Natural killer cell lineage derived from pluripotent cells

Abstract

The present disclosure provides efficient in vitro methods for the generation of NK cell lineages from human induced pluripotent stem cells (iPSCs). The cells produced in various embodiments according to the present disclosure are functional and / or more closely similar to corresponding lineages isolated from peripheral blood or lymphatic organs. The invention provides, in some aspects, isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 413,383, filed on October 5, 2022, the content of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in XML format via EFS-Web and is hereby incorporated 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 OF THE INVENTION

[0005] Lymphocyte natural killer (NK) cells have great promise in a variety of applications, including improving hematopoietic and solid organ transplantation, promoting anti-tumor immunotherapy, and controlling inflammation, infection, and autoimmune disorders. However, the clinical use of NK cells is hampered by the small number of functional NK cells that can be isolated from or otherwise obtained from conventional leukocyte-depleted products. Therefore, the development of large-scale, off-the-shelf NK cells would provide a powerful tool for immunotherapy. SUMMARY OF THE INVENTION

[0006] In various aspects and embodiments, the present disclosure provides methods for generating immunocompatible NK cell lineages for cell therapy, including adaptive NK cells, cytotoxic NK cells, immature NK cells, monopotent NK cell precursors, lymphoid precursors with the ability to generate NK cells (including lymphoid-primed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs)), and CD34+CD7 bright progenitor cells. In various embodiments, the present invention provides effective in vitro methods for developing such hematopoietic lineages from human induced pluripotent stem cells (iPSCs) (including gene-edited iPSCs). The cells generated in various embodiments according to the present disclosure are functional and / or more closely resemble the 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.

[0007] On the one hand, the present disclosure provides a method for preparing a cell population comprising NK cells. The method includes preparing a population of pluripotent stem cells (PSCs), such as a population of induced pluripotent stem cells (iPSCs) that differentiate into embryoid bodies, and enriching CD34+ cells to prepare a CD34+-enriched population. Endothelial-to-hematopoietic transition (EHT) is induced in the CD34+-enriched population to prepare a population of hematopoietic stem cells (HSCs), and optionally further enriching CD34+ cells thereafter. The resulting HSC population (or a fraction thereof) can differentiate into the NK cell lineage (including lymphoid-primed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs)). In some embodiments, the present disclosure provides a method for generating various types of NK cells in vitro from an HSC population.

[0008] 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.

[0009] In various embodiments, iPSCs are gene-edited to assist in HLA matching, such as deleting one or more HLA class I and / or class II alleles. For example, iPSCs can be gene-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 the 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 gene-edited to be HLA-A neg homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg . In some embodiments, iPSCs are further homozygous for HLA-DRB1.

[0010] In various embodiments, iPSCs are prepared and amplified using a culture system. The amplified iPSCs can be recovered from the culture to generate embryoid bodies (EBs). EBs differentiated from iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively two or one) germ layers (depending on the differentiation method). In some embodiments, the method includes harvesting CD34+-enriched cells from the EBs and inducing endothelial-to-hematopoietic differentiation.

[0011] 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 between day 7 and day 14 of iPSC differentiation. Differentiation of iPSCs can be carried out according to known techniques. In some embodiments, iPSC differentiation involves a combination of factors such as, but not limited to, bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1.

[0012] Induction of EHT can be carried out by any known method. In some embodiments, induction of EHT produces a population of hematopoietic stem cells (HSCs) that includes LT-HSCs. In some embodiments, EHT produces HSCs from endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., by stimulation, inhibition, and / or genetic modification). In some embodiments, EHT produces a stem cell population that includes one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and hematopoietic stem cell progenitors.

[0013] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from cultures that undergo the endothelial-to-hematopoietic cell transition between day 10 and day 20 of iPSC differentiation, such as between day 12 and day 17 of iPSC differentiation. Hematopoietic stem cells (HSCs) capable of giving rise to the innate myeloid, erythroid, and lymphoid lineages can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-).

[0014] In various embodiments, the HSC population or a fraction thereof differentiates into hematopoietic lineages, which can be selected from, including but not limited to, therapeutic human NK cells, including engineered NK cells, or their precursors (e.g., lineages that give rise to natural killer cells (NK cells)). The NK cell lineage includes engineered NK cells, cytotoxic NK cells, immature NK cells, monopotent NK cell precursors, and lymphoid precursors with the ability to give rise to NK cells. Such precursors include lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and CD34+CD7 bright progenitors, or modified forms thereof (e.g., genetically modified cells such as NK-CAR cells). In some embodiments, NK cells can express a chimeric antigen receptor (CAR).

[0015] In other aspects, the present invention provides a cell population comprising NK lineage or its precursors, or a pharmaceutically acceptable composition thereof, which can be produced by the methods described herein. In some embodiments, the cell population is a progenitor NK lineage cell population capable of engrafting into 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, which comprises the cell population and a pharmaceutically acceptable excipient. 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.

[0016] In other aspects, the present invention provides a method of cell therapy, comprising administering to a human subject in need thereof the cell population or a pharmaceutically acceptable composition thereof described herein. In various embodiments, the methods described herein are used to treat blood (malignant and non-malignant), bone marrow, immune diseases, and infectious diseases. In various embodiments, the human subject suffers from a condition comprising one or more of lymphopenia, cancer (hematological malignancies or solid tumors), immunodeficiency, autoimmune diseases, viral infections, skeletal dysplasia, or bone marrow failure syndromes.

[0017] Other aspects and embodiments of the present disclosure will become apparent from the following detailed disclosure and working examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Showing that overexpression (OE) of ETV2 does not affect pluripotency. Figure 1 Showing FACS plots representative of the transduction efficiency of iPSCs with an adenoviral vector overexpressing the ETV2 and GFP sequences. As shown by the expression of the TRA-1-60 stem cell marker, overexpression of ETV2 does not affect the stem cell properties of iPSCs.

[0019] Figure 2 Showing that overexpression (OE) of ETV2 increases the yield of hematopoietic endothelial cells. Representative flow cytometric analysis and relative quantification of hematopoietic endothelial cells (described as CD235a-CD34+CD31+) indicate that ETV2-OE enhances the formation of hematopoietic endothelial cells.

[0020] Figure 3 Showing that overexpression (OE) of ETV2 enhances the formation of CD34+ cells during iPSC differentiation. Representative flow cytometric analysis of CD34+ cells, and relative quantification indicate that ETV2-OE enhances the formation of CD34+ cells.

[0021] Figure 4A and Figure 4B Show that iPSC-derived HSCs with Piezo1 activation source undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 4A FACS plots of the differentiation efficiency of BM HSCs and iPSC-HSCs with Piezo1 activation source into CD34+CD7+ pro-T cells. Figure 4B Quantification of CD34+CD7+ cells (%) from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation) sources. Figure 4B Show the mean of three experiments.

[0022] Figure 5A and Figure 5B Show that iPSC-derived HSCs generated with Piezo1 activation undergo T cell differentiation and can be activated by CD3 / CD28 beads similar to BM-HSCs. Figure 5A FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs generated with Piezo1 activation. Figure 5B Quantification of CD3+CD69+ cells (%) from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation) sources. Figure 5B Show the mean of three experiments.

[0023] Figure 6 Show that iPSC-derived HSCs (activated with Piezo1 in this example) can differentiate into functional T cells. IFNγ expression is the result of T cell activation after stimulation of the T cell receptor (TCR) via CD3 / CD28 beads. The expression of IFNγ in T cells differentiated from iPSC-derived HSCs generated after Piezo1 activation enhanced the ability of HSCs to further differentiate into functional T cells. Figure 6 Show the mean of three experiments.

[0024] Figure 7 Show that HSCs differentiated from iPSCs (D8+7 with Yoda1 (“Y”)) are more readily activated by drug stimulation than NK cells derived from D8 iPSC-CD34+ cells, as measured by the activity of the functional marker CD107a.

[0025] Figure 8A and Figure 8B Show that NK cells derived from HSCs (D8+7, +Y) are superior to NK cells derived from bone marrow in killing tumor cells.

[0026] Figure 9A and Figure 9BShow phenotypic analysis of HLA-edited (e.g., triple knockout) cells by FACS and immunofluorescence. Figure 9A Show the overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface, where HLA-edited cells are positive for overall HLA class I expression to a similar extent as wild-type cells. Figure 9B Show the cellular expression of HLA-A by immunofluorescence, where HLA-A is not expressed in HLA-edited clones.

[0027] Figure 10 Show that HLA-edited clones retain their pluripotency (maintaining trilineage differentiation), as shown 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.

