Natural killer cell lines derived from pluripotent cells

Abstract

The present disclosure provides an efficient ex vivo process for generating NK cell lineages from human induced pluripotent stem cells (iPSCs). In various embodiments, the cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. In some aspects, the present invention provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 413,383, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 26, 2023, is named GRU-014PC_Sequence_Listing.xml and is 30,049 bytes in size. [Background technology]

[0003] Lymphoid natural killer (NK) cells hold promise for a variety of applications, including improving hematopoiesis and solid organ transplantation, promoting antitumor immunotherapy, and controlling inflammatory, infectious, and autoimmune disorders. However, clinical use of NK cells is hindered by the low number of functional NK cells that can be isolated or otherwise obtained from routine leukapheresis products. Therefore, the development of large-scale, commercially available NK cells would provide a powerful immunotherapeutic tool. Summary of the Invention [Means for solving the problem]

[0004] In various aspects and embodiments, the present disclosure provides lymphoid progenitor cells capable of generating NK cells, including adaptive NK cells, cytotoxic NK cells, immature NK cells, unipotent NK cell precursors, lymphoid-committed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs), as well as CD34+CD7 brightMethods are provided for generating immunocompetent NK cell lineages, including progenitor cells, for cell therapy. In various embodiments, the present invention provides an efficient ex vivo process for developing such hematopoietic lineages from human induced pluripotent stem cells (iPSCs), including gene-edited iPSCs. In various embodiments, cells generated according to the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0005] In one aspect, the present disclosure provides a method for preparing a cell population comprising NK cells. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34+-enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+-enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. The resulting HSC population (or a fraction thereof) can be differentiated into NK cell lineages (including lymphoid-committed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs). In some embodiments, the present disclosure provides methods for generating various types of NK cells ex vivo from an HSC population.

[0006] In various embodiments, iPSCs are prepared by reprogramming somatic cells. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood.

[0007] In various embodiments, iPSCs are genetically edited to support HLA matching, such as deletion of one or more HLA class I and / or class II alleles. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C alleles, and one or more of HLA-DP, HLA-DQ, and HLA-DR alleles. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C alleles. neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0008] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be harvested from the culture to generate embryoid bodies (EBs). EBs, generated by differentiation of iPSCs, are three-dimensional aggregates of iPSCs that contain three (or alternatively, two or one) embryonic germ cell layers, depending on the differentiation method(s). In some embodiments, the process includes harvesting CD34+ enriched cells from the EBs and inducing endothelial and hematopoietic differentiation.

[0009] In some embodiments, iPSC differentiation proceeds until the cells are at least about 20% CD34+ or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7-14 of iPSC differentiation. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, a combination of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1.

[0010] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells.

[0011] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition between days 10 and 20 of iPSC differentiation, such as between days 12 and 17 of iPSC differentiation. Hematopoietic stem cells (HSCs), which can give rise to innate myeloid, erythroid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (termed Lin-).

[0012] In various embodiments, the HSC population or a fraction thereof is differentiated into a hematopoietic lineage, which may be selected from, but is not limited to, therapeutic human NK cells, including adaptive NK cells or their progenitors (e.g., lineages that give rise to natural killer cells (NK cells)). NK cell lineages include adaptive NK cells, cytotoxic NK cells, immature NK cells, unipotent NK cell precursors, and lymphoid progenitors capable of generating NK cells. Such progenitor cells include lymphoid-committed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and CD34+CD7 bright These include progenitor cells, or modified forms thereof (e.g., genetically engineered cells such as NK-CAR cells). In some embodiments, the NK cells may express a chimeric antigen receptor (CAR).

[0013] In another aspect, the present invention provides a cell population or a pharmaceutically acceptable composition thereof, comprising NK lineage or progenitor cells thereof, which may be produced by the methods described herein. In some embodiments, the cell population is a progenitor NK lineage cell population capable of engraftment in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In various embodiments, a composition for cell therapy is prepared comprising the cell population and a pharmaceutically acceptable vehicle. In some embodiments, the cell population is HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the cell population is further homozygous for HLA-DRB1.

[0014] In another aspect, the present invention provides methods for cell therapy comprising administering a cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the methods described herein are used to treat hematological (malignant and non-malignant), bone marrow, immune, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer (hematologic malignancy or solid tumor), immunodeficiency, autoimmune disease, viral infection, skeletal dysplasia, or bone marrow failure syndrome.

[0015] Other aspects and embodiments of the present disclosure will be apparent from the following detailed disclosure and examples. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows a FACS plot showing the transduction efficiency of iPSCs using an adenoviral vector to overexpress ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness, as indicated by the expression of the TRA-1-60 stemness marker. [Figure 2]ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (described as CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Figure 3] Figure 1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4A] Figure 1 shows that iPSC-derived HSCs derived by Piezo1 activation undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 2 shows FACS plots of the differentiation efficiency of bone marrow (BM)-HSCs and iPSC-HSCs derived by Piezo1 activation into CD34+CD7+ pre-T cells. [Figure 4B] Figure 4B shows that iPSC-derived HSCs derived by Piezo1 activation undergo pre-T cell differentiation similar to bone marrow (BM)-HSCs. Quantification (%) of CD34+CD7+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Average values ​​from three experiments are shown. [Figure 5A] Figure 1 shows that iPSC-derived HSCs generated by Piezo1 activation can undergo T cell differentiation similar to BM-HSCs and can be activated with CD3 / CD28 beads. Figure 2 shows FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs generated by Piezo1 activation. [Figure 5B] Figure 5B shows that iPSC-derived HSCs generated by Piezo1 activation can undergo T cell differentiation similar to BM-HSCs and can be activated with CD3 / CD28 beads. Figure 5B shows the quantification (%) of CD3+CD69+ cells derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1-activated). Figure 5B shows the average of three experiments. [Figure 6]We demonstrate that iPSC-derived HSCs (in this example, using Piezo1 activation) can differentiate into functional T cells. IFNγ expression is a consequence of T cell activation after T cell receptor (TCR) stimulation via CD3 / CD28 beads. IFNγ expression in T cells differentiated from iPSC-derived HSCs generated upon Piezo1 activation enhances the HSC capacity to further differentiate into functional T cells. Figure 6 shows the average of three experiments. [Figure 7] We show that HSCs derived from iPSC differentiation (D8+7 with Yoda1 (“Y”)) are activated by pharmacological stimulation better than NK cells derived from D8 iPSC-CD34+ cells, as measured by activity of the functional marker CD107a. [Figure 8A] We show that HSC-derived NK cells (D8+7, +Y) outperform bone marrow-derived NK cells in killing tumor cells. [Figure 8B] We show that HSC-derived NK cells (D8+7, +Y) outperform bone marrow-derived NK cells in killing tumor cells. [Figure 9A] Figure 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. The overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface is shown, with HLA-edited cells being positive for overall HLA class I expression to a similar extent as wild-type cells. [Figure 9B] FIG. 1 shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells performed by FACS and immunofluorescence. Cellular expression of HLA-A via immunofluorescence is shown, where HLA-A is not expressed in HLA-edited clones. [Figure 10] We show that the HLA-edited clones retain their pluripotency (maintain tri-lineage differentiation) as illustrated by immunofluorescence, where ectodermal differentiation is indicated by NESTIN-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining. [Figure 11]Immunocompatibility of HLA-edited HSCs: HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were cocultured with peripheral blood mononuclear cells (PBMCs) bearing HLA-B and HLA-C matched but mismatched HLA-A, and PBMC-mediated cytotoxicity was measured by Annexin V staining assay. [Figure 12] Figure 1 shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs were mixed for competitive transplantation into mice, and bone marrow (BM) and peripheral blood samples were assessed by FACS to compare the relative amounts of each cell type present in the samples. [Figure 13] Figure 1 shows the ability of HSCs to effectively differentiate into NK cells as evidenced by fluorescence-activated cell sorting (FACS) experiments gated on the basis of CD56 expression. [Figure 14A] 1 shows that HSC-derived NK cells effectively kill tumor cells. A schematic diagram of an experiment is shown in which HSC-derived NK cells are co-cultured with K562 HLA-null cells and the degree of NK cell degranulation is measured using Annexin V staining and a cytotoxicity assay. [Figure 14B] Figure 1 shows that HSC-derived NK cells effectively kill tumor cells. Figure 2 shows the results of NK cell degranulation as measured by fluorescence-activated cell sorting (FACS) using Annexin V staining. [Figure 14C] Figure 1 shows that HSC-derived NK cells effectively kill tumor cells. Figure 2 shows the results of a tumor cell cytotoxicity assay using lactate dehydrogenase (LDH) as a measure of cell death.

[0017] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0018] The terms "wild-type" (WT), "unedited," and "non-HLA edited" are used interchangeably herein to refer to the non-gene-edited cells of the present disclosure.

[0019] EB34+ cells refer to embryonic body-derived CD34+ cells, which contain hemogenic endothelial cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure, in various aspects and embodiments, provides methods for generating hematopoietic lineages for cell therapy, particularly NK cell lineages, including their progenitors and progeny. In various embodiments, the lineages include lymphoid progenitors with the potential to generate NK cells, including adaptive NK cells, cytotoxic NK cells, immature NK cells, unipotent NK cell precursors, lymphoid-committed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs), as well as CD34+CD7 bright These include progenitor cells. In various embodiments, the present invention provides an efficient ex vivo process for developing such NK cell lineages from human induced pluripotent stem cells (iPSCs). In various embodiments, the cells produced in accordance with the present disclosure are functional and / or more closely resemble corresponding lineages isolated from peripheral blood, bone marrow, or lymphoid organs. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0021] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate an essentially unlimited number of pluripotent stem cells (PSCs) is exploited to generate an unlimited supply of NK cell lineages, including, but not limited to, therapeutic human NK cells, including adaptive NK cells or their progenitors (e.g., lineages that give rise to natural killer (NK) cells). NK cell lineages include adaptive NK cells, cytotoxic NK cells, immature NK cells, unipotent NK cell precursors, and lymphoid progenitors capable of generating NK cells. Such progenitor cells include lymphoid-committed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and CD34+CD7 brightThese include progenitor cells or modified forms thereof (e.g., genetically engineered cells such as NK-CAR cells). The use of NK cells as therapeutic lymphocytes is limited by their limited availability, cell number, limited proliferation capacity, and histocompatibility issues. Furthermore, compared to primary cells, hiPSCs undergo genetic modification more easily in vitro, thereby providing opportunities for improving cell target specificity and cell number, while also avoiding HLA matching issues, for example. Additionally, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). Furthermore, unlike human embryonic stem cells (hESCs), hiPSCs are of non-embryonic origin, eliminating ethical concerns and ensuring consistent quality. Therefore, the use of hiPSCs according to the present disclosure offers several advantages over primary cells for generating therapeutic hematopoietic lineages, such as NK lymphocytes.

[0022] In one aspect, the present disclosure provides a method for preparing a cell population of NK cell lineage. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34+ enriched population. Endothelial-hematopoietic transition (EHT) is induced in the CD34+ enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. The resulting HSC population (or a fraction thereof) can be differentiated into an NK cell lineage according to the present disclosure.

