, or condition. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects who have undergone chemotherapy or radiation therapy, subjects having or at risk of having a hyperproliferative disorder or cancer, e.g., a hyperproliferative disorder or cancer of the hematopoietic system, subjects having or at risk of developing a tumor, e.g., a solid tumor, and subjects having or at risk of having a viral infection or a disease associated with a viral infection.

[0169] Accordingly, aspects of the present invention further provide pharmaceutical compositions comprising hematopoietic lineage cells derived from pluripotent cells produced by the methods and compositions disclosed herein, the pharmaceutical composition further comprising a pharmaceutically acceptable medium. In one embodiment, the pharmaceutical composition comprises T cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the pharmaceutical composition comprises NK cells derived from pluripotent cells produced by the methods and compositions disclosed herein.

[0170] Additionally, aspects of the present invention provide for the therapeutic use of the above pharmaceutical compositions by introducing / administering the composition to a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0171] As one skilled in the art will appreciate, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions provided herein can be used for cell therapy. Autologous cell therapy may have fewer infections, a lower probability of GvHD, and rapid immune reconstitution. Allogeneic cell therapy may have an immune-mediated graft-versus-malignancy (GVM) effect and a low relapse rate. In some embodiments, in the case of autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA-matched to the patient. In other embodiments, the derived hematopoietic lineage cells are not HLA-matched to the subject, and the derived hematopoietic lineage cells are NK cells or T cells with HLA I and / or HLA II deficiency. Based on the specific condition of the patient or subject in need of cell therapy, one skilled in the art can determine which specific type of therapy to administer.

[0172] In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is at least 0.1 x 10 per dose. 5 cells, at least 1 x 10 5 Cells, at least 5 x 10 5 cells, at least 1 x 10 6 Cells, at least 5 x 10 6 cells, at least 1 x 10 7 Cells, at least 5 x 10 7 cells, at least 1 x 10 8 Cells, at least 5 x 10 8 cells, at least 1 x 10 9 cells, or at least 5 x 10 9 In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1 x 10 per dose. 5 cells ~ approx. 1 x 10 6 Cells, per dose, approximately 0.5 x 10 6 cells ~ approx. 1 x 10 7 Cells, per dose, approximately 0.5 x 10 7 cells ~ approx. 1 x 10 8 Cells, per dose, approximately 0.5 x 10 8 cells ~ approx. 1 x 10 9 Cells, per dose, approximately 1 x 10 9 Cells ~ approx. 5 x 10 9Cells, per dose, approximately 0.5 x 10 9 cells ~ approx. 8 x 10 9 Cells, per dose, approximately 3 x 10 9 cells ~ approx. 3 x 10 10 cells, or any range in between. Roughly 1 x 10 for a 60 kg patient. 8 Cells / dose is approximately 1.67 x 10 6 Converted to cells / kg.

[0173] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a fraction or single umbilical cord blood, or at least 0.1 x 10 5 At least 0.5 × 10 cells / kg body weight 5 At least 1 x 10 cells / kg body weight 5 At least 5 x 10 cells / kg body weight 5 At least 10 x 10 cells / kg body weight 5 At least 0.75 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 At least 1.5 x 10 cells / kg body weight 6 At least 1.75 x 10 cells / kg body weight 6 At least 2 x 10 cells / kg body weight 6 At least 2.5 x 10 cells / kg body weight 6 At least 3 x 10 cells / kg body weight 6 At least 4 x 10 cells / kg body weight 6 At least 5 x 10 cells / kg body weight 6 At least 10 x 10 cells / kg body weight 6 At least 15 x 10 cells / kg body weight 6 At least 20 x 10 cells / kg body weight 6 At least 25 x 10 cells / kg body weight 6 At least 30 x 10 cells / kg body weight 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight, or 1 x 10 9 cells / kg body weight.

[0174] In one embodiment, a dose of derived hematopoietic lineage cells is delivered to the subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2×10 6 cells / kg, at least 3 x 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 x 10 6 cells / kg or more cells / kg, including all intervening cell doses.

[0175] In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a single-dose treatment. In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a multiple-dose treatment. In some embodiments, the multiple-dose treatment is one dose every 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, every 50 days, or any number of doses in between. In some embodiments, the multiple-dose treatment includes three, four, or five weekly doses. In some embodiments of the multiple-dose treatment including three, four, or five, the weekly doses further include an observation period to determine whether additional single or multiple doses are required.

[0176] Some variation in dosage, frequency, and protocol will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose, frequency, and protocol for the individual subject.

[0177] Compositions comprising a population of derived hematopoietic lineage cells according to embodiments of the present invention may be sterile, suitable for administration to a human patient / subject, and ready to administer (i.e., administered without further treatment). A cell-based composition that is ready to administer means that the composition does not require further processing or manipulation before transplantation or administration to a subject. In other embodiments, the present invention provides isolated populations of derived hematopoietic lineage cells that are expanded and / or conditioned prior to administration of one or more factors, including small chemical molecules. Compositions and methods for modulating immune cells, including iPSC-derived effector cells, are described in detail, for example, in International Publication No. WO 2017 / 127755, the relevant disclosure of which is incorporated herein by reference. For derived hematopoietic lineage cells engineered to express a recombinant TCR or CAR, the cells can be activated and expanded using methods described, for example, in U.S. Patent No. 6,352,694. In certain embodiments, primary and costimulatory signals for the derived hematopoietic lineage cells can be provided by different protocols. For example, the factors providing each signal can be in solution or bound to a surface. When surface-bound, the factors may be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one factor may be bound to a surface and the other factor may be in solution. In one embodiment, the factor providing the costimulatory signal may be bound to the cell surface, and the factor providing the primary activation signal may be in solution or bound to a surface. In certain embodiments, both factors may be in solution. In another embodiment, the factors may be in soluble form and then crosslinked to a surface, such as a cell expressing Fc receptors or antibodies or other binding factors that bind to the factors, such as those disclosed in U.S. Patent Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen-presenting cells (aAPCs) contemplated for use in T lymphocyte activation and expansion, in accordance with the present disclosure.