[0028] Figure 11 Show the immunocompatibility of HLA-edited HSCs. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were co-cultured with peripheral blood mononuclear cells (PBMCs) that were HLA-B and HLA-C matched but HLA-A mismatched, and PBMC-mediated cytotoxicity was measured by annexin V staining assay.

[0029] Figure 12 Show 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, where bone marrow (BM) and peripheral blood samples were evaluated by FACS to compare the relative amounts of each cell type present in the samples.

[0030] Figure 13 Show the ability of HSCs to efficiently differentiate into NK cells, as confirmed by fluorescence-activated cell sorting (FACS) experiments (gated based on CD56 expression).

[0031] Figures 14A to 14C Show that HSC-derived naive NK cells efficiently kill tumor cells. Figure 14A Show a schematic of the experiment where HSC-derived NK cells were co-cultured with K562 HLA-null cells, and the extent of NK cell degranulation was measured using annexin V staining and cytotoxicity assays. Figure 14B Show the results of NK cell degranulation, as measured by fluorescence-activated cell sorting (FACS) using annexin V staining. Figure 14C Show the results of tumor cell cytotoxicity assays, where lactate dehydrogenase (LDH) was used as a measure of cell death.

[0032] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0033] The terms "wild type" (WT), "unedited", "non-HLA edited" are used interchangeably herein to refer to the non-gene edited cells of the present disclosure.

[0034] EB34+ cells refer to CD34+ cells derived from embryoid bodies. These include hematopoietic endothelial cells. Summary of the Invention

[0036] The present disclosure provides, in various aspects and embodiments, methods for generating hematopoietic cell lineages for use in cell therapy, and in particular NK cell lineages, including their progenitors and progeny. In various embodiments, the lineages include adaptive NK cells, cytotoxic NK cells, immature NK cells, monopotent NK cell precursors, lymphoid precursors with the ability to generate NK cells (including lymphoid-primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLP)), and CD34+CD7 bright progenitor cells. In various embodiments, the present invention provides efficient in vitro methods for developing such NK cell lineages from human induced pluripotent stem cells (iPSCs). The cells generated in various embodiments according to the present disclosure are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood, bone marrow, or lymphoid organs. The present invention also provides isolated cells and cell compositions generated by the methods disclosed herein, as well as methods for cell therapy.

[0037] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate substantially unlimited pluripotent stem cells (PSCs) is used to generate an unlimited supply of NK cell lineages, including but not limited to therapeutic human NK cells, including adaptive NK cells or their precursors (e.g., lineages that generate natural killer cells (NK cells)). NK cell lineages include adaptive NK cells, cytotoxic NK cells, immature NK cells, monopotent NK cell precursors, and lymphoid precursors with the ability to generate NK cells. Such precursors include lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), and CD34+CD7 brightProgenitor cells, or modified forms thereof (e.g., genetically modified cells such as NK-CAR cells). The use of NK cells as therapeutic lymphocytes is limited by their limited availability, cell number, restricted expansion potential, and histocompatibility issues. Additionally, compared to primary cells, hiPSCs can be more easily genetically modified in vitro, thus providing improved cell targeting specificity, cell number, and bypassing issues such as HLA matching. Moreover, compared to primary cells, fully engineered hiPSC clones can serve as a stable and safe source (Nianias and Themeli, 2019). Furthermore, since hiPSCs are non-embryonic in origin, unlike human embryonic stem cells (hESCs), they also have no ethical concerns and are of consistent quality. Thus, using hiPSCs according to the present disclosure has several advantages over primary cells for generating therapeutic hematopoietic cell lineages such as NK lymphocytes.

[0038] In one aspect, the present disclosure provides a method for preparing a cell population of the NK cell lineage. The method includes preparing a population of pluripotent stem cells (PSCs), such as a population of induced pluripotent stem cells (iPSCs) differentiated into embryoid bodies, and enriching CD34+ cells to prepare a CD34+-enriched population. Inducing an endothelial-to-hematopoietic cell transition (EHT) in the CD34+-enriched population to prepare a hematopoietic stem cell (HSC) population, and optionally further enriching CD34+ cells thereafter. The resulting HSC population (or a fraction thereof) can be differentiated into the NK cell lineage according to the present invention.

[0039] In some embodiments, the method produces CD3−CD56+ NK cells developed from CD34+ hematopoietic progenitor cells (i.e., the HSC population or a fraction thereof) or derivatives of the population. For example, NK cells produced from the HSC population can be produced in a culture conditioned with Notch ligand and / or a serum-free expansion medium (SFEM) supplemented with cytokines (e.g., SCF, TPO, Flt3L, and IL-7). These conditions promote the expansion of the HSC population and its differentiation into CD7 + CD5 + lymphoid progenitor cells. Then, the cells can be further cultured in SFEM supplemented with cytokines including IL-15 (e.g., SCF, Flt3L, IL-7, IL-15) to promote differentiation into NK cells. After culturing, most cells (e.g., on average about 65% or 70% or 75% or 80% of the cells) express the NK cell marker CD56 and can co-express NKp46 (an NK cell activation receptor or other NK cell lineage markers described herein). At a more mature stage of development, mature CD56 + CD16 +NK cell subsets emerge in these cultures. Acquisition of CD94 marks commitment to CD56 bright and CD56 bright NK cells then differentiate into CD56 dim NK cells, which upregulate CD16 and killer immunoglobulin-like receptors (KIR). In some embodiments, the NK cells are cytotoxic NK cells capable of killing cancer cells (e.g., leukemia cells) or virus-infected cells, etc. In some embodiments, the NK cells express CD107a as a functional marker for identifying natural killer cell activity.

[0040] NK cells can be divided into CD56 dim or CD56 bright NK cell subsets. Approximately 90% of peripheral blood and spleen NK cells are CD56 dim CD16+ and express perforin. These CD56 dim NK cells are cytotoxic and produce IFN-γ when interacting with, for example, tumor cells in vitro. In contrast, most NK cells in lymph nodes and tonsils are CD56 bright CD16- and lack perforin. These cells are prone to produce cytokines (such as IFN-γ) in response to stimulation by interleukin (IL)-12, IL-15, and IL-18. Thus, in some embodiments, these NK cell properties can be manipulated to establish their functions, such as improving engraftment or elimination of tumors (malignant or non-malignant, solid or otherwise), or elimination of virus infections.

[0041] 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 and represent unipotent NK cell precursors lacking the potential to differentiate into 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 the positive or negative expression of both IL-1β and IL-2β receptors. In some embodiments, NK cells can be identified as CD34-CD117+ / -CD94+HLADR-CD10-CD122+CD94+NKp44 lowNKG2D+CD161+, namely mature NK cells, which can then be further divided into two final developmental stages according to the expression of CD56 and CD16. In some embodiments, NK cells express CD107a as a functional marker for identifying natural killer cell activity.

[0042] Multiple NK-specific markers can be used to identify and isolate or enrich NK cells. For example, NK cells, which are typically defined as CD3-CD56+ cells, can also be CD7+CD127-NKp46+T-bet+Eomes+. Different subtypes of human NK cells can be identified as CD3-CD56 dim CD16+ or CD3-CD56 bright CD16-. The CD56 of NK cells dim CD16+ subset is mainly found in the blood and is highly cytotoxic, while the CD56 bright CD16- subset is the main subtype found in the lymph nodes and has only weak cytotoxic potential. 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-2Rβ+; CD127 / IL-7Rα-; FcγRIII / CD16+ / -; KIR family receptors+; NKG2A+; NKG2D+; NKp30+; NKp44+; NKp46+; or NKp80+.

[0043] Traditionally, hematopoietic lineages (such as NK cells) are prepared by differentiating iPSCs into embryoid bodies (e.g., on day 8) to harvest CD34+ cells. CD34 is commonly used as a marker for hematopoietic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been found that inducing the endothelial-to-hematopoietic cell transition (EHT) of a CD34+ cell population, which can be derived from iPSC embryoid bodies, can be used to generate superior hematopoietic lineages, including the NK cell lineage, in vitro.

[0044] In some embodiments, CD34+ cells (i.e., from EB dissociation) are contacted with 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 Yoda1, Jedi1, single-stranded (ss) RNA (e.g., ssRNA40), and Jedi2. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Other ways for inducing EHT can be used and are described herein. In some embodiments, after inducing EHT, the cells (HSC or its progeny) differentiate into the NK cell lineage.