[0023] In some embodiments, the method generates CD3-CD56+ NK cells developed from CD34+ hematopoietic progenitor cells (i.e., HSC populations or fractions thereof) or derivatives of this population. For example, NK cells generated from HSC populations can be generated in Notch ligand-regulated cultures and / or cultured in serum-free growth medium (SFEM) supplemented with cytokines (e.g., SCF, TPO, Flt3L, and IL-7). These conditions promote the expansion of HSC populations and the development of CD7 + CD5+ The cells can then be further cultured in SFEM supplemented with cytokines, including IL-15 (e.g., SCF, Flt3L, IL-7, IL-15), to promote their differentiation into NK cells. After culture, the majority of the cells (e.g., an average of about 65%, 70%, 75%, or 80% of the cells) express the NK cell marker CD56 and may also co-express NKp46 (an NK cell activating receptor described herein or other NK cell lineage markers). At more mature developmental stages, mature CD56 + CD16 + NK cell subsets emerge in these cultures. Acquisition of CD94 is associated with the acquisition of CD56 bright CD56 marks the commitment to bright NK cells then upregulate CD16 and the killer immunoglobulin-like receptor (KIR) CD56 dim The NK cells differentiate into NK cells. In some embodiments, the NK cells are cytotoxic NK cells capable of killing, inter alia, cancer cells (e.g., leukemia cells) or virus-infected cells. In embodiments, the NK cells express CD107a as a functional marker for identifying natural killer cell activity.

[0024] NK cells express CD56 dim or CD56 bright NK cells can be divided into subsets. Approximately 90% of peripheral blood and splenic NK cells are CD56 dim These CD56 dim NK cells are cytotoxic and, for example, produce IFN-γ upon interaction with tumor cells in vitro. In contrast, most NK cells in lymph nodes and tonsils express CD56 brightThese NK cells are CD16- and lack perforin. These cells readily produce cytokines such as IFN-γ in response to stimulation with interleukin (IL)-12, IL-15, and IL-18. Thus, in some embodiments, these NK cell properties can be manipulated to establish their function, such as improved transplantation or elimination of tumors (malignant or non-malignant, solid or otherwise), or in elimination of viral infections.

[0025] In some embodiments, the present disclosure generates NK cell precursors. NK cell precursors are identified as Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+CD127- cells, representing unipotent NK cell precursors lacking the ability to commit to other lymphoid lineages. In some embodiments, NK cell precursors are identified as Lin-CD34+CD38+CD123-CD45RA+CD7+CD10-CD127+. In some embodiments, NK cell precursors are identified as Lin-CD34+CD38+CD123-CD45RA+CD7+CD10+CD127+ precursor cells (generating lymphoid lineages). In some embodiments, NK precursor cells are identified based on their negative or positive expression of both IL-1β and IL-2β receptors. In some embodiments, NK cells are CD34-CD117+ / -CD94+HLADR-CD10-CD122+CD94+NKp44 low NKG2D+CD161+, i.e., can be identified as mature NK cells, which can then be further differentiated into two final developmental stages according to the expression of CD56 and CD16, respectively. In embodiments, NK cells express CD107a as a functional marker for the identification of natural killer cell activity.

[0026] Several NK-specific markers can be used to identify, isolate, or enrich NK cells. For example, NK cells, typically defined as CD3-CD56+ cells, can also be CD7+CD127-NKp46+T-β+Eomes+. Different subtypes of human NK cells can be identified using CD3-CD56 dimCD16+ or CD3-CD56 bright NK cells can be identified as either CD16- or CD56- dim The CD16+ subset is found primarily in the blood and is highly cytotoxic, while the CD56 bright The CD16- subset is the predominant subtype found in lymph nodes and has only weak cytotoxic activity. Other cell surface markers (or combinations thereof) can be used to characterize the NK cells of the present invention. These include, but are not limited to, CD3-, CD56 / NCAM-1+, CD94+, CD122 / IL-2 Rβ+, CD127 / IL-7 Rα-, FcγRIII / CD16+ / -, KIR family receptor+, NKG2A+, NKG2D+, NKp30+, NKp44+, NKp46+, or NKp80+.

[0027] Traditionally, hematopoietic lineages (such as NK cells) are prepared by differentiation (e.g., day 8) of iPSCs into embryoid bodies to harvest CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that inducing endothelial-to-hematopoietic transition (EHT) of CD34+ cell populations, which may be derived from iPSC embryoid bodies, can be used for the ex vivo generation of superior hematopoietic lineages, including NK cell lineages.

[0028] In some embodiments, CD34+ cells (i.e., derived from EB dissociation) are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. Exemplary Piezol agonists include Yodal, Jedil, single-stranded (ss)RNA (e.g., ssRNA40), and Jedi2. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Other modes for inducing EHT can be used and are described herein. In some embodiments, after inducing EHT, the cells (HSCs or their progeny) are differentiated into an NK cell lineage.

[0029] In some embodiments, HSCs are differentiated into a CD7+ progenitor T cell population, which can be further differentiated into an NK cell lineage. For example, a CD7+ progenitor T cell population can be generated from a hematopoietic stem cell (HSC) population, including human long-term hematopoietic stem cells (LT-HSCs) generated from iPSCs (e.g., hiPSCs). For example, an HSC population (or cells isolated therefrom) is cultured with a partial or complete Notch ligand, sonic hedgehog (SHH), retronectin (or other extracellular matrix component(s)), and / or combinations thereof to produce a population comprising a CD7+ progenitor T cell or derivative cell population.

[0030] The Notch signaling pathway regulates the formation, differentiation, and function of NK cells. For example, Notch signaling induces CD34+ cells to give rise to CD7+ and cytoplasmic (cy) CD3+ cells expressing CD56, precursor T cells, pro-T cells, and mature T lymphocytes. In vivo, NK and T cell development proceeds after lymphoid progenitor cells differentiate from bone marrow hematopoietic stem cells and migrate to the thymus. Specialized thymic epithelial cells induce the development of T cells and NK cells along a regulated pathway. Notch signaling plays a critical role during T lineage commitment in the thymus. As lymphoid progenitor cells enter the thymus, they encounter high-density expression of Notch ligands on the thymic epithelium, which promotes thymopoiesis. The present disclosure provides HSC populations generated ex vivo from iPSCs that respond to Notch ligands, SHH, and / or extracellular matrix components by robustly producing ex vivo T progenitor cells and T cell lineages, including NK cell lineages.

[0031] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin or blood cells, that have been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes (e.g., NK cells, T cells, B cells, etc.), umbilical cord blood cells, PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of the treatment described herein). In various embodiments, iPSCs can be genetically edited to support HLA matching (such as deletion of one or more HLA class I and / or HLA class II alleles or their master regulators, including, but not limited to, beta-2-microglobulin (B2M), CIITA, etc.), or to delete or express other functions. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus.

[0032] In various embodiments, iPSCs are gene-edited to be one of: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-, (iv) HLA-AB-C+DP-DR+DQ+, (v) HLA-AB-C+DP+DR+DQ-, or (vi) HLA-AB-C+DP-DR+DQ-. For retained HLA (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or can retain only a single copy of the gene. For example, the modified cells are identified as at least (a) HLA-C+ and HLA-DR+, and optionally as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0033] In some embodiments, the iPSCs are HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0034] As used herein, the term "neg," (-), or "negative" with respect to a particular HLA class I or HLA class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and therefore the cell line or population does not exhibit significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion or disruption, or alternatively by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a targeted gene refers to the elimination of functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption of a coding sequence or deletion of a critical cis-acting expression control sequence.

[0035] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. Nos. 10,676,165, 9,580,689, and 9,376,664, which are incorporated by reference in their entireties. In various embodiments, reprogramming factors are expressed using well-known viral vector systems, such as lentivirus, Sendai, or measles virus systems. Alternatively, reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into somatic cells. Furthermore, iPSCs can be generated by introducing non-integrating episomal plasmids expressing reprogramming factors, i.e., for the generation of transgene-free and virus-free iPSCs. Known episomal plasmids that have limited replicative capacity and are therefore lost over several cell generations can be employed.

[0036] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing can include, but is not limited to, modification of HLA genes (e.g., deletion of one or more HLA class I and / or HLA class II genes), deletion of β2 microglobulin (β2M), deletion of CIITA, deletion of an NK cell receptor gene, or addition of a chimeric antigen receptor (CAR) gene. Exemplary CARs can target tumor-associated antigens. Exemplary CAR NK cells can target CD19, CD38, CD33, CD47, CD20, etc. For example, iPSCs can be NK cell receptor-transduced iPSCs. Such embodiments enable the large-scale production of regenerative lymphocytes with desired antigen, tissue, or cell specificity. Alternatively, engineered iPSCs with one or more HLA knockouts can be placed in a bioreactor for feeder-free and serum-free differentiation under GMP-grade conditions to generate fully functional histocompatible NK cells.

[0037] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNAs comprise modifications at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some embodiments, the modified gRNAs exhibit increased resistance to nucleases. In some embodiments, the gRNAs comprise two separate RNA molecules (i.e., "duplex gRNAs"). Duplex gRNAs comprise two separate RNA molecules, "crispr RNA" (or "crRNA") and "tracr RNA," and are well known to those of skill in the art.

[0038] Generally, various gene editing technologies are known that can be applied in accordance with various embodiments of the present disclosure, including, but not limited to, zinc finger (ZF), transcription activator-like effector (TALE), etc. Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of a Fokl endonuclease can be used to generate double-stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, for example, US8,697,359, US8,906,616, and US8,999,641, each of which is incorporated herein by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are guide RNA dependent. Design of gRNAs to guide desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE, et al., CRISPR guide RNA design for research applications, FEBSJ. 2016 Sep; 283(17):3232-3238.In still other embodiments, non-canonical Type II or Type V Cas endonucleases with homology (albeit low primary sequence homology) to S. pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives, Int J Mol Sci. 2021 Apr;22(7):3327. In still other embodiments, gene editing employs base editing or prime editing to incorporate mutations without creating double-strand breaks. See, for example, Antoniou P, et al., Base and Prime Editing Technologies for Blood Disorders, Front. Genome Ed., 28 January 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA, Front. Genet., 9 June 2020. Various other gene editing processes are known, including the use of dead Cas (dCas) systems (e.g., Cas fusion proteins) to target DNA-modifying enzymes to desired targets using dCas as a guide RNA-dependent system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications, Int J Mol Sci. 2019 Dec;20(23):6041.

[0039] Base editors that can introduce precise genome modifications without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., designing gene therapy vectors). Base editors inherently cannot create DSBs and contain a catalytically inactive nuclease, such as Cas9 nickase (nCas9), fused to a nucleic acid base deaminase enzyme, possibly a DNA glycosylase inhibitor. Currently, there are two major categories of base editors: cytidine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G transversions. Base editors can be delivered, for example, via HDAd5 / 35++ vectors, to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699, 10,167,457, 10,113,163, 11,306,324, 11,268,082, 11,319,532, and 11,155,803. Also contemplated are prime editors comprising a reverse transcriptase conjugated to (e.g., fused with) a Cas endonuclease and a polynucleotide useful as a DNA synthesis template conjugated to (e.g., fused with) a guide RNA, as described in WO2020 / 191153.