[0178] Sterile, therapeutically acceptable compositions suitable for administration to a patient can include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., a pharmaceutically acceptable medium, e.g., cell culture medium), or other pharmaceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents are determined, in part, by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition. Accordingly, there is a wide variety of suitable formulations of the therapeutic compositions of the present disclosure (see, e.g., Remington, Pharmaceutical Sciences, 17 th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0179] These pharmaceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. As such, the buffer can be as much as about 5% on a weight-to-weight basis of the total composition. Electrolytes, such as, but not limited to, sodium chloride and potassium chloride, can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH within one of these pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0180] In some embodiments, the present invention also provides, in part, the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures disclosed herein. Such compositions are suitable for administration to human subjects. Generally speaking, any 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 certain embodiments, a pharmaceutically acceptable cell culture medium is serum-free and / or feeder-free. In various embodiments, serum-free media are animal-component-free and optionally protein-free. Optionally, media may contain biopharmaceutical-acceptable recombinant proteins. "Animal component-free media" refers to media in which components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal component-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, protein-free media are defined as being substantially free of protein. Those skilled in the art will appreciate that the above media examples are illustrative and in no way limit the formulation of media suitable for use in the present invention, and that there are many suitable media known and available to those skilled in the art. [Example]

[0181] The following examples are offered by way of illustration and not by way of limitation.

[0182] Example 1 - Materials and Methods Maintenance of hiPSCs in small-cell culture: hiPSCs were routinely passaged as single cells once the cultures reached 75%–90% confluence. For single-cell dissociation, hiPSCs were washed once with PBS (Mediatech) and treated with Accutase (Millipore) for 3–5 minutes at 37°C, followed by pipetting to ensure single-cell dissociation. The single-cell suspension was then mixed with an equal volume of normal medium, centrifuged at 225 × g for 4 minutes, resuspended in FMM, and plated on a Matrigel-coated surface. Subcultures were typically 1:6–1:8, transferred to Matrigel-coated tissue culture plates for 2–4 hours at 37°C, and fed with FMM every 2–3 days. Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2.

[0183] Manipulation of human iPSCs with ZFNs and CRISPR for targeted editing of the desired modality: For ZFN-mediated genome editing, 2 million iPSCs were transfected with a mixture of 2.5 μg of ZFN-L (FTV893) and 2.5 μg of ZFN-R (FTV894) and 5 μg of donor construct for targeted insertion of AAVS1. For CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 μg of ROSA26-gRNA / Cas9 (FTV922) and 5 μg of donor construct for targeted insertion of ROSA26. Transfection was performed using the Neon transfection system (Life Technologies) with the following parameters: 1500 V, 10 ms, 3 pulses. On days 2 or 3 post-transfection, transfection efficiency was measured using flow cytometry to assess whether the plasmid contained an artificial promoter-driver GFP and / or RFP expression cassette. On day 4 post-transfection, targeted cells were selected by adding puromycin to the medium at a concentration of 0.1 μg / mL for the first 7 days and 0.2 μg / mL from day 7 onwards. During puromycin selection, cells were passaged onto fresh Matrigel-coated wells on day 10. After day 16 of puromycin selection, surviving cells were identified as GFP- and / or RFP-positive. + The percentage of iPS cells was analyzed by flow cytometry.

[0184] Bulk and clonal sorting of iPSCs: iPSCs with or without genome-targeted editing were selected to express GFP after 20 days of puromycin selection. + SSEA4 + TRA181 +iPSCs were bulk-sorted and clonal-sorted. A pool of targeted iPSCs dissociated into single cells was resuspended in staining buffer containing Hank's balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1x 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 per million cells in 100 μL of staining buffer. The solution was washed once in staining buffer, sedimented, and resuspended in staining buffer containing 10 μM thiazovivin for flow cytometric sorting. Flow cytometric sorting was performed on a FACS Aria II (BD Biosciences). For bulk sorting, GFP was used. + SSEA4 + TRA181 +Cells were gated and sorted into FMM. For clonal sorting, sorted cells were directly discharged into a 96-well plate, each well of which was coated with 5x Matrigel and pre-filled with 200 μL of FMM supplemented with 5 μg / mL fibronectin and 1x penicillin / streptomycin (Mediatech). After sorting was completed, the 96-well plate was centrifuged at 225 g for 1-2 minutes before incubation. The plate was left undisturbed for 7 days. On day 7, 150 μL of medium was removed from each well and replaced with 100 μL of FMM. The wells were refed with an additional 100 μL of FMM on day 10 after sorting. Colony formation was detected as early as day 2, and most colonies expanded between days 7 and 10 after sorting. For the first passage, wells were washed with PBS and dissociated with 30 μL of Accutase at 37°C for approximately 10 minutes. After the cells appeared dissociated, dissociated colonies were transferred to separate wells of a 96-well plate pre-coated with 5x Matrigel, centrifuged at 225g for 2 minutes, and then incubated. Subsequent passages were routinely performed by treating with Accutase for 3-5 minutes and expanding 1:4-1:8 at 75-90% confluency into larger wells pre-coated with 1x Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence and TRA1-81 expression levels. GFP levels approaching 100% were significantly higher than those observed in the control group. + and TRA1-81 + A clonal line of 100 clones was selected for further PCR screening and analysis and cryopreserved as a master cell bank.