[0045] In some embodiments, HSCs differentiate into a CD7+ progenitor T cell population, which can further differentiate into the NK cell lineage. For example, the CD7+ progenitor T cell population can be generated from a population of hematopoietic stem cells (HSCs) comprising human long-term hematopoietic stem cells (LT-HSCs) generated from iPSCs (e.g., hiPSCs). For example, a population of HSCs (or cells isolated therefrom) is cultured with a partial or full complement of Notch ligands, Sonic Hedgehog (SHH), recombinant human fibrin fragments (or other extracellular matrix components), and / or combinations thereof to generate a population comprising a CD7+ progenitor T cell or derivative cell population.

[0046] 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, progenitor T cells, pre-T cells, and mature T lymphocytes that express CD56. In vivo, the development of NK and T cells occurs after lymphoid progenitors 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 controlled pathway. Notch signaling plays a key role during thymic T cell lineage commitment. When lymphoid progenitors enter the thymus, they encounter dense expression of Notch ligands on thymic epithelium, driving thymopoiesis. The present disclosure provides populations of HSCs generated in vitro from iPSCs, and these HSC populations respond to Notch ligands, SHH, and / or extracellular matrix components by massively generating T progenitors and T cell lineages (including the NK cell lineage) in vitro.

[0047] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cell" or "iPSC" refers to a cell derived from a somatic cell, such as a skin or blood cell that has 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 human primary tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, the iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of treatment as described herein). In various embodiments, the iPSCs can be gene edited to facilitate HLA matching (such as deleting one or more HLA class I and / or class II alleles or their master regulators, including but not limited to β-2-microglobulin (B2M), CIITA, etc.), or gene edited to cause other loss-of-function or expression of other functions. For example, the iPSCs can be gene 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, the iPSCs retain the expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, the iPSCs are homozygous for at least one retained class I and class II locus.

[0048] In various embodiments, the iPSCs are gene edited to be one of the following: (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-A-B-C+DP-DR+DQ+; (v) HLA-A-B-C+DP+DR+DQ-, (vi) HLA-A-B-C+DP-DR+DQ-. For the retained HLAs (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or retain only a single copy of the gene. For example, the modified cells are at least identified as (a) HLA-C+ and HLA-DR+, and optionally identified as (b) HLA-B-, (c) HLA-DP-, and (d) one or more of HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0049] In some embodiments, the iPSCs are gene edited for HLA-A neg, for those that are homozygous for both HLA - B and HLA - C, and HLA - DPB1 neg and HLA - DQB1 neg . In some embodiments, the iPSC is further homozygous for HLA - DRB1.

[0050] As used herein, the term "negative" (-) or "negative" with respect to a particular HLA class I or class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and thus 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 target gene refers to the abolition of the functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption of the coding sequence, or deletion of critical cis - acting expression control sequences.

[0051] Somatic cells can be reprogrammed by expressing 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. Patent 10,676,165; U.S. Patent 9,580,689; and U.S. Patent 9,376,664, which are hereby incorporated by reference in their entireties. In various embodiments, well - known viral vector systems such as lentivirus, Sendai virus, or measles virus systems are used to express the reprogramming factors. Alternatively, the reprogramming factors can be expressed by introducing mRNA encoding the reprogramming factors into somatic cells. In addition, iPSCs can be generated by introducing non - integrating episomal plasmids that express the reprogramming factors, i.e., for generating transgene - free and virus - free iPSCs. Known episomal plasmids can be employed, which have limited replication ability and thus are lost after several generations of cells.

[0052] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing includes, but is not limited to, modification of, for example, HLA genes (e.g., deletion of one or more HLA class I and / or class II genes), deletion of β2-microglobulin (β2M), deletion of CIITA, deletion or addition of NK cell receptor genes, 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 generation 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 and differentiated under GMP-grade conditions without feeder layers and serum-free to produce fully functional and tissue-compatible NK cells.

[0053] In some embodiments, the 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 gRNA contains a modification at or near the 5'-end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of the 5'-end) and / or a modification at or near the 3'-end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of the 3'-end). In some embodiments, the modified gRNA exhibits increased resistance to nucleases. In some embodiments, the gRNA comprises two separate RNA molecules (i.e., “dual gRNA”). The dual gRNA comprises two separate RNA molecules: “crispr RNA” (or “crRNA”) and “tracr RNA” and is well known to those skilled in the art.

[0054] Generally, various gene editing techniques are known and can be applied according to various embodiments of the present disclosure. Gene editing techniques include, but are not limited to, zinc fingers (ZFs), transcription activator-like effectors (TALEs), etc. Fusion proteins containing one or more of these DNA-binding domains and the cleavage domain of the FokI endonuclease can be used to generate double-strand breaks in the desired region of DNA in a cell (see, e.g., U.S. Patent Application Publication No. US2012 / 0064620, U.S. Patent Application Publication No. US2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. US 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. US2011 / 0301073, U.S. Patent Application Publication No. US2011 / 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 hereby incorporated by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, e.g., US 8,697,359, US 8,906,616, and US 8,999,641, each of which is hereby incorporated 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 RNA-guided. gRNA design for guiding desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, e.g., Mohr SE et al., CRISPR guide RNA design for research applications ,FEBS J. September 2016; 283(17):3232–3238. In other embodiments, non-canonical type II or type V Cas endonucleases that are homologous (although with low primary sequence homology) to Streptococcus 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 oftheCurrent Achievements, Applications, and Future Research Perspectives ,Int J MolSci. April 2021; 22(7):3327. In other embodiments, gene editing employs base editing or prime editing to incorporate mutations without causing double-strand breaks. See, e.g., Antoniou P et al.,Base and Prime Editing Technologies for Blood Disorders , Front. Genome Ed., January 28, 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double - Strand Breaks or Donor DNA , Front. Genet., June 9, 2020. Various other gene editing processes are known, including using a dead Cas (dCas) system (e.g., Cas fusion proteins) to direct DNA-modifying enzymes to a desired target, using dCas as a guide RNA-directed system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications , Int J Mol Sci. December 2019; 20(23):6041.

[0055] Base editors that can install precise genomic alterations without generating double-strand DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., engineering gene therapy vectors). Base editors essentially comprise catalytically disabled nucleases, such as Cas9 nickase (nCas9), which cannot generate DSBs, and are fused to a nucleobase deaminase and, in some cases, to a DNA glycosylase inhibitor. Currently, there are two main types of base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G conversions. Base editors can be delivered, for example, via an HDAd5 / 35++ vector to efficiently edit promoters and enhancers, thereby activating or inactivating 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. Primer editors, which comprise a reverse transcriptase conjugated (e.g., fused) to a Cas endonuclease and a polynucleotide conjugated (e.g., fused) to a guide RNA and used as a DNA synthesis template, are also envisioned, as described in WO 2020 / 191153.

[0056] Exemplary vectors useful for genome editing applications include, but are not limited to, 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, coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses (including herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., canarypox virus, vaccinia virus, or modified vaccinia virus)). A vector comprising a nucleic acid molecule of interest can be delivered to a cell (e.g., iPS cell, endothelial cell, hematopoietic endothelial cell, HSC (ST-HSC or LT-HSC)) by any method known in the art, including but not limited to transduction, transfection, infection, and electroporation. Any of these vectors can include transposable elements (such as piggyBac transposons or Sleeping Beauty transposons). Transposons insert specific DNA sequences into the genome of vertebrates. Once excised from the transposon, the gene can be integrated into the genome of mammalian cells by catalytic cleavage of a similar excision site present in the nuclear genome by a transposase.

[0057] To increase efficiency, in some embodiments, Cas and gRNA can be combined before being delivered to the cell. The Cas-gRNA complex is referred to as a ribonucleoprotein (RNP). Many methods have been developed to deliver RNPs directly to cells. For example, RNPs can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, and this procedure allows rapid entry of RNPs into the nucleus, so genome cleavage can begin immediately. See, e.g., Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR / Cas9 genome editing .Theranostics. January 1, 2021;11(2):614 - 648, which is hereby incorporated by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNPs.

[0058] Typically, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage, and the protospacer adjacent motif is typically found 3 to 4 nucleotides downstream of the cleavage site. A PAM is a short DNA sequence (usually 2 to 6 base pairs in length) that lies after 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 of the HLA locus does not include four Gs, four Cs, a GC repeat sequence, or a combination thereof.