[0040] Exemplary vectors that can be used for genome editing applications include plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors), herpes simplex virus vectors, baculovirus vectors, negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses. Examples of vectors that can be used include, but are not limited to, double-stranded DNA viruses, including viruses (e.g., canarypox, vaccinia, or modified vaccinia viruses). Vectors containing a nucleic acid molecule of interest can be delivered to cells (e.g., iPS cells, endothelial cells, hemogenic endothelial cells, HSCs (ST-HSCs or LT-HSCs) via any method known in the art, including, but not limited to, transduction, transfection, infection, and electroporation. Any of these vectors may contain a transposable element (such as a piggyBac transposon or a Sleeping Beauty transposon). Transposons insert specific sequences of DNA into the vertebrate genome. A gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome.

[0041] For increased efficiency, in some embodiments, Cas and gRNA can be combined before delivery into cells. The Cas-gRNA complex is known as a ribonucleoprotein (RNP). Several methods have been developed for direct delivery of RNP into cells. For example, RNP can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, which allows RNP to rapidly enter the cell's nucleus and immediately begin cleaving the genome. See, for example, Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing. Theranostics. 2021 Jan 1;11(2):614-648, incorporated herein by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNP.

[0042] Generally, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage and is typically found 3-4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2-6 base pairs in length) following the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism at an HLA locus does not contain four Gs, four Cs, a GC repeat, or a combination thereof.

[0043] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed by using the gRNAs described herein to design a single gRNA that targets each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). To perform gene knockout, the gRNA targets the Cas9 protein to the appropriate site for editing. The Cas9 protein can then perform a double-strand break (DSB), which repairs the DNA through a non-homologous end joining (NHEJ) mechanism, generating an indel that results in a frameshift mutation and terminates the function of the resulting protein. However, off-target gene recombination can occur, altering the function of an otherwise intact gene. For example, the Cas9 endonuclease can generate DSBs at undesired off-target locations, even in the presence of some degree of mismatch. This off-target activity can result in genomic instability events such as point mutations and genomic structural perturbations. In various embodiments, an sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, an sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, an sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0044] gRNAs can be used to develop clonal iPSCs. Such iPSC lines can be assessed for (i) on-target editing, (ii) off-target editing, and (iii) translocation editing, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR using primers designed to target and enrich for the region of interest, followed by next-generation sequencing (e.g., Amplicon sequencing, AMP-seq). On-target and translocation panels can amplify the intended edited region and allow for the selection of iPSC clones with the expected edits that do not contain chromosomal translocations resulting from unintended DSB cleavage site fusions. Off-target panels can enrich for any potential off-target regions identified through sequencing, allowing for the selection of iPSC clones with negligible off-target mutations. Together, these assays enable the screening of iPSC clones to select clones with the desired edits while ruling out potential CRISPR / Cas9-related genome integrity issues.

[0045] In some embodiments, to further ensure the genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that have not undergone translocation and mutation events and have not integrated the episomal vector. For example, whole genome sequencing (WGS) can be performed on CD34+ cells and iPSC clones after reprogramming, and the genomes can be compared for differences resulting from editing. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, allowing for the informed selection of iPSC clones that have not undergone mutations during reprogramming.

[0046] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that did not develop indels or translocations during reprogramming, as described, for example, in Ramme AP, et al., "Supporting dataset of two integration-free induced pluripotent stem cell lines from related human donors," Data Brief. 2021 May 15;37:107140, incorporated herein by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal abnormalities with a resolution similar to G-banding karyotyping. The size of structural abnormalities that can be detected is >2 Mb for chromosome gains and >1 Mb for chromosome losses. The KARYOSTAT array is functionalized for balanced whole-genome coverage with low-resolution DNA copy number analysis, and the array covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay allows for the detection of aneuploidy, submicroscopic abnormalities, and mosaic events.

[0047] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that have not developed copy number aberrations (CNAs) during reprogramming, as described, for example, in Wiesner et al. "Molecular Techniques," Editor(s): Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, "Pathology of Melanocytic Tumors," Elsevier, 2019, pp. 364-373, ISBN 9780323374576, and Hussein SM, et al. "Copy number variation and selection during reprogramming to pluripotency," Nature. 2011 Mar 3;471(7336):58-62, which are incorporated herein by reference in their entireties. aCGH is a technique that analyzes the entire genome for CNAs by comparing sample DNA with reference DNA.

[0048] In some embodiments, a targeted hematologic malignancy NGS panel analysis is used to select iPSC clones that did not develop hematologic malignancy mutations during reprogramming. For example, a targeted hematologic malignancy NGS panel focuses on genes associated with myeloid leukemia, lymphoma, and / or other hematologic malignancies, generating smaller, more manageable datasets than more broad methods. A targeted hematologic malignancy NGS panel analysis involves the use of highly multiplexed PCR to amplify regions associated with hematologic malignancies, followed by next-generation sequencing.

[0049] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that have not integrated the episomal vector and have been passaged sufficiently for episomal vector clearance. As discussed herein, iPSC reprogramming of CD34+ cells can be achieved by delivering episomal vectors encoding reprogramming factors. However, episomal vectors can randomly integrate into the cellular genome, although this is rare, which can disrupt developmental processes, homeostasis, and the like. Therefore, ddPCR can be used to detect residual episomal vectors in iPSC cultures, allowing for the selection of iPSC clones that have not integrated the episomal vector.

[0050] In some embodiments, after assessing that selected clones do not contain editing-related genomic abnormalities, the clones can be further tested for spontaneous mutations that may arise during expansion, such as mutations affecting hematologic malignancies, indels, translocations, and numerical abnormalities, as described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancies NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0051] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be recovered from the culture to generate embryoid bodies (EBs). EBs generated by differentiation of iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively, two or one) embryonic germ cell layers based on the differentiation method(s). EB preparation is described, for example, in US 2019 / 0177695, which is incorporated herein by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, as described, for example, in Abecasis B. et al., "Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches." J. of Biotechnol. 246 (2017) 81-93. EBs can be used to generate any desired cell type. Other methods involving 3D suspension culture for the growth or differentiation of EBs are described in WO2020 / 086889, which is incorporated herein by reference in its entirety.

[0052] In some embodiments, processes according to each aspect can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial-hematopoietic transformation. HSCs, including relatively high frequencies of LT-HSCs, can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or local stimuli, as well as factors such as extracellular matrix, niche factors, cell-extrinsic factors, induction of cell-intrinsic properties, and including pharmacological and / or genetic means.

[0053] In some embodiments, the method includes preparing hematopoietic endothelial cells from pluripotent stem cells prior to induction of EHT. In some embodiments, combined overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can lead to the formation of hematopoietic endothelial cells from PSC sources. In some embodiments, the method includes overexpression of the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. ETV2 can be expressed by introduction of an encoding non-integrating episomal plasmid for constitutive or inducible expression of ETV2 and for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of ETV2-expressing cells can include the addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA. Sci. Adv. Vol. 6 (2020). Cells generated in this manner can be used to produce CD34+ cells and induce EHT according to embodiments of the present disclosure.

[0054] After CD34+ enrichment, HSCs are then generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0055] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7-14 of iPSC differentiation, such as on days 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, a combination of bFGF, Y27632 (or other ROCK inhibitors), WNT agonists (e.g., CHIR99021), BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated using feeder-free, serum-free, and / or GMP-compliant materials, such as pomalidomide or Revlimid. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells, such as the OP9 or MS5 cell line, in serum-containing medium. The culture can contain growth factors and cytokines to support differentiation into embryoid bodies or monolayers. The OP9 co-culture system can be used to generate multipotent HSPCs that can further differentiate into several hematopoietic lineages, including T lymphocytes, B lymphocytes, megakaryocytes, monocytes or macrophages, and erythrocytes. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells, Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using defined conditions in conjunction with specific signals can be used. For example, expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into multipotent CD34+ / CD45+ progenitor cells.Furthermore, expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs. See Doulatov S. et al., Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors, Cell Stem Cell. 2013 Oct 3;13(4).

[0056] Exemplary ROCK inhibitors used in the establishment and differentiation of iPSCs include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, RevitaCell™ supplement, 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, H-100, and the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated by reference in its entirety.

[0057] Differentiation of iPSCs (e.g., into EBs) can employ a WNT agonist, such as CHIR99021. A WNT agonist is a molecule that mimics or increases WNT signaling. Non-limiting examples of WNT agonists include the small molecule CHIR-99021 (CAS 252917-06-9), 2-amino-4,6-disubstituted pyrimidines such as BML 284 (CAS 853220-52-7), SKL 2001 (CAS 909089-13-0), WAY 262611 (CAS 1123231-07-1), WAY 316606 (CAS 915759-45-4), SB 216763 (CAS 280744-09-4), IQ 1 (CAS 331001-62-8), QS 11 (CAS 944328-88-5), deoxycholic acid (CAS 83-44-3), BIO (CAS 667463-62-9), Kenpaullone (CAS 142273-20-9), or (hetero)arylpyrimidines. In some embodiments, the WNT agonist is an agonist antibody or functional fragment thereof, or an antibody-like polypeptide.

[0058] Induction of EHT can be by any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population, including LT-HSC. In some embodiments, EHT generates HSCs through endothelial or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., via stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population, including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells. In some embodiments, EHT cultures include one or more (e.g., combinations) of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

[0059] In some embodiments, the method comprises increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34-enriched cells, ECs, HECs, or HSCs, which may be by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method comprises increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO2019 / 236943 and WO2021 / 119061, which are incorporated by reference in their entireties. In some embodiments, inducing EHT comprises increasing the expression or activity of dnmt3b.

[0060] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. An exemplary Piezol agonist is Yodal. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yodal (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezol. Syeda R, Chemical activation of the mechanotransduction channel Piezol. eLife (2015).

[0061] Derivatives of Yodal can be employed in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are employed in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., "Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation," British Journal of Pharmacology 175(1744-1759):2018. Still other Piezo1 agonists include Jedi1, Jedi2, single-stranded (ss) RNA (e.g., ssRNA40), and derivatives and analogs thereof. See Wang Y., et al., "A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel," Nature Communications (2018) 9:1300; and Sugisawa, et al., "RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis," Cell, Volume 182, Issue 3, 2020, Pages 609-624, which are incorporated herein by reference in their entireties. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the Piezo1 agonist or derivative ranges from about 1 μM to about 500 μM, or from about 5 μM to about 200 μM, or from about 5 μM to about 100 μM, or in some embodiments, from about 25 μM to about 150 μM, or from about 25 μM to about 100 μM, or from about 25 μM to about 50 μM.

[0062] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34-enriched cells). In certain embodiments, pharmacological Piezo1 activation may further be applied to iPSCs, embryoid bodies, ECs, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s). In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, allows for superior generation of T cell progenitors compared to other methods for inducing EHT.