[0185] Hematopoietic Differentiation: To initiate differentiation into hematopoietic lineages, hiPSCs were seeded as monolayers on day (D) 0 in maintenance medium and allowed to adhere and proliferate for approximately 24 hours. The monolayers were maintained until approximately D5-D6, at which point they were dissociated into single cells and seeded as low-density monolayers until differentiation at approximately D10. The dissociated single cell populations at D10 were sorted by FACS for further analysis and marker profiling for characterization of secondary HE. Alternatively, or in addition, as disclosed herein, one or more of anti-CD82, anti-CD34, and anti-CD43 antibodies were used for cell sorting of the D10 cell population to identify CD82. + , CD34 + CD82 + or CD34 + CD43 - CD82 + Secondary HE cells containing the phenotype were obtained and were optionally cryopreserved in cryopreservation medium or continued to differentiate into effector cells.

[0186] Differentiation of HE cells into T and NK lineages: Sorted D10 HE cells were further differentiated into iT and iNK lymphoid lineages. After sorting, specifically for iT cells, HE cells were transferred to low-adherence tissue culture plates for serum-free differentiation. After approximately 10 days of culture (after HE isolation), cell cultures were assessed for the development of T cell precursors by co-expression of the cell surface markers CD34 and CD7. After approximately 15-20 days of further differentiation, these CD34 + CD7 + T cell precursors gave rise to distinct populations of mature iT cells, as evidenced by CD4 and CD8 expression.

[0187] After sorting, specifically for iNK cells, HE cells were cultured in serum-free differentiation medium for approximately 10-15 days. Cell cultures were assessed for the development of NK cell precursors. After an additional 10-15 days of culture and expansion, and optional conditioning as further disclosed herein, the presence of activated / mature NK cells was identified using markers including CD56, CD122, NKp30, CD94, CD16, NKG2D, and KIR.

[0188] Example 2 - Screening for surface marks on secondary hemogenic endothelial cells Cell markers serve as monograms to aid in the identification and classification of cells. Most markers are molecules or antigens within the plasma membrane of the cell. Many surface markers are classified by their clusters of differentiation (CDs), which are recognized by specific antibodies. Generally, specific combinations of markers are unique to different cell types.

[0189] During in vitro differentiation of pluripotent stem cells (ECs) through endothelial mesodermal origin, heterogeneous endothelial cells (ECs) acquire arterial, venous, or hematopoietic fates, forming phenotypically and functionally specialized subtypes of endothelial cells. These cell subtypes form in close spatial and temporal proximity and are currently distinguished primarily by gene expression profiling. Hematopoietic cells arise from a unique population of endothelial cells known as hemogenic endothelium (HE) through endothelial-hematopoietic transition (EHT). EHT is a continuous process in which cells with endothelial characteristics gradually acquire hematopoietic morphology and phenotype. Endothelial progenitor cells that undergo such hematopoietic transition are called secondary hemogenic endothelium. Currently, secondary HE cells express CD34 but not CD43, CD73, CD93, and CXCR4. + It has been identified as a CD34 cell. + CD43 in cells - CD73 - CD93 - CXCR4 - Compared to this negative screening for cell subpopulations, identifying one or more positive markers specific for secondary HE cell subtypes greatly improves the efficiency and accuracy of isolating initial cell populations with the desired quality and purity for subsequent hematopoietic cell differentiation.

[0190] To effectively screen for novel positive markers of HE, we used Applicant's proprietary hiPSC platform, which allows single cell passaging and high-throughput flow cytometry sorting, allowing for the derivation of clonal hiPSCs. To identify additional markers for enrichment of secondary HE cells with the potential to give rise to hematopoietic cells, hiPSCs were plated as monolayers and differentiated into hematopoietic cells using the methods and compositions disclosed herein.

[0191] Flow cytometry analysis of D10 cells differentiated under control conditions was compared with cells differentiated under cytokine conditions containing BMP4. + HE specification was found to be cytokine-induced, as shown in Figure 1. RUNX1 is a transcription factor expressed in cells and is thought to be primarily related to HE identity. 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™. The screen subsequently showed increased expression of CD34 compared to control cells. + RUNX1 + We identified several surface marker candidates that were expressed at higher percentages within this subset (Figure 2). As shown in Figure 2, CD82, CD61, CD44, CD143, and CD226, among other candidate markers, were significantly higher than CD34. - CD34 rather than the population + It is more highly expressed in the CD34 population + This higher expression in the population also correlates with cytokine-induced RUNX1 + Of the above candidate markers, CD143 and CD82 are the most promising based on their differential expression and close correlation with RUNX1 expression.

[0192] Example 3 - Verification of surface marks on secondary hemogenic endothelial cells Cytokine-induced D10 cells were purified using CD34 magnetic-activated cell sorting. Figure 3A shows scRNA-seq analysis of curated signature gene expression using Uniform Manifold Approximation and Projection (UMAP) visualization, which was then used to identify cell clusters expressing each signature gene (CD34, RUNX1, SPN, or ITGA2B). As shown in Figure 3A, each dot represents one cell, and color intensity indicates the expression level of the signature gene.