[0059] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed using the gRNAs described herein by designing a single gRNA targeting each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). For gene knockout, the gRNA targets the Cas9 protein to a suitable site for editing. Next, the Cas9 protein can perform a double-strand break (DSB), where the DNA is repaired by the non-homologous end joining (NHEJ) mechanism, which generates indels that result in frameshift mutations and terminate the function of the resulting protein. However, off-target gene modification can occur and alter the function of other intact genes. For example, even in the presence of a certain degree of mismatch, the Cas9 endonuclease can generate DSBs at unwanted off-target locations. Such off-target activity can produce genomic instability events such as point mutations and genomic structural variations. In various embodiments, the sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, the sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, the sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0060] gRNAs can be used to develop cloned iPSCs. (i) On-target editing, (ii) off-target editing, and (iii) translocation editing of such iPSC lines can be evaluated, for example using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR that utilizes primers designed to target and enrich regions of interest, followed by next-generation sequencing (e.g., amplicon sequencing, AMP-seq). The on-target and translocation sets can amplify the expected edited regions, allowing selection of iPSC clones with the expected edits that do not have chromosomal translocations caused by fusion of unintended DSB cleavage sites. The off-target set can enrich any potential off-target regions identified by sequencing and allow selection of iPSC clones with negligible off-target mutations. Collectively, these assays enable screening of iPSC clones to select clones with desired edits while excluding potential CRISPR / Cas9-related genomic integrity issues.

[0061] In some embodiments, to further ensure genomic stability and integrity of the reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones that, for example, have not undergone translocation and mutation events and do not have integrated episomal vectors. For example, whole-genome sequencing (WGS) is performed on CD34+ cells and the reprogrammed iPSC clones, where genomic differences resulting from editing are compared. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, enabling informed selection of iPSC clones that do not generate mutations during reprogramming.

[0062] In some embodiments, karyotype analysis using a system such as the KARYOSTAT assay is used to select iPSC clones that do not generate indels and translocations during reprogramming, such as described in Ramme AP, et al., “ Supporting dataset of two integration - free induced pluripotent stem cell lines from related human donors ,” Data Brief. May 15, 2021; 37:107140, which is hereby incorporated by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal aberrations with a resolution similar to that of G-banded karyotype analysis. For chromosomal gains, the size of the structural abnormalities detectable is >2 Mb, and for chromosomal losses, the size of the structural abnormalities detectable is >1 Mb. The KARYOSTAT array is functionalized to achieve balanced whole-genome coverage by low-resolution DNA copy number analysis, where the assay covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay is capable of detecting aneuploidy, submicroscopic aberrations, and mosaic events.

[0063] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that do not generate copy number aberrations (CNAs) during reprogramming, such as as described in Wiesner et al. “ Molecular Techniques ”, edited by Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, “Pathology of Melanocytic Tumors,” Elsevier, 2019, pages 364 - 373, ISBN 9780323374576; and Hussein SM, et al. “Copy number variation and selection during reprogramming to pluripotency,” Nature. March 3, 2011; 471(7336):58 - 62, which are hereby incorporated by reference in their entirety. aCGH is a technique for analyzing the entire genome for CNAs by comparing sample DNA and reference DNA.

[0064] In some embodiments, targeted hematological malignancy NGS panel analysis is used to select iPSC clones that do not generate hematological malignancy mutations during reprogramming. For example, the targeted hematological malignancy NGS panel can focus on myeloid leukemia, lymphoma, and / or other genes associated with hematological malignancies to generate a smaller, more manageable dataset than a more extensive approach. Targeted hematological malignancy NGS panel analysis includes using highly multiplexed PCR to amplify regions associated with hematological malignancies, followed by next - generation sequencing.

[0065] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that do not integrate episomal vectors and have been passaged sufficiently for episomal vector clearance. As described herein, iPSC reprogramming of CD34+ cells can be achieved by delivery of episomal vectors encoding reprogramming factors. However, although rare, episomal vectors can randomly integrate into the cell genome, which may disrupt developmental processes, homeostasis, etc. Thus, the ddPCR method can be used to detect residual episomal vectors in iPSC cultures and is capable of selecting iPSC clones that do not integrate episomal vectors.

[0066] In some embodiments, after evaluating that the selected clone has no genome aberrations associated with editing, the clone can be additionally tested for spontaneous mutations that may occur during expansion. For example, mutations affecting blood malignancy genes, indels, translocations, numerical aberrations, such as those 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 hematological malignancy NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0067] 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 differentiated from iPSCs are three-dimensional aggregates of iPSCs and contain three (or two or one) embryonic germ layers based on the differentiation method. For example, the preparation of EBs is described in US2019 / 0177695, which is hereby incorporated by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, such as described by 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 for the expansion or differentiation of EBs, including 3D suspension culture, are described in WO 2020 / 086889, which is hereby incorporated by reference in its entirety.

[0068] In some embodiments, the methods according to each aspect can include generating CD34+ enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial cell to hematopoietic cell differentiation. HSCs containing a relatively high frequency of LT-HSCs can be generated from a cell population using: various stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimuli, and factors such as induction of extracellular matrix, niche factors, extracellular extrinsic factors, cell intrinsic properties; and including pharmacological and / or genetic means.

[0069] In some embodiments, the method includes preparing endothelial cells with hematopoietic potential from pluripotent stem cells prior to EHT induction. In some embodiments, overexpression of a combination of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can result in the formation of endothelial cells with hematopoietic potential from PSC sources. In some embodiments, the method includes overexpressing the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. ETV2 can be expressed by introducing a plasmid encoding a non-integrating episome for constitutive or inducible expression of ETV2 and for generating transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. Any available method for introducing mRNA can be used, including electroporation or lipofection. Differentiation of cells expressing ETV2 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). According to embodiments of the present disclosure, cells generated in this manner can be used to generate CD34+ cells and induce EHT.

[0070] After CD34+ enrichment, HSCs are generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0071] 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 to 14 of iPSC differentiation (such as, for example, day 8, day 9, day 10, day 11, day 12, day 13, or day 14). Differentiation of iPSCs can be carried out according to known techniques. In some embodiments, iPSC differentiation involves a combination of factors such as, but not limited to, 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, feeder-free, serum-free, and / or GMP-compatible materials (such as pomalidomide or lenalidomide) are used to differentiate hPSCs. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells (such as the OP9 or MS5 cell lines) in a serum-containing medium. The culture can contain growth factors and cytokines to support the differentiation of embryoid bodies or monolayer systems. The OP9 co-culture system can be used to generate pluripotent HSPCs, which can be further differentiated into several hematopoietic lineages, including T lymphocytes, B lymphocytes, megakaryocytes, monocytes or macrophages, and red blood cells. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells , Stem Cell Research & Therapy 11, article number 481 (2020). Alternatively, a stepwise process using defined conditions with specific signals can be used. For example, the expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into CD34+ / CD45+ progenitor cells with multilineage potential. In addition, the 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. October 3, 2013; 13(4).

[0072] Exemplary ROCK inhibitors for establishing and differentiating iPSCs include, but are not limited to: thiazovivin, Y27632, fasudil, AR122-86, RevitaCell.TM. Supplement, H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, H-100, and the ROCK inhibitor disclosed in U.S. Patent No. 8,044,201, which is hereby incorporated by reference in its entirety.

[0073] Differentiation of iPSCs (e.g., differentiation into EBs) can employ WNT agonists 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), SKL2001 (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)aryl pyrimidines. In some embodiments, the WNT agonist is an agonist antibody or a functional fragment thereof or an antibody-like polypeptide.

[0074] The induction of EHT can be carried out by any known method. In some embodiments, the induction of EHT generates a population of hematopoietic stem cells (HSCs) that includes LT-HSCs. In some embodiments, EHT generates HSCs from endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., by stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population that includes one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and hematopoietic stem cell progenitors. In some embodiments, the EHT culture contains one or more (e.g., in combination) of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

[0075] In some embodiments, the method includes increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34-enriched cells, ECs, HECs, or HSCs, which can be carried out by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO 2019 / 236943 and WO 2021 / 119061, which are hereby incorporated by reference in their entireties. In some embodiments, the induction of EHT includes increasing the expression or activity of dnmt3b.

[0076] In some embodiments, the cells are contacted with an effective amount of a mechanosensor or a mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensor is Piezol. An exemplary Piezol agonist is Yoda1. In some embodiments, the mechanosensor 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 against the mechanosensitive ion channel Piezol. Syeda R, Chemical activation of the mechanotransduction channel Piezol .eLife (2015).