[0063] Alternatively, or in addition, Dnmt3b activity or expression can be increased directly in cells, for example, in CD34-enriched cells. For example, Dnmt3b mRNA expression can be increased by transgene-free methods, including but not limited to, delivering a transcript encoding Dnmt3b to cells, or by introducing a transgene encoding Dnmt3b, or by introducing a non-integrating episome into cells. In some embodiments, gene editing is employed to introduce genetic modifications into Dnmt3b-expressing elements in cells, such as, but not limited to, increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0064] In some embodiments, the method includes increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up- or down-regulated upon cyclic strain or Piezol activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell; a transgene-free approach can be employed, including, but not limited to, introducing an episome into the cell; or, alternatively, a transgene encoding Gimap6 can be introduced. In some embodiments, gene editing is employed to introduce genetic modifications (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or to affect RNA splicing) into a Gimap6-expressing element in the cell.

[0065] In embodiments of the present disclosure employing mRNA delivery to cells, known chemical modifications can be used to circumvent the innate immune response in cells. For example, synthetic RNAs containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can result in translation block, secretion of inflammatory cytokines, and cell death. RNAs containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into proteins with high efficiency. See US Pat. No. 9,181,319, incorporated herein by reference, particularly for nucleotide modifications to circumvent the innate immune response.

[0066] In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired level of overexpression (with varying promoter strength or other selection of expression control elements). Various viral vectors or transfection reagents (including lipid nanoparticles) known in the art can be used to introduce the transgene. In some embodiments, expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, expression or activity of Dnmt3b and / or Gimap6, or other genes disclosed herein, is increased using gene editing techniques, for example, to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0067] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to cells. In various embodiments, the cells subjected to cyclic 2D, 3D, or 4D stretch are selected from one or more of CD34-enriched cells, iPSCs, ECs, and HECs. For example, the cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is incorporated herein by reference in its entirety. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, the mechanical means applies a stretching force to the cells or to a cell culture surface having cells (e.g., ECs or HECs) cultured thereon. For example, cyclic 2D, 3D, or 4D stretching can be applied to cells ex vivo under defined and controlled cyclic strain conditions using a computer-controlled vacuum pump system or other means for providing a stretching force (e.g., the FlexCell™ Tension System, CytoStretcher System) attached to a flexible, biocompatible and / or biomimetic surface. For example, the applied cyclic stretching can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0068] Alternatively, or in addition, EHT is stimulated by Trpv4 activation, which can be by contacting cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0069] When a cell population is described herein as having a particular phenotype, it is understood that the phenotype represents a substantial portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Furthermore, in various steps, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an undesired phenotype, such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence-activated cell sorter or magnetic beads that bind to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the proliferation of CD34+ cells, thereby producing an expanded population of stem cells. In some embodiments, the cell population is cultured under conditions that promote the proliferation of CD34+ cells, thereby producing an expanded population of stem cells. Additionally, NK cells produced according to the present disclosure can be enriched for desired markers, such as expression or expression levels of CD56.

[0070] In various embodiments, CD34+ cells (e.g., suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic transition, such as on days 8-20 (e.g., days 10-17) of iPSC differentiation.

[0071] In various embodiments, the HSCs or CD34+ enriched cells are further expanded. For example, the HSCs or CD34+ enriched cells can be expanded according to the methods disclosed in US8,168,428, US9,028,811, US10,272,110, and US10,278,990, which are incorporated by reference in their entireties. In some embodiments, the ex vivo expansion of the HSCs or CD34 enriched cells employs prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0072] Hematopoietic stem cells (HSCs), which give rise to erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in US9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include sorting a cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. CD34 + , CD90 + , CD38 - , and CD43 - In some embodiments, a stem cell population for differentiation into hematopoietic lineages is selected for further differentiation from a fraction that is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + is.

[0073] In some embodiments, stem cell populations, or CD34+ enriched cell or fraction thereof, or derivative populations, are expanded as described in US 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55, and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0074] In some embodiments, CD34 + Compounds that promote cell proliferation include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, incorporated herein by reference).

[0075] In some embodiments, the stem cell population or CD34+ enriched cells are further enriched for or modified to express periostin and / or platelet-derived growth factor receptor alpha (pdgfra), as described in WO2020 / 205969 (incorporated herein by reference in its entirety). Such expression can be by transgene-free methods, including but not limited to, by delivering an encoding transcript to the cells, by introducing an encoding transgene, or by introducing a non-integrating episome into the cells. In some embodiments, gene editing is employed to introduce genetic modifications into expression elements in the cells, such as to increase promoter activity or strength, ribosome binding, RNA stability, or affect RNA splicing.

[0076] In yet another embodiment, the stem cell population or CD34-enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated, or transiently silenced, in the stem cell population. Inhibition of EZH1 can direct myeloid progenitor cells (e.g., CD34+CD45+) to the lymphoid lineage. See WO2018 / 048828, which is incorporated herein by reference in its entirety. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0077] The HSC population or a fraction thereof is differentiated into the NK cell lineage.

[0078] In vivo, NK cell development can be divided into approximately six stages based on bone marrow (BM) and lymph node (LN) development. See Abel AM et al: Natural Killer Cells: Development, Maturation, and Clinical Utilization. Front Immunol. 2018 Aug 13;9:1869. NK cells corresponding to these stages can be generated according to the present disclosure. CD3ε-CD7+CD127+ cells mark the earliest stage of committed NKPs (stage 2a). CD7 expression persists throughout development. Expression of IL-1R, the receptor for IL-1β, defines stage 2b. Expression of activating receptors, including NKG2D, CD335 (natural cytotoxicity receptor, NCR1, NKp46), and CD337 (NCR3, NKp30), marks the transition of NK cells from stage 2b to stage 3. Stage 4 of human NK cell development is subdivided into two parts based on the expression of the activating receptor NKP80 (KLRF1, a type II transmembrane protein). The main distinction of NK cells in stage 4a is that they express abundant amounts of CD56 (CD56 brightThese NK cells are NKP80- and express maximal levels of NKG2D, CD335, CD337, inhibitory NKG2A, and CD161 (NK1.1, KLRB1, NKR-P1A). In stage 4b, human NK cells become positive for NKP80 and express their CD56 bright Maintain the condition. CD56 dim CD56 to become bright Downregulation of CD56 expression, and expression of the immunoglobulin superfamily member CD16 (FcγRIII) on a subset of NK cells, defines stage 5. The expression level of CD56 provides a functional classification of human NK cells. Most human NK cells in peripheral blood express CD56 dim CD56 bright NK cells are considered less mature and are present mainly in the SLT, whereas CD56 dim This subset represents the majority of circulating NK cells. The majority of immature NK cells (iNK) express high amounts of CD56 dim (>90%) low-converting CD56 population bright CD56 population (approximately 5%). bright While NK cells are potent producers of proinflammatory cytokines, the cytolytic function of human NK cells is primarily mediated by CD56 dim Because CD56 is present in the NK cell population, downregulation of CD56 during human NK cell maturation is strongly associated with the acquisition of antitumor cytotoxicity. dim Terminal maturation of NK cells (stage 6) is defined by the expression of CD57 (HNK-1, Leu-7). Additional subtypes, such as "antigen-experienced" or "adaptive" CD2+ NK cells, are defined by higher expression of NKG2C (KLRC2, CD159c).

[0079] Thus, in some embodiments, generating natural killer (NK) cells from pluripotent stem cells (e.g., iPSCs) can include (i) preparing an HSC population comprising CD34+ cells as described; (ii) culturing the HSC population under conditions sufficient to differentiate the cells into NK cells, which can optionally be a culture medium comprising factors such as FGF2, VEGF, TPO, SCF, IL-3, and FLT3L; and (iii) during differentiation, factors such as IL-7 and IL-15 can be included in the culture medium. NK cells can be identified by NK cell markers such as CD3-CD56, and based on their level of differentiation, can be identified by CD56. bright NK cells or CD56 dim They can be further characterized as NK cells.

[0080] In some embodiments, HSC populations or fractions thereof are differentiated into NK cells or their precursors or derivatives, with or without the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1. In some embodiments, the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1, is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population to prepare a CD34+ enriched population. Endothelial-hematopoietic transformation of the CD34+ enriched cell population is induced for at least 2 days but not more than 12 days, optionally with the use of agonists of mechanosensitive receptors or mechanosensitive channels, such as Yoda1, jedi1, jedi2, or ssRNA40. HSCs and / or HSPCs are differentiated into progenitor NK cell populations or NK cell populations.

[0081] In some embodiments, endothelial-to-hematopoietic transformation of the CD34+ enriched cell population is induced over a period of at least 2 days, further for 4 hours, or 8 hours, or 12 hours, or 16 hours, or 20 hours, or 24 hours, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or 10 days, or 11 days, or 12 days, but not more than 12 days.

[0082] In some embodiments, the NK cell lines generated in accordance with the present disclosure are predominantly (e.g., at least about 50% or at least about 75%) CD56 bright In some embodiments, the NK cell lineages generated in accordance with the present disclosure are predominantly (e.g., at least about 50% or at least about 75%) CD56 dim In some embodiments, the NK cells are cytotoxic innate lymphoid natural killer (NK) cells. Upon activation, cytolytic NK cells share similar effector functions with T cells, including the production of cytotoxic granules and proinflammatory cytokines. NK cells can also shape the adaptive immune system by influencing T cells at different stages of their lifespan. For example, during T cell priming, NK cells indirectly modify T cell responses by influencing dendritic cells (DCs).

[0083] In some embodiments, NK cells produced according to the present disclosure secrete cytokines at levels comparable to endogenous NK cells, including, but not limited to, IFN-γ, TNF-α, GM-CSF, IL-10, IL-5, and IL-13, as well as chemokines such as MIP-1α, MIP-1β, IL-8, and RANTES. As an example, IFN-γ secreted by NK cells is a potent effector cytokine that plays an important role in antiviral, antibacterial, and antitumor responses.

[0084] In some embodiments, NK cells prepared according to the present disclosure secrete chemokines, such as, but not limited to, XCL1, CCL2, CCL3, CCL4, CCL5, CCL22, CXCL8, MIP-1α, MIP-1β, IL-8, and RANTES, or any combination thereof. Such chemokines secreted by NK cells can recruit other effector cells during an immune response. Cytokines and chemokines can be routinely measured by known methods, such as quantitative polymerase chain reaction (q-PCR), enzyme-linked immunosorbent assay (ELISA), or flow cytometry analysis.

[0085] During differentiation, the HSC population and its progeny differentiate into CD7 + To differentiate into precursor T cells and then into the NK cell lineage, the cells are cultured ex vivo with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or a combination thereof. Furthermore, according to known processes, xenogeneic OP9-DL1 or irradiated K562-mbIL21-41BBL cells can be employed for hematopoietic differentiation into T cells and NK cells. The OP9-DL1 coculture system uses a bone marrow stromal cell line (OP9) transduced with the Notch ligand Delta-like-1 (DLL1) or DLL-4 to support T cell development from a stem cell source. The OP9-DL1 system limits the potential of the cells for clinical use. There is a need for a feeder-free system capable of generating cells such as NK cells from hiPSCs for clinical use, and in some embodiments, the present invention fulfills this goal. In a non-limiting example, to generate mature NK cells using a Notch ligand, iPSC expansion is performed for 6 days, followed by embryoid body formation, which takes approximately 8 days. The cells are further cultured for approximately 5 days to allow the development of CD34+ hemogenic endothelial cells from which the HSCs were derived. The HSCs are then cultured in T cell or NK cell specific medium supplemented with retronectin and DLL-4 for the generation of T cell progenitors, identified as CD34+CD7+CD5+ / -. The T cell progenitors can then be differentiated to generate NK cells.