[0193] Next, we used the UMAP visualization of the curated signature genes in Figure 3A to define each cell cluster in Figure 3B. The cell clusters in Figure 3B include: (1) non-hematopoietic CD34 + RUNX1 cells express the endothelial marker CD34 but do not express RUNX1 or the hematopoietic markers SPN (CD43) or ITGA2B (CD41) - EC, (2) RUNX1, which expresses CD34 and RUNX1 but not SPN or ITGA2B, which are desirable HE cells. + ECs, (3) all cells that are (differentiated) hematopoietic cells and express SPN or ITGA2B.

[0194] CD34 on D10 + After identifying the three cell clusters, we used a violin plot to show the CD82 expression in each of the three subsets of the D10 cell population (Figure 3C). As shown in Figure 3C, the width of the violin plot represents the number of cells at each CD82 expression level, and the mean and median CD82 expression levels are labeled for each cell subset. From the violin plot, it was clear that hematopoietic cells express both RUNX1 and CD82, but RUNX1 alone did not. - RUNX1 compared with non-hematopoietic populations + It was shown that CD82 gene expression was significantly higher in the EC (HE) population, which supports the hypothesis that CD82 is a marker that can distinguish HE from non-HE.

[0195] Subsequently, flow cytometry analysis of cytokine-induced D10 cells was performed to compare the expression of other selected HE candidate markers with RUNX1 and CD82. RUNX1 is a transcription factor and is not expressed on the cell surface, making it unsuitable as a marker for enriching intact HE cells by flow sorting. As shown in Figure 4A, the first column of the flow plot shows that the candidate marker is associated with the endothelial population (CD34 + ), all tested markers except CD44 are primarily restricted to the endothelial population. In column 2 (Figure 4A), CD34 is shown as a candidate HE marker. + CD43 - The third and fourth columns (Figure 4B) show CD34 relative to CD43 to identify the population. + CD43 - The candidate HE markers gated by population are shown. As shown, all candidate markers were RUNX1 + CD61 and CD226 are expressed in the entire RUNX1 + We observed that CD82 and CD143 are not expressed by all endothelial populations, suggesting that they mark only a subset of HE at D10 of differentiation. Both CD82 and CD143 are restricted to the endothelial population, and RUNX1 + Because they are expressed within the population, the data support each of them as valid HE candidate surface markers.

[0196] The addition of a p38 MAPK (mitogen-activated protein kinase) inhibitor to the composition for obtaining secondary HE cells from iPSCs is beneficial for maintaining the expression of CD82 and CD34. + CD82 in HE cells + RUNX1 + It was further observed that this resulted in the maintenance of a subpopulation of CD82. Therefore, to increase the efficiency of obtaining secondary HE cells from differentiation of iPSCs, it is contemplated that the composition may optionally further include a p38 MAPK inhibitor, such as DBM1285 (an exemplary small molecule inhibitor that was tested), in addition to the BMP4 cytokine, and the secondary HE cells may be further enriched in CD82.+ , and optionally CD34 + , CD43 - , CD93 - , CXCR4 - , and CD73 - have a phenotype that includes one or more of the following:

[0197] Example 4 - Application of surface marks on secondary hemogenic endothelial cells Current methods for identifying secondary HE involve the identification of endothelial populations (CD34) that express more mature endothelial markers, including CD73, CD93, and CXCR4. + CD43 - ) exclusion, and as a result, selected HEs are generally CD34 + CD43 - CD73 - CD93 - CXCR4 - However, this phenotype is also shared by endothelial progenitor cells, which precede HE in cell development. These cells typically differentiate slowly and therefore have a higher percentage of CD34 + CD43 - CD73 - CD93 - CXCR4 - The population with is more focused on the development of immature endothelial progenitor cells rather than secondary HE, making negative marker screening an unreliable method for detecting secondary HE cells that have the potential to develop T lineage cells in addition to NK, NKT, and B cell lineages.

[0198] For the CD82 and CD143 markers from the above screen, CD82 was used as a proof of concept to demonstrate their validity as secondary HE markers. - CD93 - CXCR4 - CD82 within the endothelial population + To demonstrate cell enrichment, flow cytometry analysis of cytokine-induced D10 cells was performed, and cells were pre-gated for single / live events. As shown in Figure 5, CD82 expression was significantly higher than CD34 expression on cells.+ CD43 - CD73 - CD93 - CXCR4 - population, and therefore the data support CD82 as a reliable alternative to triple-negative markers for identifying secondary HE.

[0199] In a separate experiment, the prevalence of secondary HE within cytokine-induced D10 populations was calculated using extreme limiting dilution analysis (ELDA) software. Cytokine-induced D10 populations were subjected to fluorescence-activated cell sorting (FACs) based on the markers indicated along the x-axis (mean ± SD), and the mean prevalence of secondary HE is shown above each column in Figure 6. Importantly, CD34 + CD43 - CD82 + 1 out of 38 cells in the population and CD82 + One in 44 cells in the population was secondary HE, further supporting the use of CD82 as a reliable positive marker for secondary HE identification. Meanwhile, the data also demonstrate that the single marker CD82 was not sufficient for the selection of D10 cells for secondary HE. + Using CD43 - CD82 + Using (CD43 - elimination of differentiated hematopoietic cells by CD34 + CD43 - CD82 + Using CD34 + This indicates that improved and satisfactory secondary HE rates can be obtained for each cell population.