[0077] Derivatives of Yodal can be used in various embodiments. For example, in some embodiments, derivatives containing a 2,6-dichlorophenyl nucleus are employed. Exemplary agonists are disclosed in Evans EL, et al., Yoda1 analogue (Dooku1)which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation , in British J. of Pharmacology 175(1744 - 1759):2018. Other Piezo1 agonists include Jedi1, Jedi2, single - stranded (ss) RNA (e.g., ssRNA40) and their derivatives and analogs. 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; Sugisawa, et al., RNA Sensing by Gut Piezo1Is Essential for Systemic Serotonin Synthesis , Cell, Volume 182, Issue 3, 2020, pp. 609 - 624, which is hereby incorporated by reference in its entirety. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the Piezo1 agonist or derivative is in the range of about 1 μM to about 500 μM, or about 5 μM to about 200 μM, or about 5 μM to about 100 μM, or in some embodiments, in the range of about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM.

[0078] In various embodiments, pharmacological Piezo1 activation is applied to CD34 + cells (i.e., CD34 + - enriched cells). In certain embodiments, pharmacological Piezo1 activation can be further applied to iPSCs, embryoid bodies, ECs, hematopoietic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineages. In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs, CD34 + cells isolated from EBs, and / or combinations thereof, which allows for better generation of T progenitor cells compared to other methods for inducing EHT according to various embodiments.

[0079] Alternatively or additionally, the activity or expression of Dnmt3b can be directly increased in cells (e.g., in CD34 - enriched cells). For example, the mRNA expression of Dnmt3b can be increased by delivering the transcript encoding Dnmt3b to the cells, or by introducing a transgenic encoding Dnmt3b, or by non - transgenic methods (not limited to introducing non - integrating episomes into the cells). In some embodiments, gene editing is employed to introduce genetic modifications to the Dnmt3b expression element in the cells, such as but not limited to increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0080] In some embodiments, the method includes increasing the activity or expression of Gimap6 in cells, alone or in combination with Dnmt3b and / or other genes that are upregulated or downregulated upon cyclic strain or piezoelectric activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cells, or a transgene-free method can be employed, including but not limited to introducing an episome into the cells; or alternatively a transgene encoding Gimap6. In some embodiments, gene editing is used to introduce genetic modifications into the Gimap6 expression element in the cells (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability or affect RNA splicing).

[0081] In embodiments of the present disclosure that employ mRNA delivery to cells, known chemical modifications can be used to avoid the innate immune response in the cells. For example, synthetic RNA containing only canonical nucleotides can bind pattern recognition receptors and can trigger an effective immune response in the cells. Such a response can lead to translation blockade, secretion of inflammatory cytokines, and cell death. RNA containing certain non-canonical nucleotides can evade detection by the innate immune system and can be efficiently translated into proteins. See US 9,181,319, which is hereby incorporated by reference, particularly with respect to nucleotide modifications to avoid the innate immune response.

[0082] In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into the cells, which can direct the desired overexpression level (other options with different promoter strengths or expression control elements). A variety of viral vectors or transfection reagents known in the art (including lipid nanoparticles) can be used to introduce the transgene. In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by a transgene-free method (e.g., episomal delivery). In some embodiments, gene editing techniques are used to increase the expression or activity of Dnmt3b and / or Gimap6 or other genes disclosed herein, e.g., to introduce one or more modifications to increase promoter strength, ribosome binding or RNA stability.

[0083] 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, a cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is hereby incorporated by reference in its entirety. Cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, mechanical means apply a stretching force to the cells or to the cell culture surface on which the cells (e.g., ECs or HECs) are cultured. For example, a computer-controlled vacuum pump system or other components for providing a stretching force attached to a flexible biocompatible and / or biomimetic surface (e.g., FlexCell TM Tension System, Cytostretcher System) can be used to apply in vitro cyclic 2D, 3D, or 4D stretch to cells under defined and controlled cyclic strain conditions. For example, the cyclic stretch applied can be a cyclic strain of about 1% to about 20% (e.g., a cyclic strain of about 6%), for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about one hour, at least about two hours, at least about six hours, at least about eight 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.

[0084] Alternatively or additionally, EHT is stimulated through Trpv4 activation. Trpv4 activation can be achieved by contacting the cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, which are optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0085] When a cell population is described herein as having a certain phenotype, it is understood that the phenotype represents a significant 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. In addition, at each step, the cell population can be enriched for cells of the desired phenotype, and / or depleted of cells of the 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, fluorescence-activated cell sorting or magnetic beads that bind cells to certain cell surface antigens (including those described herein) can be used based on cell surface antigens to sort cells. A negative selection column can be used to remove cells that express undesired cell surface markers. In some embodiments, the 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 expansion of CD34+ cells, thereby generating an expanded stem cell population. In some embodiments, the cell population is cultured under conditions that promote the expansion of CD34+ cells, thereby generating an expanded stem cell population. In addition, NK cells generated according to the present disclosure can be enriched for the expression or expression level of desired markers, such as CD56.

[0086] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from a culture undergoing endothelial-to-hematopoietic transition, such as between day 8 and day 20 (e.g., day 10 to day 17) of iPSC differentiation.

[0087] In various embodiments, HSCs or CD34+-enriched cells are further expanded. For example, HSCs or CD34-enriched cells can be expanded according to methods disclosed in US 8,168,428; US 9,028,811; US10,272,110; and US10,278,990, which patents are hereby incorporated by reference in their entireties. In some embodiments, in vitro expansion of HSCs or CD34+-enriched cells uses prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs contain 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.

[0088] Hematopoietic stem cells (HSCs) that 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 stem cell population comprising HSCs is enriched, e.g., as described in US 9,834,754, which is hereby incorporated by reference in its entirety. For example, the method can include sorting a cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. One or more components that are CD34 + , CD90 + , CD38 - , and CD43 - can be selected for further differentiation. In some embodiments, the stem cell population for differentiation into hematopoietic lineages is at least about 80% CD34 + , or at least about 90% CD34 + , or at least about 95% CD34 + .

[0089] In some embodiments, the stem cell population, or CD34+-enriched cells or components thereof, or a derived cell population is expanded as described in US2020 / 0308540, which is hereby incorporated by reference in its entirety. For example, cells are expanded by exposing the cells to an aryl hydrocarbon receptor antagonist including, e.g., 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 September 10, 2010;329(5997):1345–1348.

[0090] In some embodiments, compounds that promote CD34 + cell expansion include pyrimidoindole derivatives, including, e.g., UM171 or UM729 (see US2020 / 0308540, which is hereby incorporated by reference).

[0091] In some embodiments, a stem cell population or CD34+-enriched cells are further enriched for cells expressing periostin and / or platelet-derived growth factor receptor alpha (pdgfra), or are modified to express periostin and / or pdgfra, as described in WO 2020 / 205969, which is hereby incorporated by reference in its entirety. Such expression can be carried out by delivering a coding transcript to the cell, or by introducing a coding transgene, or by a transgene-free method (not limited to introducing non-integrating episomes into the cell). In some embodiments, gene editing is employed to introduce genetic modifications into the expression elements in the cell, such as to modify promoter activity or strength, ribosome binding, RNA stability or to affect RNA splicing.

[0092] In other embodiments, a stem cell population or CD34-enriched cells are cultured with an EZH1 inhibitor, a histone methyltransferase. Alternatively, EZH1 is partially or completely absent or inactivated or transiently silenced in the stem cell population. Inhibition of EZH1 can direct bone marrow progenitors (e.g., CD34+CD45+) to the lymphoid lineage. See WO 2018 / 048828, which is hereby incorporated by reference in its entirety. In other embodiments, EZH1 is overexpressed in the stem cell population.

[0093] The HSC population or a fraction thereof differentiates into the NK cell lineage.

[0094] In vivo, NK cell development can be divided into approximately six stages based on the development of the bone marrow (BM) and lymph nodes (LN). See Abel AM et al.: Natural Killer cells: Development,Maturation,and Clinical Utilization .Front Immunol. August 13, 2018; 9:1869. In accordance with the present disclosure, NK cells conforming to these stages can be generated. CD3ε-CD7+CD127+ cells mark the earliest stage of committed NKPs (stage 2a). CD7 is expressed throughout development. Expression of IL-1R (IL-1β receptor) 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. The fourth stage 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 high levels of CD56 (CD56 bright)。These NK cells are NKP80-negative and express maximal levels of NKG2D, CD335, CD337, inhibitory NKG2A, and CD161 (NK1.1, KLRB1, NKR-P1A). At stage 4b, human NK cells are positive for NKP80 and maintain their CD56 bright status. CD56 bright expression is downregulated to CD56 dim , and the immunoglobulin superfamily member CD16 (FcγRIII) is expressed in the NK cell subset, defined as stage 5. The expression level of CD56 provides a functional classification of human NK cells. Most human NK cells in peripheral blood are CD56 dim . CD56 bri bright NK cells are considered less mature and are mainly present in the SLT, while the CD56 dim subset represents most of the NK cells in the circulation. Most immature NK cells (iNK) are converted into a minority CD56 bri bright population (about 5%), which is then converted back into the majority CD56 dim population (>90%). Since CD56 bri bright NK cells are potent producers of inflammatory cytokines, while the cytolytic function of human NK cells is mainly present in the CD56 dim population, the downregulation of CD56 is closely associated with the acquisition of anti-tumor cytotoxicity during human NK cell maturation. The terminal maturation of CD56 dim NK cells (stage 6) is defined by the expression of CD57 (HNK-1, Leu-7). Other classifications (such as "antigen-experienced" or "adapted" CD2+ NK cells) are defined by higher expression of NKG2C (KLRC2, CD159c).