[0086] The term "Notch ligand" as used herein refers to a ligand capable of binding to a Notch receptor polypeptide present on the membrane of a hematopoietic stem cell or progenitor T cell. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate, and L-Lag2) containing 20-22 amino acids at the amino terminus and 3-8 EGF repeats on the extracellular surface. In various embodiments, Notch is a ligand of Delta-like-1 (DLL1), Delta-like-4 (DLL4), SFIP3, Delta-like-5 (DLL5), SFIP4, SFIP5, SFIP6, SFIP7, SFIP8, SFIP9, SFIP10, SFIP11, SFIP12, SFIP13, SFIP14, SFIP15, SFIP16, SFIP17, SFIP18, SFIP19, SFIP20, SFIP21, SFIP22, SFIP23, SFIP24, SFIP25, SFIP26, SFIP27, SFIP28, SFIP29, SFIP30, SFIP31, SFIP32, SFIP33, SFIP34, SFIP35, SFIP36, SFIP37, SFIP38, SFIP39 ...Max (disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are incorporated by reference in their entireties), or a functional portion thereof, including at least one of Jagged1 (JAG1), Jagged2 (JAG2), Delta-like ligand 3 (DLL3), and X-delta 2. A key signal delivered by thymic stromal cells to invading lymphoid progenitors in vivo is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0087] As used herein, "Notch ligand" also includes intact (full-length), partial (truncated), or modified (containing one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands or fragments thereof from any species that retain at least one activity or function of the full-length Notch ligand. Also included are peptides that mimic Notch ligands. Notch ligands can be "canonical Notch ligands" or "non-canonical Notch ligands." Canonical Notch ligands are typically characterized by an N-terminal (NT) domain followed by a Delta / Serrate / LAG-2 (DSL) domain and an extracellular domain containing multiple tandemly arranged epidermal growth factor (EGF)-like repeats. The DSL domain, along with the adjacent NT domain and the first two EGF repeats containing Delta and OSM-11-like (DOS) motifs, are typically required for canonical ligands to bind to Notch. The intracellular domains of some canonical ligands contain a carboxy-terminal PSD-95 / Dlg / ZO-1-ligand (PDZL) motif, which plays a role independent of Notch signaling. The C. elegans DSL ligand lacks the DOS motif but has been proposed to cooperate with ligands containing only DOS to activate Notch signaling.

[0088] In various embodiments, the HSC / HSPC population is cultured in artificial thymic organoids (ATO). See Hagen, M. et al. (2019). ATO will involve culturing HSCs (or aggregates of HSCs) with a Notch ligand-expressing stromal cell line under serum-free conditions. Artificial thymic organoids are composed of naive CD3 + CD8 + and CD3 + CD4 + In some embodiments, the artificial thymus organoid comprises DLL4 and BMP2, or functional fragments thereof.

[0089] In some embodiments, progenitor NK cells or T cells are isolated by enrichment for CD7 expression. In some embodiments, progenitor T cells are expanded as described in US2020 / 0308540, which is incorporated herein by reference in its entirety. For example, cells can be expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55. In some embodiments, the proliferation-promoting compound comprises a pyrimidoindole derivative, including, for example, UM171 or UM729 (see US2020 / 0308540, which is incorporated herein by reference).

[0090] The differentiation of precursor NK cells or T cells into progenitor cells capable of generating NK cells can, in some embodiments, further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. In various embodiments, the generated CD7+ precursor T cells express CD1a. The CD7+ precursor NK cells or T cells do not express CD34 or express reduced levels of CD34 compared to the HSC population. In some embodiments, the CD7+ precursor T cells (or a portion thereof) further express CD5. Thus, the phenotype of the precursor T cells is CD7. + CD1a +In some embodiments, the phenotype of the precursor T cells may be CD7 + CD5 + In some embodiments, the progenitor T cells are CD7 + CD1a + CD5 + , optionally CD34 + In some embodiments, the progenitor T cells exhibit reduced levels of CD34 expression (compared to the HSC population), minimal CD34 expression, or no CD34 expression. In some embodiments, CD34 expression is reduced by at least about 50% or at least about 75% in the population relative to the HSC population.

[0091] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind to and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including a human or humanized antibody), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule that can activate the Notch signaling pathway.

[0092] In some embodiments, the Notch ligand is a Delta family Notch ligand. In some embodiments, the Delta family ligand is Delta-1 (Genbank Accession No. AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank Accession Nos. NM_005618 and NP_005609, Homo sapiens; Genbank Accession Nos. X80903, 148324, M. musculus), Delta-4 (Genbank Accession No. AF273454, BAB18580, Mus musculus; Genbank Accession Nos. AF279305, AAF81912, Homo sapiens), and / or Delta-like 4 (DLL4, Genbank Accession Nos. Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank Accession No. NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0093] In some embodiments, the Notch ligand is DLL4 with one or more affinity-enhancing mutations, such as one or more (or all) of the following: for hDLL4, G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling, Nature Chemical Biology (2022).

[0094] In some embodiments, the Notch ligand comprises an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical (e.g., about 100% identical) to a human DLL1 or DLL4 Notch ligand. A functional derivative of a Notch ligand (including a fragment or portion thereof) will be capable of binding to and activating a Notch receptor. Binding to a Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0095] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles that are optionally paramagnetic to enable magnetic enrichment or concentration processes. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally using other adhesion molecules such as VCAM-1. See US 2020 / 0399599, incorporated herein by reference in its entirety. In other embodiments, the beads or particles are constructed of biological materials, such as polymers (e.g., polystyrene or PLGA), gold, iron dextran, or particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or largest dimension of about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In still other embodiments, polymer scaffolds with conjugated ligands can be employed, as described in WO 2020 / 131582, incorporated herein by reference in its entirety. For example, the scaffold can be constructed from polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters of about 1 pm to 100 pm.

[0096] In some embodiments, the C-terminus of the Notch ligand is conjugated to a support of choice. In some embodiments, this can involve adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, the Notch ligand-Fc fusion is prepared such that the Fc segment can be immobilized by binding to Protein A or Protein G that has been conjugated to a support. Of course, any of the known protein conjugation methods can be employed.

[0097] Thus, in various embodiments, the Notch ligand can be immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated with components of an extracellular matrix, such as one or more selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand and / or components of the extracellular matrix are embedded in an inert material that provides 3D culture conditions. Exemplary materials include, but are not limited to, cellulose, alginate, and combinations thereof. In some embodiments, the Notch ligand, components of the extracellular matrix, or combinations thereof, are in contact with culture conditions that provide cells with a topographical pattern and / or texture (e.g., roughness) that promotes differentiation and / or proliferation.

[0098] In some embodiments, HSCs are differentiated into precursor T cells by culturing them in medium containing TNF-α and / or an antagonist of the aryl hydrocarbon / dioxin receptor (SR1) and in the presence of a Notch ligand. See US2020 / 0390817, US2021 / 0169934, and US2021 / 0169935, which are incorporated by reference in their entireties. In some embodiments, HSCs are cultured in medium containing TNF-α, IL-7, thrombopoietin (TPO), Flt3L, and stem cell factor (SCF), and optionally SR1, in the presence of immobilized delta-like-4 ligand and fibronectin fragments. In some embodiments, cells are cultured with retronectin, a recombinant human fibronectin containing three functional domains: a human fibronectin cell-binding domain (C domain), a heparin-binding domain (H domain), and a CS-1 sequence domain. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand, TNF-α, and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-1 ligand and retronectin. In some embodiments, cells are cultured in the presence of SFIP3 and retronectin. In some embodiments, cells are cultured in the presence of immobilized delta-like-4 ligand and an SHH molecule and / or a functional derivative thereof. Exemplary fibronectin fragments include one or more of RGDS, CS-1, and heparin-binding motifs. Fibronectin fragments can be free in solution or immobilized on a culture surface or particle. In some embodiments, cells are cultured for 5-7 days to prepare CD7+ precursor T cells, which can then be used to generate NK cells from T cell precursors that retain the ability to generate NK cells.

[0099] In some embodiments, the presence of cytokines and / or growth factors is desired for differentiation of HSCs or cells isolated therefrom, and these can be selected from or comprise (or consist essentially of): TPO, SCF, Flt3L, IL3, IL7, and SDF-1a. Furthermore, T cells or progenitor T cells resulting in the culture are further cultured in the presence of, for example, IL-3 and / or IL-15. IL-15 is added to support NK cell differentiation, and IL-3 is optionally omitted after the formation of initial NK cells. In some embodiments, the medium does not contain one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor to initiate differentiation of NK cell precursors or pre-NK cell precursors into NK cells. In some embodiments, NK precursor cells derived from pluripotent stem cells are CD3-CD45+CD56+CD7+. In some embodiments, pluripotent stem cell-derived NK cells are CD3-CD45+CD56+, and optionally further defined by NKp46+, CD57+, and CD16+.

[0100] In some embodiments, NK cells are generated from precursor T cells as described in US 10,266,805, which is incorporated herein by reference in its entirety. For example, precursor T cells can give rise to NK cells when cultured with IL-15. In some embodiments, NK cells express a CAR based on gene editing of iPSCs, embryonic bodies, hCD34+ cells, or NK cells, or via mRNA expression in NK cells. Additionally, or optionally, NK CARs can be engineered to express cytokines (e.g., IL-15) to make the NK-CAR more potent in targeting tumors.

[0101] In non-limiting examples, NK cells can be efficiently transduced with vectors such as, but not limited to, retroviral or non-integrating viral vectors (e.g., adenovirus, adeno-associated virus, integration-deficient retrolentivirus, poxvirus), or non-viral vectors (e.g., plasmid vectors, artificial chromosomes), or episomal or episomal hybrid vectors carrying second-generation CAR-targeting tumor antigens (e.g., CD19, CD38, CD33, CD47, CD20, etc.). CAR expression can be demonstrated across different NK cell subsets according to routine protocols. NK CAR cells (e.g., CAR.CD19-NK cells, CAR.CD38-NK cells, CAR.CD33-NK cells, CAR.CD47-NK cells, CAR.CD20-NK cells, etc.) may exhibit greater anti-tumor activity (e.g., anti-leukemic activity) against CD19, CD38, CD33, CD47, CD20 cell lines and primary blasts obtained from patients with, for example, B-cell precursor ALL, compared to unmodified NK cells.