[0200] The definitive hematopoietic potential expressed by the cell populations obtained using various marker combinations was determined by differentiation of iTs. D10 CD34 markers isolated by FACS were used. + CD43 - CD82 + and CD34 + CD43 - CD82- Cell fractions were plated into the cultures disclosed herein (iTC-A2 and iTC-B2). At approximately day 35 of iT differentiation, cultures were assessed for the presence of iT cells by expression of the pan-hematopoietic marker CD45 along with lymphoid markers CD5 and CD7. Because the iT cells in this example had a CD19-CAR knocked into the TRAC locus, preventing assembly of T cell receptors on the cell surface, the detectable intracellular T cell coreceptor CD3 (icCD3) was used as a marker for differentiated T cells. As shown in Figure 7, populations were FAC-sorted by the markers indicated above the flow plots, and differentiated iT cells from each of the D100 populations shown were primarily CD8 + Generated cytotoxic T cells but CD4 + No helper T cells were generated. Furthermore, without being bound by theory, CD8aa (CD8a + CD8b - ) compared with CD8ab (CD8a + CD8b + ) suggests that the cells are more adaptive. + CD43 - Cellular CD82 + Successful differentiation of fraction-derived T cells indicates the secondary nature of the CD82-specified HE cells.

[0201] For iNK differentiation, D30 cultures were assessed for the presence of iNK cells by expression of the pan-hematopoietic marker CD45 and the NK marker CD56. Populations were FAC sorted by the markers shown above the flow plot in Figure 8. Myeloid cells (CD11b / CD14 + Although a relatively small population of NK cells was detected, the strong expression of the lymphoid marker CD7 and the expression of the NK cell receptor NKG2A shown in Figure 8 indicates successful NK cell differentiation.

[0202] Taken together, the data demonstrate that CD82 is a reliable cell surface marker for secondary HE detection during iPSC differentiation. + or CD34+ CD82 + The cells are a secondary HE population during the early stages of directed differentiation of iPSCs into hematopoietic cells, and expression of CD82 can be used as a surrogate positive marker to replace negative markers including CD73, CD93, CXCR4, or any combination thereof in identifying HE, or more specifically, secondary HE subpopulations.

[0203] Example 5 - OP9 stromal cell-free differentiation of HE into mature functional lymphoid cells In an attempt to differentiate iPSCs in a chemically defined serum-free medium without stromal cells of mouse or human origin, sorted secondary HE cells (iCD34, CD82 + , CD34 + CD82 + , or CD34 + CD43 - CD82 + ) were seeded in culture without passaging for approximately 18-20 days, or up to 27-30 days, to prepare differentiated CD56-expressing NK cells for collection, storage, or further maturation and expansion. Notably, the differentiation process described herein does not include any feeder cells, specifically, DLL4-expressing OP9 stromal cells (OP9-DLL4) or their irradiated counterparts (irOP9-DLL4), rendering the differentiated cell product free of exogenous and unidentified cellular components, which is desirable for regulatory compliance in the manufacturing of cell therapies.

[0204] A composition that replaces OP9-DLL4 for the differentiation of hematopoietic cells, including NK cells, includes human DLL4 Fc chimeric recombinant protein (Fc-rhDLL4) and RetroNectin® (Takara Bio USA, San Jose, California). RetroNectin in this Retro / DLL4 extracellular matrix is ​​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 medium containing the Retro / DLL4 extracellular matrix further 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 can be present in the medium for the first 2 to 4 days, TPO and VEGF can be present in the medium for the first 4 to 8 days, and IL3 can be present in the medium for the first 7 to 11 days.

[0205] iNK proliferation fold and CD45 + CD56 + , CD7 + and CD1 1b + / CD14 + / CD15 + The iNK progenitor cell specification / profile, as indicated by cell abundance, was compared between stromal cell-free differentiation of secondary HE cells using irOP9-DLL4 or a commercially available NK differentiation stem cell kit (Stemcell Technologies). As shown in Figure 9A-9D, all three differentiation strategies met a minimum expansion fold (i.e., approximately 25-fold) and achieved similar iNK progenitor cell specifications (e.g., CD45 expression). + CD56 + and CD7 + Retro / DLL4 differentiation may not be as robust in cell proliferation as seen with irOP9-DLL4 differentiation, but it is superior to the Stemcell Kit in generating fewer myeloid cells (e.g., CD11b + / CD14 + / CD15 +(prevalence rate).

[0206] The collected iNK progenitor cells were matured and expanded in tissue culture vessels over two 7-day expansion periods in combination with irradiated K562 cells engineered to express 41BBL and IL21 (irK562-41BBL-IL21). After the first 7-day maturation and expansion period (R1D7), the cells were assayed for fold expansion and restimulated with additional irK562-41BBL-IL21 to continue maturation and expansion. After the second 7-day maturation and expansion period (R2D7), the cells were assayed for fold expansion and cryopreserved for in vitro cytotoxicity assays. As shown in Figures 10A-C, iNK progenitor cells differentiated using the Stemcell Kit failed to expand after R1D7, whereas those differentiated on irOP9-DLL4 and Retro / DLL4 showed similar levels of expansion, far exceeding those of the Stemcell Kit differentiation. Furthermore, after R2D7, the Retro / DLL4 group surprisingly out-proliferated both the irOP9-DLL4 group and the Stemcell Kit group, providing a much more favorable total fold expansion across both rounds of maturation and expansion (Figure 10C). + CD56 + iNK cells and those obtained using conventional irOP9-DLL4 differentiation showed similar profiles exhibited by various inhibitory, activating, and coactivating NK receptors, thereby demonstrating that the stromal cell-free Retro / DLL4 system is an effective and efficient solution for differentiating HE cells into NK lineage cells.