[0095] Thus, in some embodiments, generating natural killer (NK) cells from pluripotent stem cells (e.g., iPSCs) can include the steps of: (i) preparing a population of HSCs comprising CD34+ cells as described; (ii) culturing the population of HSCs under conditions sufficient to differentiate the cells into NK cells, which can optionally be a medium comprising factors such as FGF2, VEGF, TPO, SCF, IL-3, and FLT3L; (iii) during differentiation, factors such as IL-7 and IL-15 can be included in the medium. NK cells can be identified by NK cell markers (such as CD3-CD56) and can be further characterized as CD56 bri bright NK cells or CD56 dim NK cells.

[0096] In some embodiments, the HSC population or a fraction thereof differentiates into NK cells or their progenitors or derivatives without relying on 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 population of differentiated pluripotent stem cells to prepare a CD34+-enriched population. The CD34+-enriched cell population is induced to undergo an endothelial-to-hematopoietic cell transition for at least two days but no more than 12 days, wherein the use of agonists of mechanosensitive receptors or mechanosensitive channels (such as Yoda1, jedi1, jedi2, ssRNA40) is optional. The HSC and / or HSPC differentiate into a progenitor NK cell population or an NK cell population.

[0097] In some embodiments, the endothelial-to-hematopoietic cell transition of the CD34+-enriched cell population is induced for at least 2 days and 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 no more than 12 days.

[0098] In some embodiments, the NK cell lineage generated according to the present disclosure is predominantly (e.g., at least about 50% or at least about 75%) CD56 bright 。In some embodiments, the NK cell lineage generated according to the present disclosure is 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 cells (NK). Once activated, cytolytic NK cells have effector functions similar to T cells, including the production of cytotoxic granules and inflammatory cytokines. NK cells can also shape the adaptive immune system by influencing T cells at different stages of their life cycle. For example, during T cell priming, NK cells indirectly alter T cell responses by influencing dendritic cells (DC).

[0099] In some embodiments, the NK cells generated according to the present disclosure secrete cytokine levels comparable to endogenous NK cells. Such cytokines include, but are 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 a key role in antiviral, antibacterial, and antitumor responses.

[0100] 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.

[0101] During differentiation, the HSC population and its progeny are cultured in vitro with Notch ligand(s) (partial or full), SHH, extracellular matrix components, and / or combinations thereof to differentiate the HSCs into CD7 + progenitor T cells, and then into the NK cell lineage. Additionally, according to known methods, xenogeneic OP9-DL1 cells or irradiated K562-mbIL21-41BBL cells can be used to differentiate hematopoietic cells into T cells and NK cells. The OP9-DL1 co-culture system uses a bone marrow stromal cell line (OP9) transduced with the Notch ligand delta-like-1 (DLL1) or DLL-4 to support the development of stem cell-derived T cells. The OP9-DL1 system limits the potential for clinical application of the cells. There is a need for a feeder-free cell system capable of generating cells (such as NK cells) from hiPSCs for clinical applications, and in some embodiments, the present invention meets this objective. In a non-limiting example, to generate mature NK cells using Notch ligand, the iPSCs are expanded 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 HSCs to develop from the CD34+ hematopoietic endothelial cells from which they are derived. The HCSs are then cultured in T cell- or NK cell-specific medium supplemented with recombinant human fibrin fragment and DLL-4 to generate Tpro cells, which are identified as CD34+CD7+CD5+ / - . Then, the Tpro cells can differentiate to generate NK cells.

[0102] As used herein, the term "Notch ligand" refers to a ligand capable of binding to a Notch receptor polypeptide present in the membrane of hematopoietic stem cells or progenitor T cells. 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), which contains 20 to 22 amino acids at the amino terminus and 3 to 8 EGF repeats on the extracellular surface. In various embodiments, the Notch ligand comprises delta-like-1 (DLL1), delta-like-4 (DLL4), SFIP3, delta 最大(Disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are hereby incorporated by reference in their entireties) or a functional portion thereof, at least one of Jagged 1 (JAG1), Jagged 2 (JAG2), delta-like ligand 3 (DLL3), and X-delta2. The key signal that thymic stromal cells transmit to incoming lymphocyte progenitors in vivo is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0103] As used herein, "Notch ligand" also includes full-length (full-length), partial (truncated form), or modified (containing one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands of any species or fragments thereof 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 characterized by an extracellular domain that typically includes an N-terminal (NT) domain, followed by a delta / serrate / LAG-2 (DSL) domain and multiple tandemly arranged epidermal growth factor (EGF)-like repeats. Binding of a canonical ligand to Notch typically requires the DSL domain and flanking NT domain, as well as the first two EGF repeats containing the delta and OSM-11-like protein (DOS) motif. The intracellular domain of some canonical ligands contains a carboxyl-terminal PSD-95 / Dlg / ZO-1-ligand (PDZL) motif, which functions independently 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.

[0104] In various embodiments, the HSC / HSPC population is cultured in an artificial thymic organoid (ATO). See Hagen, M. et al. (2019). The ATO will contain a culture of HSCs (or HSC aggregates) with a stromal cell line expressing a Notch ligand under serum-free conditions. An artificial thymic organoid is a 3D system that induces the differentiation of hematopoietic precursors into naive CD3 + CD8 + and CD3 + CD4 + T cells. In some embodiments, the artificial thymic organoid contains DLL4 and BMP2, or functional fragments thereof.

[0105] In some embodiments, progenitor NK cells or T cells are isolated by enriching for CD7 expression. In some embodiments, progenitor T cells are expanded as described in US2020 / 0308540, which is hereby incorporated by reference in its entirety. For example, cells can be expanded by exposing the cells 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 compound that promotes expansion includes pyrimidoindole derivatives including, for example, UM171 or UM729 (see US 2020 / 0308540, which is hereby incorporated by reference).

[0106] In some embodiments, the progenitor cells that differentiate into progenitor NK cells or T cells that can give rise to NK cells can further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. In various embodiments, the resulting CD7+ progenitor T cells express CD1a. The CD7+ progenitor NK cells or T cells do not express CD34, or express a reduced level of CD34 compared to the HSC population. In some embodiments, the CD7+ progenitor T cells (or a portion thereof) further express CD5. Thus, the phenotype of the progenitor T cells can be CD7 + CD1a + . In some embodiments, the phenotype of the progenitor T cells is CD7 + CD5 + . In some embodiments, the progenitor T cells are CD7 + CD1a + CD5 + , and optionally CD34 + . In some embodiments, the progenitor T cells exhibit a reduced level of CD34 expression, a very low level of CD34 expression (compared to the HSC population), or no CD34 expression. In some embodiments, the CD34 expression in this population is reduced by at least about 50%, or at least about 75%, relative to the HSC population.

[0107] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including human or humanized antibodies), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule capable of activating the Notch signaling pathway.

[0108] In some embodiments, the Notch ligand is a delta family Notch ligand. In some embodiments, the delta family ligand is delta-1 (Genbank accession number AF003522, Homo sapiens), delta-like 1 (DLL1, Genbank accession numbers NM_005618 and NP_005609, Homo sapiens; Genbank accession numbers X80903, 148324, Mus musculus), delta-4 (Genbank accession numbers AF273454, BAB18580, Mus musculus; Genbank accession numbers AF279305, AAF81912, Homo sapiens) and / or delta-like 4 (DLL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM 019454, Mus musculus). The Notch ligand is commercially available or can be produced by, for example, recombinant DNA technology.

[0109] In some embodiments, the Notch ligand is DLL4 having one or more affinity-enhancing mutations with respect to hDLL4, such as one or more (or all) of the following: G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4 ligands as broad-spectrum modulators ofNotch signaling , Nature Chemical Biology (2022).

[0110] 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. Functional derivatives (including fragments or portions thereof) of the Notch ligand will be capable of binding and activating the Notch receptor. Binding to the Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cellular signaling assays.