[0102] CARs are designed to enhance the ability of cells to recognize, bind to, and kill tumor cells. In some embodiments, a CAR enhances the ability of NK cells to recognize tumor cells. In some embodiments, a CAR enhances the anti-tumor activity of NK cells. In some embodiments, the CAR is a G-protein coupled receptor 87 (GPR87) CAR, solute carrier family 7 member 11 (SLC7A11(xCT)) CAR, TNF receptor superfamily member 17 (BCMA) CAR, CD30 CAR, CD19 CAR, CD22-CAR, CD33 CAR, CD133-CAR, NKG2D CAR (or a CAR or receptor containing an NKG2D ectodomain), mesothelin-CAR, CD70 CAR, NKp30 CAR, CD73 CAR, or CAR-NK cell that targets the following tumors or tumor antigens: (i) human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma; (ii) epidermal growth factor receptor (EGFR) - non-small cell lung cancer, epithelial carcinoma, and glioma; (iii) mesothelin-mesothelioma, ovarian cancer, and pancreatic adenocarcinoma; (iv) prostate-specific membrane antigen (PSMA)-prostate cancer; (v) Carcinoembryonic antigen (CEA) - pancreatic adenocarcinoma, breast cancer, and colorectal cancer; (vi) glypican-3-hepatocellular carcinoma; (vii) epidermal growth factor receptor variant III (EGFRvIII)-glioblastoma; (viii) Disialoganglioside 2 (GD2)—neuroblastoma and melanoma; (ix) Carbonic anhydrase IX (CAIX) - Renal cell carcinoma; (x) Interleukin-13Ra2-glioma, (xi) fibroblast activation protein (FAP) - malignant pleural mesothelioma; (xii) L1 cell adhesion molecule (L1-CAM)—neuroblastoma, melanoma, and ovarian; (xiii) Cancer antigen 125 (CA125)-epithelial ovarian cancer, (xiv) Cluster of differentiation antigen 133 (CD133) - glioblastoma, cholangiocarcinoma, adenocarcinoma, (xv) Cancer / Testis Antigen 1B (CTAG1B) - melanoma and ovarian cancer; (xvi) Mucin 1 - seminal vesicle cancer, (xvii) folate receptor-a (FR-a)-ovarian cancer; (xviii) a growth factor receptor selected from one or more of ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β), or FGFR1-4.

[0103] The cell population or cells derived therefrom (e.g., progeny) can be used with FDA-approved CAR-T therapies such as tisagenlecleucel, also known as tisa-cel (Kymriah), axi-cel (Yescarta), axi-cel (Yescarta), brexucabutagen autorucel, also known as brexu-cel (Tecartus), lisocabtagenemaraleucel, also known as liso-cel (Breyanzi), idecbutagenbicelucel, also known as ide-cel (Abecma), cilta-cel (Carvykti), or any other CAR-based therapy that damages normal cells during their therapeutic use.

[0104] Thus, in some aspects and embodiments of the invention, genetically modified NK cell lines, or their progenitors or progeny, are engineered to express a chimeric antigen receptor (CAR) on the cell surface, particularly a CAR that specifically binds to a growth factor receptor. Most typically, the CAR comprises the intracellular domain from Fcε receptor γ (FcεRIγ). However, in further contemplated embodiments, the CAR may also comprise the T cell receptor (TCR) CD3ζ (CD3ζ) intracellular domain, alone or in combination with additional components from second- or third-generation CAR constructs (e.g., CD28, CD134, CD137, and / or ICOS).

[0105] In one embodiment, the NK cells or their precursors or progeny are modified to express an autocrine growth-stimulating cytokine or a variant thereof. Additionally, or optionally, the genetically modified NK cells also express recombinant CD16 or a high-affinity variant thereof, conferring target-specific ADCC to the cells.

[0106] In some embodiments, NK cells or their progenitors or progeny are modified to express a homing receptor. A "homing receptor" refers to a receptor that directly or indirectly activates a cellular pathway that directs cells to migrate toward a target cell or tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissues via high endothelial venules. Homing receptors can also be used by cells to migrate toward the source of a chemical gradient, such as a chemokine gradient. Examples of homing receptors include G protein-coupled receptors such as chemokine receptors, including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, and DARC; cytokine receptors; and cell adhesion molecules such as selectins, including L-selectin (CD62L), and integrins, such as α4β7 integrin, LPAM-1, and LFA-1. Homing receptors generally bind to cognate ligands on target tissues or cells. In some embodiments, the homing receptor binds to an addressin on the endothelium of venules, such as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1).

[0107] In another aspect, the present invention provides a cell population or a pharmaceutically acceptable composition thereof produced by the methods described herein. In some embodiments, the cell population is a progenitor NK cell population capable of engraftment in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need thereof. In various embodiments, a composition for cell therapy is prepared comprising the cell population and a pharmaceutically acceptable vehicle. The pharmaceutical composition is at least about 10 2 cells, or at least about 10 3 or at least about 10 4 or at least about 10 5 or at least about 10 6 or at least about 10 7 or at least about 10 8cells, or at least about 10 9 cells, or at least about 10 10 cells, or at least about 10 11 cells, or at least about 10 12 cells, or at least about 10 13 cells, or at least about 10 14 For example, in some embodiments, pharmaceutical compositions containing about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg) are administered. In other embodiments, the cells are administered at a concentration of about 10 per kilogram of recipient body weight. 5 ~Approx. 5×10 5 cells (e.g., approximately 2.5 x 10 5 cells / kg), or approximately 10 per kilogram 6 ~Approx. 5×10 6 cells (e.g., approximately 2.5 x 10 6 cells / kg), or approximately 5 x 10 cells per kilogram 6 ~about 10 7 cells (e.g., approximately 5 x 10 6 cells / kg), or approximately 10 per kilogram 7 ~about 10 8 cells (e.g., approximately 5 x 10 7 cells / kg), or approximately 10 per kilogram 8 ~about 10 9 cells (e.g., approximately 5 x 10 8 cells / kg), or approximately 10 per kilogram 9 ~about 10 10 cells, or approximately 10 10 ~about 10 11 cells, or approximately 10 per kilogram 11 ~about 10 12 cells, or approximately 10 per kilogram 12 ~about 10 13 cells, or approximately 10 per kilogram 13 ~about 10 14 It is administered in individual cells.

[0108] In some embodiments, the NK cells are derived from the HLA-edited iPSCs described. For example, in some embodiments, the NK cells are derived from HLA-A neg , homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg In some embodiments, the NK cells are further homozygous for HLA-DRB1.

[0109] The cell compositions of the present disclosure (e.g., prepared according to the present disclosure) may further comprise a pharmaceutically acceptable excipient or carrier. Such excipient or carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer refers to a solution or liquid whose chemical composition neutralizes acids or bases without a significant change in pH. Examples of buffers contemplated by the present invention include, but are not limited to, physiological saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate-buffered saline (PBS), and Ringer's solution. The compositions may include a vehicle suitable for intravenous infusion or other administration routes, and the compositions may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). Other carriers may include dimethoxyethane (DME), N,N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell composition may be provided in an appropriate volume within an implantable device (eg, a scaffold), or within a bag, or within a vial, tube, or container, and stored frozen until use.

[0110] Pharmaceutical compositions for use in the disclosed methods may also contain additional therapeutic agents for the treatment of specific target disorders. For example, pharmaceutical compositions may also include cytokines and growth factors (e.g., interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical compositions to provide the benefits of the therapeutic approaches disclosed herein, i.e., improved therapeutic efficacy with reduced systemic toxicity.

[0111] The NK cells or CAR-NK cells can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease or disorder being treated, the particular mammal (e.g., human) being treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to physicians. The therapeutically effective amount of cells administered will be governed by such considerations.

[0112] The formulations herein may also contain more than one active agent as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide a cytotoxic agent, cytokine, or immunosuppressant. The effective amount of such other agents will depend on the amount of acceptable carriers, excipients, or stabilizers present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and by the same route of administration as used herein above, or at about 1-99% of the dosages previously employed.

[0113] In other aspects, the present disclosure provides methods for cell therapy comprising administering a cell population described herein or a pharmaceutically acceptable composition thereof to a human subject in need thereof. In various embodiments, the subject has cancer or an infectious disease, such as a viral infection. In various embodiments, the methods described herein are used to treat hematological (malignant and non-malignant), bone marrow, immune disorders, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer, an immunodeficiency, or an autoimmune disease.

[0114] Exemplary diseases include various autoimmune diseases, including, but not limited to, alopecia, 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, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's granulomatosis). Hematologic malignancies that can be treated include, but are not limited to, acute and chronic leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome. Infectious diseases that can be treated include, but are not limited to, HIV (human immunodeficiency virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), adenovirus, and BK polyomavirus-associated disorders. Other conditions include anemia, bone marrow failure syndromes, including, but not limited to, skeletal dysplasia, hemoglobinopathies, iron deficiency anemia, pernicious anemia, aplastic anemia, sickle cell anemia, vitamin deficiency anemia, and hemolytic anemia, as well as certain genetic disorders (e.g., those affecting the immune system).

[0115] In some embodiments, the subject has cancer, such as a hematological malignancy, including but not limited to, leukemia, lymphoma, and multiple myeloma, or a solid tumor, including but not limited to, a tumor of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testicle, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus.

[0116] In some embodiments, the subject has a condition selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistopathy, inborn errors of metabolism, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency.

[0117] For the use of HLA-edited NK cells (e.g., derived from gene-edited iPSCs), subjects can be matched at retained HLA loci, such as one or more (or all) of HLA-B, HLA-C, and HLA-DRB1.

[0118] Agents such as hormones, growth factors, and cytokine antibodies that may be co-administered with derivatives of the cell line(s) of the invention or banks of expanded primary cells (e.g., iPSC-derived HSCs or their progenitors or progeny) include molecules such as renin, growth hormones including human growth hormone and bovine growth hormone, growth hormone-releasing factor, parathyroid hormone, thyroid-stimulating hormone, lipoproteins, alpha-1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle-stimulating hormone, calcitonin, luteinizing hormone, glucagon, VMC factor, factor I, Coagulation factors such as factor X, tissue factor (TF), and von Willebrand factor, anticoagulants such as protein C, atrial natriuretic factor, pulmonary surfactant, plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factors, tumor necrosis factor-α and -β, enkephalinase, RANTES (regulated by activation and expressed and secreted by normal T cells), human macrophage inflammatory protein (MIP-1-α), serum albumins such as human serum albumin, murine leukemia virus (VEV) and leukemia virus (LEV). Laryngeal duct inhibitory substance, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, microbial proteins such as β-lactamase, DNase, IgE, cytotoxic T lymphocyte-associated antigen (CTLA) such as CTLA-4, inhibin, activin, vascular endothelial growth factor (VEGF), hormone or growth factor receptors, protein A or D, rheumatoid factor, neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or NGF-β nerve growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, fibroblast growth factor receptor 2 (FGFR2), transforming growth factors (TGFs) such as epidermal growth factor (EGF), TGF-α, and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, bone morphogenetic proteins (BMPs), including BMP1, BMP6, BMP7, and BMP receptor 2, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I),Insulin-like growth factor binding protein, hepatocyte growth factor (HGF), EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLR1, mesothelin, cripto, alphavbeta6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD CD proteins such as CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, TNFα, IFNα, GM-CSF, IL-3, or antibodies that bind to one or more tumor-associated antigens or cell surface receptors, erythropoietin, bone morphogenetic factors, immunotoxins, bone morphogenetic proteins (BMPs), interferons such as interferon-α, -β, and -γ, colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF, interleukins (ILs) such as IL-2, IL-6, IL-12, IL-23, IL-12 / 23 p40, IL-17, IL-15, IL-21, IL-1a, IL-1b, IL-18, IL-8, IL-4, IL-3, and IL-5, superoxide dismutase, T cell receptors, surface membrane proteins, degradation-promoting factors, viral antigens such as portions of the HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM, tumor-associated antigens such as HER2, HER3, or HER4 receptors, endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, LGR5, B7H4, TAG72 (tumor-associated glycoprotein 72), and fragments of any of the above-listed polypeptides.