[0207] To demonstrate the functionality of mature iNK cells differentiated from secondary HE cells using the stromal cell-free method, cryopreserved iNK cells from the irOP9-DLL4 and Retro / DLL4 groups were thawed and cocultured with Nalm6 target cells or Nalm6 cells engineered to express an exemplary surface antigen (e.g., Nalm6-KLK2). After 4 hours of coculture, cell apoptosis was measured using a caspase 3 / 7 activity assay. As shown in Figures 11A and 11B, even though the iNK cells from the Retro / DLL4 group exhibited lower antigen-independent spontaneous killing of Nalm6 target cells than those from the irOP9-DLL4 group across a range of effector:target ratios, the iNK cells from both groups possessed equally effective antigen-specific killing directed by the CAR. The lower antigen-independent spontaneous killing of iNK cells differentiated from the stromal cell-free Retro / DLL4 method may be advantageous for therapeutic effector cells as a desirable safety feature by reducing occult innate NK killing.

[0208] In separate experiments, thawed iNK cells from the irOP9-DLL4 and Retro / DLL4 groups were cocultured with PC3 cells or PC3 cells engineered to express antigens (e.g., PC3-KLK2) on their surface. After 16 hours, cytokine release in each group was measured by ELISA in the supernatants. As shown in Figures 12A and 12B, iNK cells from both groups exhibited similar levels of maximal release of IFNγ and TNFα under PMA / ionomycin stimulation, as well as similar levels of antigen-dependent cytokine release of IFNγ and TNFα, confirming that the stromal cell-free Retro / DLL4 differentiation system supports the functionality of differentiated effector cells.

[0209] To demonstrate the persistence of differentiated effector cells, mature expanded iNK cells from the irOP9-DLL4 and Retro / DLL4 groups were further subjected to an IncuCyte® serial restimulation assay. In this assay, differentiated iNK cells from each group were combined with PC3 parental cells or PC3 cells expressing cell surface antigens (e.g., PC3-KLK2) at multiple effector:target ratios. The number of surviving target cells was monitored by hourly fluorescence imaging over 48 hours. The number of viable cells was quantified and normalized to the number of viable cells remaining in the target cell-only control group. After 48 hours, iNK cells from R1 were restimulated with fresh PC3 parental cells or PC3-KLK2 target cells to generate a normalized viable cell count (R2 re-stim). iNK cells from R2 were restimulated again 48 hours later to generate a normalized viable cell count (R3 re-stim). As shown in Figure 13, iNK cells from the Retro / DLL4 group maintained the same antigen-dependent continuous killing ability of target cells as those from the irOP9-DLL4 group, further confirming that the stromal cell-free Retro / DLL4 differentiation system supports long-term effector cell function.

[0210] Example 6 - Modified Growth Composition for Mature Lymphoid Cells To regulate effector cell activation during the cell proliferation phase of iPSC differentiation, AhR (aryl hydrocarbon receptor) inhibitors were found to improve effector cell function, including antitumor efficacy. iPSCs were differentiated into secondary HE cells as previously described, followed by differentiation into pre-expansion NK cells, at which point they were expanded by co-culture with IL21 and 41BBL expressed by irK562 feeder cells for approximately 7 days. An exemplary AhR inhibitor, CH-223191 (e.g., UM729, UM171, SRI, among others), was added to the expansion cultures on day 2 at a final concentration of approximately 3 μM to enhance the proliferation signal. Figure 14 shows that in the presence of CH-223191, greater fold expansion and yield of differentiated NK cells were obtained compared to iNK cells expanded without the small molecule AhR inhibitor.

[0211] Cells treated with CH-223191 were cryopreserved long-term in liquid nitrogen. To assess cell recovery from cryopreservation, cells were thawed and cultured in B0 medium for 7 days, with cell numbers counted over the course of the culture. Cells treated with AhR inhibitors demonstrated greater post-thaw recovery and persistence over time. Furthermore, cells treated with CH-223191 and cryopreserved in liquid nitrogen were further evaluated for in vitro and in vivo function. Cells treated with CH-223191 and cryopreserved in liquid nitrogen showed enhanced antitumor efficacy at endpoint compared with untreated or control cells, demonstrating that treatment with AhR inhibitors confers better antitumor efficacy over time (Figure 15).

[0212] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as limitations on the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0213] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0214] The present disclosure illustratively described herein can be practiced in the absence of any element, limitation, or limitations not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms. The terms and expressions used are used as terms of description, not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather recognizes that various modifications are possible within the scope of the present disclosure as claimed. Thus, while the present disclosure has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may occur to those skilled in the art, and that such modifications and variations are within the scope of the present invention as defined by the appended claims.

Claims

1. A cell population comprising: (i)CD82 ＋ 、 (ii) CD34 ＋ CD82 ＋ and / or (iii) CD34 ＋ CD43 － CD82 ＋ comprising a cell having a phenotype of A cell population, wherein the cells comprise secondary hemogenic endothelial (HE) cells, the cells being derived from differentiation of iPSCs in vitro.