[0111] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles, which are optionally paramagnetic to allow for magnetic enrichment or concentration processes. In other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally together with other adhesion molecules such as VCAM-1. See US2020 / 0399599, which is hereby incorporated by reference in its entirety. In other embodiments, the beads or particles are polymers (e.g., polystyrene or PLGA), gold, dextran iron, or are composed of biomaterials such as particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or maximum dimension ranging from about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In other embodiments, a polymeric scaffold with conjugated ligands can be employed as described in WO 2020 / 131582, which is hereby incorporated by reference in its entirety. For example, the scaffold can be composed of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-ε-caprolactone, polyphosphazene, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic polyols, poloxamers, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold includes pores with diameters between about 1 pm and 100 pm.

[0112] In some embodiments, the C-terminus of the Notch ligand is conjugated to a selected support. In some embodiments, this can include adding a sequence at the C-terminus of the Notch ligand that can be conjugated to the support enzymatically, for example, via a biotin molecule. In another embodiment, a Notch ligand-Fc fusion is prepared such that the Fc fragment can be immobilized by binding to protein A or protein G, which is conjugated to the support. Of course, any known protein conjugation method can be employed.

[0113] Thus, in various embodiments, the Notch ligand is immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated together with components of the extracellular matrix, such as one or more selected from fibronectin, recombinant human fibrin fragments, 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 a combination thereof are in contact with the culture conditions, providing a topographical pattern and / or texture (e.g., roughness) that aids in cell differentiation and / or expansion.

[0114] In some embodiments, HSCs are cultured in a medium containing TNF-α and / or an aryl hydrocarbon / dioxin receptor antagonist (SR1) and differentiated into progenitor T cells in the presence of a Notch ligand. See US2020 / 0390817, US2021 / 0169934, and US2021 / 0169935, which are hereby incorporated by reference in their entireties. In some embodiments, the HSCs are cultured in a 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 fragment. In some embodiments, the cells are cultured with a recombinant human fibrin fragment, which is recombinant human fibronectin and contains three functional domains: the human fibronectin cell-binding domain (C domain), the heparin-binding domain (H domain), and the CS-1 sequence domain. In some embodiments, the cells are cultured in the presence of immobilized delta-like-4 ligand and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of immobilized delta-like-4 ligand, TNF-α, and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of immobilized delta-like-1 ligand and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of SFIP3 and the recombinant human fibrin fragment. In some embodiments, the cells are cultured in the presence of immobilized delta-like-4 ligand and SHH molecule and / or its functional derivatives. Exemplary fibronectin fragments contain one or more RGDS, CS-1, and heparin-binding motifs. The fibronectin fragment can be free in solution or immobilized on the culture surface or on particles. In some embodiments, the cells are cultured for 5 to 7 days to prepare CD7+ progenitor T cells, and then these CD7+ progenitor T cells can be utilized to generate NK cells from T cell progenitors that retain the potential to produce NK cells.

[0115] In some embodiments, cytokines and / or growth factors are required for the differentiation of HSCs or cells isolated therefrom, and these factors may be selected from or include (or consist essentially of): TPO, SCF, Flt3L, IL3, IL7, and SDF-1a. In addition, T cells or progenitor T cells generated in 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 may optionally be excluded after early NK cell formation. In some embodiments, the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor to initiate the differentiation of pre-NK cell progenitors into NK cell progenitors or NK cells. In some embodiments, pluripotent stem cell-derived NK progenitors are CD3-CD45+CD56+CD7+. In some embodiments, pluripotent stem cell-derived NK cells are CD3-CD45+CD56+ and are optionally further defined by NKp46+, CD57+, and CD16+.

[0116] In some embodiments, NK cells are generated from progenitor T cells as described in US 10,266,805, which is hereby incorporated by reference in its entirety. For example, NK cells can be generated when progenitor T cells are cultured with IL-15. In some embodiments, NK cells express CAR based on iPSCs, embryoid bodies, hCD34+ cells, or gene editing of NK cells, or via mRNA expression in NK cells. Additionally, or optionally, NK CARs can be engineered to express cytokines (such as IL-15) to make NK-CARs more effective in targeting tumors.

[0117] In non-limiting examples, NK cells can be efficiently transduced by vectors such as, but not limited to, retroviruses or non-integrating viral vectors (e.g., adenovirus, adeno-associated virus, integrase-deficient lentivirus, poxvirus) or non-viral vectors (e.g., plasmid vectors, artificial chromosomes) or episomal or episomal hybrid vectors carrying second-generation CARs targeting tumor antigens (e.g., CD19, CD38, CD33, CD47, CD20, etc.). Expression of CAR can be demonstrated in different NK cell subsets according to conventional 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 higher tumor activity (e.g., anti-leukemia activity) against CD19, CD38, CD33, CD47, CD20 cell lines and primary blasts obtained from patients (e.g., patients with B-cell precursor ALL) compared to unmodified NK cells.

[0118] The CAR is designed to enhance the ability of cells to recognize, bind to, and kill tumor cells. In some embodiments, the CAR enhances the ability of NK cells to recognize tumor cells. In some embodiments, the CAR enhances the anti-tumor activity of NK cells. In some embodiments, but not limited thereto, the CAR is a G protein-coupled receptor 87 (GPR87) CAR, a solute carrier family 7 member 11 (SLC7A11 (xCT)) CAR, a TNF receptor superfamily member 17 (BCMA) CAR, a CD30 CAR, a CD19 CAR, a CD22-CAR, a CD33 CAR, a CD133-CAR, an NKG2D CAR (or a CAR or receptor comprising the NKG2D extracellular domain), a mesothelin-CAR, a CD70 CAR, an NKp30 CAR, a CD73 CAR, or a CAR-NK cell, which targets the following tumors or tumor antigens:

[0119] (i) Human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma;

[0120] (ii) Epidermal growth factor receptor (EGFR) - non-small cell lung cancer, epithelial cancer, and glioma;

[0121] (iii) Mesothelin - mesothelioma, ovarian cancer, and pancreatic adenocarcinoma;

[0122] (iv) Prostate-specific membrane antigen (PSMA) - prostate cancer;

[0123] (v) Carcinoembryonic antigen (CEA) - pancreatic adenocarcinoma, breast cancer, and colorectal cancer;

[0124] (vi) Glypican-3 - hepatocellular carcinoma;

[0125] (vii) Epidermal growth factor receptor variant III (EGFRvIII) - glioblastoma;

[0126] (viii) Disialoganglioside 2 (GD2) - neuroblastoma and melanoma;

[0127] (ix) Carbonic anhydrase IX (CAIX) - renal cell carcinoma;

[0128] (x) Interleukin-13Ra2 - glioma;

[0129] (xi) Fibroblast activation protein (FAP) - malignant pleural mesothelioma;

[0130] (xii) L1 cell adhesion molecule (L1-CAM) - neuroblastoma, melanoma, and ovarian cancer;

[0131] (xiii) Cancer antigen 125 (CA 125) - epithelial ovarian cancer;

[0132] (xiv) Cluster of differentiation 133 (CD 133) - glioblastoma, cholangiocarcinoma, adenocarcinoma;

[0133] (xv) Cancer / testis antigen 1B (CTAG1B) - melanoma and ovarian cancer;

[0134] (xvi) Mucin 1 - seminal vesicle cancer;

[0135] (xvii) Folate receptor - a (FR - a) - ovarian cancer;

[0136] (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 to 4.

[0137] The cell population or cells derived therefrom (e.g., progeny) can be used in conjunction with FDA - approved CAR - T therapies such as Tisagenlecleucel (also known as tisa - cel (Kymriah)), Axicabtagene ciloleucel (also known as axi - cel (Yescarta)), Brexucabtagene autoleucel (also known as brexu - cel (Tecartus)), Lisocabtagene maraleucel (also known as liso - cel (Breyanzi)), Idecabtagene vicleucel (also known as ide - cel (Abecma)), Ciltacabtegene autoleucel (also known as cilta - cel (Carvykti)), or any other CAR - based therapy that damages normal cells during their therapeutic application.

[0138] Thus, in some aspects and embodiments of the present invention, the genetically modified NK cell line or its precursors or progeny are engineered to express a chimeric antigen receptor (CAR) on the cell surface, and in particular a CAR that specifically binds to a growth factor receptor. Most typically, the CAR comprises the intracellular domain from the Fcε receptor γ (FcεRIγ). However, in further contemplated embodiments, the CAR may also comprise the intracellular domain of the T - cell receptor (TCR) CD3ζ (CD3ζ), alone or in combination with additional components from second - or third - generation CAR constructs (e.g., CD28, CD134, CD137, and / or ICOS).