[0119] Examples of antibodies or fragments thereof that may be administered include anti-PD-L1 antibodies, abciximab (Reopro), adalimumab (Humira, Amjevita), alefacept (Amevive), alemtuzumab (Campath), basiliximab (Simulect), belimumab (Benlysta), bezlotoximab (Zinplava), canakinumab (Ilaris), certolizumab pegol (Cimzia), cetuximab (Erbitux), daclizumab (Zenapax, Zinbryta), denosumab (Prolia, Xgeva), efalizumab (Raptiva), golimumab (Simponi, Simponi), and others. Aria), Inflectra (Remicade), ipilimumab (Yervoy), ixekizumab (Taltz), natalizumab (Tysabri), nivolumab (Opdivo), olaratumab (Lartruvo), omalizumab (Xolair), palivizumab (Synagis), panitumumab (Vectibix), pembrolizumab (Keytruda), and rituximab (Rituxan), tocilizumab (Actemra), trastuzumab (Herceptin), secukinumab (Cosentyx), ranibizumab, abciximab, raxivacumab, caplacizumab, infliximab, bevacizumab, dabigatran, idarucizumab, or ustekinumab (Stelara), or a combination thereof.Further, the antibodies include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-EGFR antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis antibodies, X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD1 antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody , anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-C The antibody may be selected from D41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody.

[0120] Concomitant administration does not require that the therapeutic agents be administered simultaneously, provided that the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the active ingredient(s) in the pharmaceutical composition overlap in time, thereby exerting a combined therapeutic effect. In general, each agent will be administered at a dose, and on a time and schedule determined for that agent.

[0121] The pharmaceutical composition may be administered at any dosage appropriate to achieve the desired result. In some embodiments, the desired outcome is a reduction in the intensity, severity, frequency, and / or delay in the onset of one or more symptoms of an infection. In some embodiments, the desired outcome is inhibition or prevention of an infection. The required dosage will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the infection being prevented or treated, the particular composition being used, and its mode of administration.

[0122] In some embodiments, the pharmaceutical compositions according to the present disclosure are administered in single or multiple doses, hi some embodiments, the pharmaceutical compositions are administered in multiple doses administered on different days.

[0123] As used herein, the term "about" means ±10% of the associated numerical value.

[0124] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. [Example]

[0125] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation, but does not affect pluripotency. method iPSCs were developed from hCD34+ cells by episomal reprogramming, as known in the art and essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid bodies and hemogenic endothelial differentiation were performed essentially as described in R. Sugimura, et al., Hematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0126] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low-attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. Cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0127] result Induced pluripotent stem cells (iPSCs) were transduced using an adenoviral vector containing both ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot demonstrating the transduction efficiency of iPSCs using an adenoviral vector for overexpressing ETV2 and GFP sequences.

[0128] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and then into hemogenic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 significantly increased the expression of CD235a. - CD34 in the population + and CD31 +These results suggest that ETV2-OE enhances the formation of hemogenic endothelial cells, as evidenced by the expression of markers (Figure 2). Specifically, Figure 2 shows representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), demonstrating that ETV2-OE enhances the formation of hemogenic endothelial cells compared to controls.

[0129] Furthermore, the results showed that ETV2-OE expresses CD34 + This suggests that ETV2-OE enhances CD34+ cell formation (Figure 3). Figure 3 shows representative flow cytometry analysis and relative quantification of CD34+ cells demonstrating that ETV2-OE enhances CD34+ cell formation.

[0130] Overall, these data indicate that ETV2 overexpression in iPSCs does not affect their pluripotent properties, but promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation.

[0131] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs. method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells had adhered to the bottom of the well (by visual inspection) for approximately 4-18 hours, Yoda1 was added to the cultures for some experiments. After 4-7 days, cells were collected for analysis.

[0132] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells from the iPSC-derived embryoid bodies were harvested and cultured for an additional 5–7 days to induce endothelial-hematopoietic (EHT) transition. CD34+ cells were then harvested from the EHT cultures on days 5–7 for further hematopoietic lineage differentiation.

[0133] CD34+ cells harvested from EHT cultures on days 5–7 (or days 13–21 total of differentiation from iPSCs) were seeded onto 48-well plates pre-coated with rhDL4 and retronectin. T lineage differentiation was induced in medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1α, and SB203580.

[0134] 80% of the medium was changed every other day from days 2 to 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of pre-T cells (CD34+CD7+CD5+ / -).

[0135] 80% of the medium was changed every other day from days 8 to 13. On day 14, 100,000 cells / well were transferred to new coated plates, and cells were analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0136] 80% of the medium was changed every other day from days 15 to 20. Cells were harvested on day 21 and analyzed via FACS for CD3, CD8, CD5, CD7, TCRab expression on behalf of T cells, and / or activated using CD3 / CD28 beads to assess their functional properties.

[0137] After 21 days of differentiation, cells were harvested and approximately 80,000 cells were replated into new 96-well culture plates in RPMI 1640 (without L-glutamine, without phenol red) with FBS, L-glutamine, and IL-2, and then activated with 1:1 CD3 / CD28 beads. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for CD3, CD69, and CD25 expression by FACS and for IFN-γ expression using RT-qPCR. Supernatants were analyzed by ELISA.

[0138] result Figures 4A and 4B show that iPSC-derived HSCs derived by Piezo1 activation undergo pre-T cell differentiation, similar to bone marrow (BM)-HSCs. Furthermore, Figures 5A and 5B show that iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation, similar to BM-HSCs, and can be activated with CD3 / CD28 beads. Figure 6 shows that iPSC-derived HSCs generated by Piezo1 activation can differentiate into functional T cells, as demonstrated by INFγ expression upon stimulation with CD3 / CD28 beads. Together, these results demonstrate that Piezo1 activation during HSC formation enhances the HSC capacity to further differentiate ex vivo into progenitor and functional T cells, as well as related lineages such as NK cells.

[0139] Figure 7 shows that HSCs derived from iPSC differentiation (D8+7) differentiate into NK cells and are activated by pharmacological stimulation. Figure 7 shows that HSCs derived from iPSC differentiation (D8+7 with Yoda1 or "Y") are pharmacologically activated (as measured by activity of the functional marker CD107a).

[0140] Figures 8A and 8B show that HSC-derived NK cells (D8+7) outperform bone marrow-derived NK cells in killing tumor cells. Figures 8A and 8B show superior tumor cell killing by HSC-derived NK cells compared to BM-derived NK cells. The NK cell to tumor cell ratio is 5:1.

[0141] Example 3 - Evaluation of off-target editing in HLA knockout HSCs HLA typing of HLA-edited HSC clones was performed to check for unwanted editing and ensure that major editing events, such as deletion(s), did not occur within other regions of chromosome 6. Sequencing methods and analyses were performed to assess the extent of gRNA off-target activity and select gRNAs that represent a low risk of affecting non-target HLA genes.

[0142] Sequencing was performed using in situ cleavage labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to end-prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequent DNA sequencing of the DNA library enriched for the DNA-labeled fragments, eliminating all irrelevant non-functional DNA. Because the library preparation was PCR-free, each resulting sequencing read was equivalent to a single labeled DSB end from the cell. This generated a DNA cleavage readout, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction, and allowed for the unambiguous mapping of off-target mutations.

[0143] Table 1 below summarizes the results of the editing strategy in two representative HLA-edited clones relative to wild-type cells (gHSC). [Table 1]

[0144] Table 2 provides non-limiting examples of gRNAs used in experiments that can be used to knock out expression of the indicated HLA genes. [Table 2]

[0145] The results show that the editing strategy was successful in selectively targeting the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0146] These results were confirmed by phenotypic analysis of the HLA-edited clones by FACS and immunofluorescence. As shown in Figures 9A and 9B, the HLA-edited cells tested positive for overall expression of HLA class I molecules, comparable to that of wild-type cells. Specific expression of HLA-A via immunofluorescence confirmed that HLA-A was not expressed in the HLA-edited cells, supporting the finding that the gene editing strategy successfully deleted only the HLA-A gene. Specifically, Figure 9A shows that all HLA-edited cells were positive for HLA-like class I molecules to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that despite the deletion of HLA-A, other class I molecules, such as HLA-B and HLA-C, were expressed and were not affected by the gene editing strategy.

[0147] To confirm that the HLA-A gene had been deleted, the specific expression of HLA-A was analyzed by immunofluorescence. As can be seen in Figure 9B, HLA-A was not expressed in the HLA-edited clones, indicating that the gene editing strategy was effective in specifically deleting only the HLA-A gene. Such preservation of overall class I expression with deletion of HLA-A would facilitate patient matching while avoiding NK cell-mediated rejection.

[0148] Example 4 - Evaluation of pluripotency and immune compatibility of HLA-edited HSCs The ability of HLA-edited cells to preserve pluripotency was assessed. As shown in Figure 11, immunofluorescence evaluation of HLA-edited iPSC clones showed that they maintained tri-lineage differentiation, with ectoderm differentiation indicated by NESTIN-488 and PAX6-594 staining, mesoderm differentiation indicated by GATA-488 staining, and endoderm differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0149] HLA class I molecules are expressed on the surface of all nucleated cells, and if HLA class I molecules are mismatched between donor and recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatches can lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). Conversely, complete deletion of HLA-I molecules via B2M KO would render the cells targets for NK cell-mediated cytotoxicity. Preservation of overall class I expression with deletion of HLA-A could facilitate patient matching while avoiding NK cell-mediated rejection. Therefore, the immune compatibility of HLA-edited HSCs was tested by coculture with peripheral blood mononuclear cells (PBMCs) to assess whether immune cells would reject grafts of HLA-edited and wild-type HSCs (gHSCs).