2. the secondary HE cells (i) concentrated, and / or (ii) The cell population of claim 1, which is capable of differentiating into hematopoietic lineage cells including T cell precursors and T cells in addition to NK precursor cells, NK cells, NKT cells, or B cells.

3. 2. The cell population of claim 1, wherein the iPSCs are clonal iPSCs, iPSCs dissociated into single cells, cells of an iPSC cell line, or cells of an iPSC Master Cell Bank (MCB).

4. The cell population of claim 3 , wherein the iPSCs are naive iPSCs.

5. The iPSCs further comprise one or more genetic imprints introduced into the iPSCs by genome editing during or after reprogramming of a non-pluripotent cell into the iPSCs, the genetic imprints comprising: (i) one or more genetic modification modalities introduced by genomic insertion, deletion, or substitution in the genome of said iPSCs; or (ii) comprising one or more retainable therapeutic properties of source-specific immune cells specific to a donor, a disease, or a therapeutic response, wherein the iPSCs are reprogrammed from the source-specific immune cells; The cell population of claim 3 , wherein the cells also contain one or more identical genetic imprints.

6. differentiation of the iPSCs to obtain the cell population, (i) differentiating iPSCs to obtain hemogenic endothelial (HE) cells; (ii) CD82 ＋ The HE cells were sorted for cells that were CD82 ＋ and obtaining secondary HE cells that express a cell marker comprising: The cell population of claim 1 , wherein the secondary HE cells are capable of differentiating into hematopoietic lineage cells.

7. The step of differentiating iPSCs to obtain HE cells comprises: (a) differentiating iPSCs to obtain mesodermal progenitor cells; The cell population of claim 6 , further comprising the step of (b) differentiating the mesodermal progenitor cells to obtain HE cells.

8. The cell marker is CD34 ＋ , CD43 － , RUNX1 ＋ or any combination thereof, wherein the resulting secondary HE cells are CD34 ＋ CD82 ＋ , CD34 ＋ CD43 CD82 ＋ , CD34 ＋ CD82 ＋ RUNX1 ＋ , or CD34 ＋ CD43 － CD82 ＋ RUNX1 ＋ The cell population of claim 6, comprising a phenotype of:

9. Differentiation of the iPSCs by: (i) a cytokine that results in a higher percentage of RUNX1-expressing HE cells compared to the absence of said cytokine, and / or (ii) contacting the cell population with a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor, wherein the small molecule p38 MAPK inhibitor results in improved maintenance of CD82 expression in the HE cells compared to the absence of the inhibitor.

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

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

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

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

14. A method for producing iPSC-derived secondary HE cells, comprising the steps of differentiating iPSCs to obtain iPSC-derived hemogenic endothelial (HE) cells, and transfecting the HE cells with CD82 ＋ and thereby select for cells that are CD82 ＋ and obtaining secondary HE cells that express a cell marker comprising: The method, wherein the secondary HE cells are capable of differentiating into hematopoietic lineage cells, including NK precursors, NK cells, NKT cells, or B cells, as well as T cell precursors and T cells.

15. The selecting step is ＋ , CD43 － , RUNX1 ＋ or any combination thereof, wherein the resulting secondary HE cells are ＋ CD82 ＋ , CD34 ＋ CD43 － CD82 ＋ , CD34 ＋ CD82 ＋ RUNX1 ＋ , or CD34 ＋ CD43 － CD82 ＋ RUNX1 ＋ The method of claim 14, comprising the phenotype of

16. (i) contacting the iPSCs with a medium comprising a BMP activator and bFGF, thereby differentiating the iPSCs to obtain mesodermal progenitor cells; 15. The method of claim 14, further comprising the step of: (ii) contacting the mesodermal progenitor cells with a 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. 17. The method of claim 16, wherein contact with the p38 MAPK inhibitor increases the maintenance of CD82 expression in HE cells compared to contact without the p38 MAPK inhibitor, the BMP activator comprises BMP4, and / or the Wnt pathway activator comprises a GSK3 inhibitor.

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

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

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

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

22. A composition for generating secondary HE (hemogenic endothelial) cells from iPSCs, comprising a BMP activator, bFGF, VEGF, a Wnt pathway activator, and optionally a p38 MAPK inhibitor.

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

24. 23. The composition of claim 22, wherein the BMP activator comprises BMP4 and / or the p38 MAPK inhibitor comprises at least one of DBM1285, VX-745, VX-702, RO4402257, SCIO-469, BIRB-796, SD-0006, PH-797804, AMG-548, LY2228820, SB-681323, GW-856553, RV568, CAS 219138-24-6, SB203580, and SB242235.

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

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

27. 1. A method for generating secondary HEs from iPSCs, comprising: (i) differentiating iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain HE cells; (iii) CD82 ＋ sorting the HE cells for cells that are CD82 ＋ and obtaining secondary hemogenic endothelial (HE) cells that express cell markers including The method, wherein the secondary HE cells are capable of differentiating into hematopoietic lineage cells, including NK precursors, NK cells, NKT cells, or B cells, as well as T cell precursors and T cells.

28. The selecting step is ＋ , CD43 － , RUNX1 ＋ or any combination thereof, wherein the resulting secondary HE cells are ＋ CD82 ＋ , CD34 ＋ CD43 － CD82 ＋ , CD34 ＋ CD82 ＋ RUNX1 ＋ , or CD34 ＋ CD43 － CD82 ＋ RUNX1 ＋ 28. The method of claim 27, comprising the phenotype of:

29. The step (ii) of differentiating the mesodermal progenitor cells into HE cells comprises: (i) a cytokine that results in a higher percentage of RUNX1-expressing HE cells compared to the absence of said cytokine, and / or 28. The method of claim 27, comprising (ii) contacting with a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor, wherein the small molecule p38 MAPK inhibitor results in improved maintenance of CD82 expression in the HE cells compared to without the inhibitor.

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

31. 28. The method of claim 27, further comprising the step of cryopreserving the obtained secondary HE cells.

32. 12. A method for producing iPSC-derived hematopoietic lineage cells by differentiating the secondary HE cells of any one of claims 1 to 11, comprising contacting the secondary HE cells with a 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 precursors and T cells in addition to NK cell precursors, NK cells, NKT cells, or B cells.

33. 33. The method of claim 32, wherein the iPSC-derived hematopoietic lineage cells comprise NK cell precursors and / or NK cells, (1) the medium composition further comprises IL15, and / or (2) the secondary HE cells comprise a genetic 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. 34. The method of claim 33, wherein the media composition does not include OP9 stromal cells.

35. 35. The method of claim 34, wherein the differentiating step is carried out in the presence of an extracellular matrix comprising recombinant human fibronectin or a fragment thereof and Fc-rhDLL4.

36. 1. A method for producing iPSC-derived hematopoietic lineage cells, comprising: iPSCs were differentiated to express CD82 ＋ Obtaining secondary hemogenic endothelial (HE) cells that express cell markers including: differentiating the secondary HE cells to obtain iPSC-derived hematopoietic lineage cells; The method, wherein the iPSC-derived hematopoietic lineage cells include T cell precursors and T cells in addition to NK cell precursors, NK cells, NKT cells, or B cells.

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

38. differentiating the iPSCs to obtain secondary hematopoietic endothelial (HE) cells; (i) differentiating the genetically engineered iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain HE cells; (iii) CD82 ＋ The HE cells were sorted for cells that were CD82 ＋ and obtaining secondary HE cells that express a cell marker comprising:

39. The selecting step is ＋ , CD43 － , RUNX1 ＋ or any combination thereof, wherein the resulting secondary HE cells are ＋ CD82 ＋ , CD34 ＋ CD43 － CD82 ＋ , CD34 ＋ CD82 ＋ RUNX1 ＋ , or CD34 ＋ CD43 － CD82 ＋ RUNX1 ＋ 39. The method of claim 38, comprising the phenotype:

40. The step of differentiating the mesodermal progenitor cells into HE cells comprises: (i) a cytokine that results in a higher percentage of RUNX1-expressing HE cells compared to the absence of said cytokine, and / or 39. The method of claim 38, comprising (ii) contacting with a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor, wherein the small molecule p38 MAPK inhibitor results in improved maintenance of CD82 expression in the HE cells compared to without the inhibitor.

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

42. 37. The method of claim 36, further comprising the step of cryopreserving the secondary HE cells, wherein the cryopreserved secondary HE cells are thawed prior to their differentiation.

43. 37. The method of claim 36, wherein the step of differentiating the secondary HE cells does not include OP9 stromal cells.

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

45. 1. A method for producing NK cells in a feeder-free environment, comprising: (a) differentiating iPSCs or secondary 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 comprise OP9 stromal cells; and (i) the culture medium comprises IL15, and / or (ii) the secondary HE cells contain a genetic insert of a polynucleotide encoding a cytokine signaling complex containing a partial or complete peptide of exogenous IL15 and / or its receptor expressed on the cell surface; (b) expanding and activating the NK lineage cells to obtain NK cells having cytotoxicity against a target.

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

47. the secondary HE cells (i)CD82 ＋ 、 (ii) CD34 ＋ CD82 ＋ 、 (iii) CD34 ＋ CD43 － CD82 ＋ and / or (iv) CD34 ＋ and CD43 － , CD93 － , CXCR4 － , CD73 － , and RUNX1 ＋ and at least one of:

48. The step of differentiating the iPSCs comprises: (i) differentiating the iPSCs to obtain mesodermal progenitor cells; (ii) differentiating the mesodermal progenitor cells to obtain hematopoietic endothelial (HE) cells; (iii) CD82 ＋ and selecting the HE cells for cells which are The secondary HE cells are CD82 ＋ 46. ​​The method of claim 45, wherein the cells express a cell marker comprising:

49. The selecting step is ＋ , CD43 － , RUNX1 ＋ or any combination thereof, wherein the resulting secondary HE cells are ＋ CD82 ＋ , CD34 ＋ CD43 － CD82 ＋ , CD34 ＋ CD82 ＋ RUNX1 ＋ , or CD34 ＋ CD43 － CD82 ＋ RUNX1 ＋ 49. The method of claim 48, comprising the phenotype:

50. The step of differentiating the mesodermal progenitor cells into HE cells comprises: (i) a cytokine that results in a higher percentage of RUNX1-expressing HE cells compared to the absence of said cytokine, and / or 49. The method of claim 48, comprising (ii) contacting with a small molecule p38 MAPK (mitogen-activated protein kinase) inhibitor, wherein the small molecule p38 MAPK inhibitor results in improved maintenance of CD82 expression in the HE cells compared to without the inhibitor.

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

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

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

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

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