[0150] Wild-type (gHSC) and HLA-edited HSCs were cocultured with PBMCs bearing HLA-B and HLA-C markers matched but mismatched HLA-A. B2M KO HSCs, which lack expression of HLA class I molecules, and CIITA KO HSCs, which lack expression of class II molecules, were used as controls to compare the degree of PBMC-mediated cytotoxicity for HLA-null and HLA-mismatched HSCs, respectively. Figure 12 shows the results of a PBMC-mediated cytotoxicity assay in the coculture, measured by Annexin V staining. The results show that deletion of HLA-A in HLA-edited HSCs protected the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO HSCs were susceptible to PBMC-mediated cytotoxicity. HSCs cocultured with sorted CD8+ T cells from the same PBMC donor protected HLA-edited and B2M KO HSCs from CD8+ T cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protected only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0151] In summary, the immunocompatibility results indicate that CD8+ T cells present in the PBMC samples were involved in killing cells bearing mismatched HLA molecules (WT and CIITA KO), while NK cells present in the PBMCs were involved in killing HLA-null cells (B2M KO). However, HLA-edited HSCs were protected from CD8+ T cell-mediated cytotoxicity (because mismatched HLA-A had been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression was largely preserved).

[0152] Example 5 - Evaluation of in vivo engraftment potential of HLA-edited HSCs To evaluate the engraftment potential of HLA-edited HSCs, the ability of the cells to engraft in vivo was assessed by competitive transplantation against wild-type HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs were mixed and transplanted into mice. Bone marrow (BM) and peripheral blood samples were collected and evaluated by FACS to compare the relative amounts of each cell type present in the samples. As shown in Figure 12, both HLA-edited HSCs and wild-type HSCs contributed to approximately equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to wild-type HSCs in their engraftment and reconstitution potential. Therefore, the properties of wild-type (unedited, parental) HSCs are expected to match those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0153] Example 6 - Evaluation of degranulation and cytotoxicity of immunocompatible HSC-derived NK cells Next, HSCs were evaluated for their ability to differentiate into NK cells that maintained their degranulation and cytotoxicity. As shown in Figure 13, HSCs effectively differentiated into NK cells, as determined by fluorescence-activated cell sorting (FACS) experiments gated based on the expression of the known NK cell surface marker CD56. HSCs also demonstrated the ability to differentiate at least CD34+ BM and iPSC-EB CD34+ cell populations. To measure the ability of HSC-derived NK cells to effectively kill tumor cells, the experimental protocol shown in Figure 14A was performed. HSC-derived NK cells were cocultured for 3.5 hours with K562 HLA-null cells, a human erythroid myeloid blastoid leukemia cell line derived from the pleural effusion of a patient with chronic myeloid leukemia. These cells express ligands for aNKR, and their lack of HLA cell surface expression also contributes to NK cell activation by suppressing negative signaling through iNKR. Thus, these HLA-null cell lines have the potential to induce distinct functional profiles in NK cells and their subsets. After coculture, the degree of NK cell degranulation was measured using FACS and Annexin V staining, as well as a cytotoxicity assay. As shown in Figure 14B, Annexin V staining demonstrated that HSC-derived NK cells exhibited a higher degree of activation from HLA-null K562 cells than from CD34+ BM and iPSC-EB CD34+ cells. This was confirmed by the cytotoxicity assay results, as shown in Figure 14C.

[0154] References 1. Nianias, A. & Themeli, M. Induced Pluripotent Stem Cell (iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep 14, 261-268 (2019). 2.Brauer,P.M.,Singh,J.,Xhiku,S.& Zuniga-Pfluecker,J.C.T Cell Genesis:In Vitro Veritas Est?Trends Immunol 37,889-901(2016). 3.Kennedy,M.et al.T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures.Cell Reports 2,1722-1735(2012). 4.Sturgeon,C.M.,Ditadi,A.,Awong,G.,Kennedy,M.& Keller,G.Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells.Nat Biotechnol 32,554-561(2014). 5.Chang,C.-W.,Lai,Y.-S.,Lamb,L.S.& Townes,T.M.Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells.PLoS One 9,(2014). 6.Nishimura,T.et al.Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation.Cell Stem Cell 12,114-126(2013). 7.Themeli,M.et al.Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.Nat Biotechnol 31,928-933(2013). 8.Vizcardo,R.et al.Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+T Cells.Cell Stem Cell 12,31-36(2013). 9.Montel-Hagen,A.et al.Organoid-induced differentiation of conventional T cells from human pluripotent stem cells.Cell Stem Cell 24,376-389.e8(2019). 10.Guo,R.et al.Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors.Cell Research 30,21-33(2020). 11.Nagano,S.et al.High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells.Molecular Therapy-Methods & Clinical Development 16,126-135(2020). 12.Iriguchi,S.et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.Nature Communications 12,430(2021).

Claims

1. 1. A method for preparing a population of NK cells or their precursors, comprising: enriching CD34+ cells from the differentiated pluripotent stem cell (PSC) population to prepare a CD34+ enriched population; inducing endothelial-to-hematopoietic transformation of the CD34+ enriched cell population for at least 2 days but not more than 12 days to produce a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs); differentiating said population comprising HSCs and / or HSPCs into a progenitor NK cell population or an NK cell population.

2. 2. The method of claim 1, wherein the PSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or human primary tissue.

3. 3. The method of claim 2, wherein the iPSC population is derived from CD34+ cells isolated from peripheral blood.

4. 4. The method of claim 2 or 3, wherein the iPSCs are homozygous for one or more HLA class I and / or HLA class II genes.

5. The method of claim 4, wherein the iPSCs are homozygous for HLA-DRB1.

6. The method of claim 4, wherein the iPSCs are homozygous for both HLA-B and HLA-C.

7. 7. The method of any one of claims 2 to 6, wherein the iPSCs have been gene-edited to delete one or more HLA class I genes, one or more class II genes, and / or one or more genes that govern HLA or MHC expression or presentation capacity.

8. The method of claim 7, wherein the iPSCs comprise a deletion of HLA-A.

9. The method of claim 7 or 8, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

10. The iPSCs are HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , and HLA-DQB1 ｎｅｇ 10. The method of any one of claims 2 to 9, optionally further comprising homozygous for HLA-DRB1.

11. The method according to claim 7, wherein the one or more genes controlling the ability to express or present HLA or MHC are β2-microglobulin and / or CIITA.

12. The method of any one of claims 1 to 11, wherein CD34+ enrichment and endothelial-hematopoietic transition are induced between days 8 and 15 of iPSC differentiation.

13. 13. The method of any one of claims 1-12, wherein the CD34+ enriched population is cultured in medium containing a combination of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

14. 14. The method of claim 13, wherein the endothelial-to-hematopoietic conversion generates a population of HSCs comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem progenitor cells.

15. The method of any one of claims 12 to 14, wherein the CD34+ cells are harvested from a culture undergoing endothelial-hematopoietic transformation, and comprises harvesting of CD34+ floating and / or adherent cells.

16. The method of claim 14 or 15, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSCs).

17. The method of any one of claims 1 to 16, wherein the induction of endothelial-hematopoietic transition comprises increasing the expression or activity of dnmt3b.

18. 18. The method of claim 17, wherein the induction of endothelial-to-hematopoietic transition comprises applying cyclic stretch to the CD34+ cells.

19. 19. The method of claim 18, wherein the cyclic stretching is 2D, 3D, or 4D cyclic stretching.

20. The method according to any one of claims 1 to 17, wherein the induction of endothelial-hematopoietic transition comprises Piezo1 activation.

21. 21. The method of claim 20, wherein the Piezo1 activation is by contacting the CD34+ enriched cells or a fraction thereof with one or more Piezo1 agonists, optionally selected from Yoda1, Jedi1, Jedi2, ssRNA40, or analogs or derivatives thereof.

22. The method of any one of claims 1 to 17, wherein the induction of endothelial-hematopoietic transition comprises Trpv4 activation.

23. 23. The method of claim 22, wherein said Trpv4 activation is by contacting said CD34+ enriched cells with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4α-PDD, or analogs or derivatives thereof.

24. 24. The method of any one of claims 1 to 23, wherein the HSC population or a fraction thereof is cultured with a partial or complete Notch ligand.

25. 25. The method of claim 24, wherein the Notch ligand comprises at least one of DLL1, DLL4, SFIP3, or a functional portion thereof.

26. 26. The method of claim 25, wherein the Notch ligand comprises DLL4 with one or more affinity-enhancing mutations.

27. 27. The method of any one of claims 24 to 26, wherein the Notch ligand is immobilized, functionalized and / or embedded in a 2D or 3D culture system.

28. 28. The method of any one of claims 24 to 27, wherein the Notch ligand is optionally incorporated with a component of the extracellular matrix selected from fibronectin, retronectin, and laminin, derivatives or analogues thereof, and / or combinations thereof.

29. 29. The method of claim 28, wherein the Notch ligand and / or extracellular matrix components are embedded in an inert material that provides 3D culture conditions, optionally selected from cellulose, alginate, and combinations thereof.

30. 30. The method of any one of claims 27 to 29, wherein the Notch ligand, extracellular matrix component, or a combination thereof, is in contact with culture conditions that provide a topographical pattern and / or roughness to cells.

31. 31. The method of any one of claims 24 to 30, wherein the Notch ligand, extracellular matrix component, topographical pattern and / or roughness, or a combination thereof, is optionally cultured with a cytokine and / or growth factor selected from one or more of TNF-α and SHH.

32. The HSC population or a fraction thereof comprises BMP2, Delta-like 1 (DLL1), Delta-like 4 (DLL4), SFIP3, and Delta Ｍａｘ The method of any one of claims 24 to 31, wherein the cells are cultured in an artificial thymus organoid, optionally comprising a Notch ligand selected from one or more of:

33. 33. The method of any one of claims 24 to 32, wherein the HSC population or a fraction thereof is cultured in the presence of one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL7, and SDF-1a.

34. The method of any one of claims 24 to 33, wherein the resulting T cells or precursor T cells are further cultured in the presence of IL-3 and / or IL-15.

35. 35. The method of claim 34, wherein IL-15 is added to support NK cell differentiation, and IL-3 is optionally omitted after formation of early NK cells.

36. 36. The method of any one of claims 1 to 35, wherein the NK cell line expresses a chimeric antigen receptor (CAR).

37. The natural killer cells are primarily CD56 ＤＩＭ The method according to any one of claims 1 to 36, wherein

38. The natural killer cells are primarily CD56 ＢＲＩＧＨＴ The method according to any one of claims 1 to 36, wherein

39. The method of any one of claims 1 to 36, wherein the natural killer cell lineage is an NK cell precursor.

40. 40. An NK cell population produced by the method of any one of claims 1 to 39, or a pharmaceutically acceptable composition thereof.

41. A NK cell population, or a pharmaceutically acceptable composition thereof, wherein the NK cell population is selected from the group consisting of HLA-A ｎｅｇ , homozygous for both HLA-B and HLA-C, HLA-DPB1 ｎｅｇ , and HLA-DQB1 ｎｅｇ , optionally further being homozygous for HLA-DRB1, or a pharmaceutically acceptable composition thereof.

42. 42. A method for cell therapy comprising administering the NK cell population of claim 40 or 41 or a pharmaceutically acceptable composition thereof to a human subject in need thereof.

43. 43. The method of claim 42, wherein the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, autoimmune disease, viral infection, skeletal dysplasia, and bone marrow failure syndrome.

44. 44. The method of claim 43, wherein the subject has cancer, which is optionally a hematological malignancy or a solid tumor.

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