[0139] In one aspect, NK cells or their precursors or progeny are modified to express an autocrine growth-stimulatory cytokine or a variant thereof. Additionally, or optionally, the genetically modified NK cells will also express recombinant CD16 or a high-affinity variant thereof to confer cell-targeted specific ADCC.

[0140] In one aspect, NK cells or their precursors or progeny are modified to express homing receptors. A "homing receptor" refers to a cellular pathway that activates receptors whose activation directly or indirectly leads to the migration of cells towards target cells or tissues. For example, homing receptors expressed by leukocytes are utilized by leukocytes and lymphocytes to enter secondary lymphoid tissues via high endothelial venules. Homing receptors can also be utilized by cells to migrate towards 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; cell adhesion molecules, such as selectins (including L-selectin (CD62L)), integrins (such as α4β7 integrin, LPAM-1, and LFA-1). Homing receptors typically bind to homologous ligands on target tissues or cells. In some embodiments, the homing receptor binds to an addressin, such as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), on the endothelial cells of venules.

[0141] In other aspects, the present invention provides a population of cells or a pharmaceutically acceptable composition thereof produced by the methods described herein. In some embodiments, the population of cells is a population of progenitor NK cells that is capable of engrafting 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, which comprises the population of cells and a pharmaceutically acceptable excipient. The pharmaceutical composition can comprise 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 8 cells, 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 1014 cells. For example, in some embodiments, a pharmaceutical composition comprising from about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg) is administered. In other embodiments, about 10 5 to about 5×10 5 cells (e.g., about 2.5×10 5 cells / kg), or from about 10 6 to about 5×10 6 cells (e.g., about 2.5×10 6 cells / kg), or from about 5×10 6 to about 10 7 cells (e.g., about 5×10 6 cells / kg), or from about 10 7 to about 10 8 cells (e.g., about 5×10 7 cells / kg), or from about 10 8 to about 10 9 cells (e.g., about 5×10 8 cells / kg), or from about 10 9 to about 10 10 cells, or from about 10 10 to about 10 11 or about 10 11 to about 10 12 cells, or from about 10 12 to about 10 13 cells, or about 10 13 to about 10 14 cells are administered per kilogram of recipient body weight.

[0142] In some embodiments, the NK cells are derived from HLA-edited iPSCs as described. For example, in some embodiments, the NK cells are 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.

[0143] 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 an acid or a base without a significant change in pH. Examples of buffers contemplated by the present invention include, but are not limited to, normal / saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate buffered saline (PBS), Ringer's solution. The composition may comprise excipients suitable for intravenous infusion or other routes of administration, and the composition may comprise 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 implantable device (e.g., a stent) or in a bag or in a vial, test tube, or container in an appropriate volume and stored frozen until use.

[0144] The pharmaceutical compositions for the disclosed methods may also contain other therapeutic agents for treating a specific target disease. For example, the pharmaceutical composition may further comprise cytokines and growth factors (interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical composition to produce the advantages of the therapeutic methods disclosed herein, namely, providing improved therapeutic efficacy and reduced systemic toxicity.

[0145] NK cells or CAR-NK cells can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context 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 delivery of the agent, the method of administration, the dosing schedule, and other factors known to the physician. The therapeutically effective amount of the cells to be administered will be governed by such considerations.

[0146] Depending on the needs of the particular indication being treated, the formulations herein may also contain more than one active agent, preferably those having complementary activities and not having an adverse effect on each other. For example, it may be desirable to further provide a cytotoxic agent, a cytokine, or an immunosuppressant. The effective amount of such other agents depends on the amount of acceptable carrier, excipient, or stabilizer in the formulation, the type of disease or disorder or treatment, and the other factors discussed above. These agents are generally used at the same dose and route of administration as used above, or about 1% to 99% of the dose employed heretofore.

[0147] In other aspects, the present disclosure provides a method of cell therapy comprising administering to a human subject in need thereof the cell population described herein or a pharmaceutically acceptable composition 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 blood (malignant and non-malignant), bone marrow, immune diseases, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, and autoimmune diseases.

[0148] Examples of diseases include various autoimmune diseases, including but not limited to alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), certain forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, certain forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, discoid lupus erythematosus, certain forms of thyroiditis, certain forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener). Blood malignancies that can be treated include but are not limited to acute and chronic leukemia, lymphoma, multiple myeloma, and myelodysplastic syndromes. 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-related disorders. Other conditions include: skeletal dysplasia, hemoglobinopathies; anemia, including but not limited to iron deficiency anemia, pernicious anemia, aplastic anemia, sickle cell anemia, vitamin deficiency anemia, and hemolytic anemia; bone marrow failure syndromes, and certain genetic disorders (e.g., genetic disorders affecting the immune system).

[0149] In some embodiments, the subject has cancer, such as a blood malignancy, including but not limited to leukemia, lymphoma, and multiple myeloma; or a solid tumor, including but not limited to brain tumor, prostate tumor, breast tumor, lung tumor, colon tumor, uterine tumor, skin tumor, liver tumor, bone tumor, pancreatic tumor, ovarian tumor, testicular tumor, bladder tumor, kidney tumor, head tumor, neck tumor, stomach tumor, cervical tumor, rectal tumor, laryngeal tumor, or esophageal tumor.

[0150] In some embodiments, the subject has a disorder selected from the following: acute myeloid leukemia; acute lymphoblastic leukemia; chronic myeloid leukemia; chronic lymphocytic leukemia; myeloproliferative disorders; myelodysplastic syndromes; multiple myeloma; non-Hodgkin lymphoma; Hodgkin 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 lymphohistiocytosis; congenital metabolic defects; severe congenital neutropenia; Shwachman-Diamond syndrome; Diamond-Blackfan anemia; and leukocyte adhesion deficiency.

[0151] Regarding the use of HLA-edited NK cells (e.g., derived from gene-edited iPSCs), the subject can be matched at the retained HLA loci (such as one or more (or all) of HLA-B, HLA-C, and HLA-DRB1).

[0152] Agents (such as hormones, growth factors, and cytokine antibodies) that can be co-administered with the cell line derivatives or expanded primary cell banks of the present invention (e.g., iPSC-derived HSCs or their progenitor cells 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; α-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; blood coagulation factors such as factor VIIIc, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulation factors 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 tumor necrosis factor-β; enkephalinase; RANTES (regulated upon activation, normal T cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Mullerian-inhibiting substance; relaxin A chain; relaxin B chain; prorelaxin; murine gonadotropin-associated peptide; microbial proteins such as β-lactamase; deoxyribonuclease; 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 brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT4, NT-5, or NT-6) or nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; bone morphogenetic proteins (BMP), including BMP1, BMP6, BMP7, and BMP receptor 2; insulin-like growth factor-I and insulin-like growth factor-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; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, TNF-α, IFN-α, GM-CSF, IL-3 or an antibody that binds to one or more tumor-associated antigens or cell surface receptors; erythropoietin; bone-inducing factor; immunotoxin; bone morphogenetic protein (BMP); interferons such as interferon-α, interferon-β and interferon-γ; colony-stimulating factor (CSF), for example M-CSF, GM-CSF and G-CSF; interleukins (IL), for example IL-2, IL-6, IL-12, IL-23, IL-12 / 23p40, 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 receptor; surface membrane protein; decay-accelerating factor; viral antigen, for example a part of the HIV envelope; transporter; homing receptor; addressin; regulatory protein; integrin such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens such as HER2, HER3 or HER4 receptor; endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, LGR5, B7H4, TAG72 (tumor-associated glycoprotein 72) and a fragment of any of the polypeptides listed above.

[0153] Examples of antibodies or fragments thereof that can be administered include, but are not limited to, anti-PD-L1 antibodies, abciximab (Reopro), adalimumab (Humira, Amjevita), alefacept (Amevive), alemtuzumab (Campath), basiliximab (Simulect), belimumab (Benlysta), bezlotoxumab (Zinplava), canakinumab (Ilaris), certolizumab (Cimzia), cetuximab (Erbitux), daclizumab (Zenapax, Zinbryta), denosumab (Prolia, Xgeva), efalizumab (Raptiva), golimumab (Simponi, Simponi Aria), infliximab (Remicade), ipilimumab (Yervoy), ixekizumab (Taltz), natalizumab (Tysabri), nivolumab (Opdivo), olaratumab (Lartruvo), omalizumab (Xolair), palivizumab (Synagis), panitumumab (Vectibix), pembrolizumab (Keytruda), rituximab (Rituxan), tocilizumab (Actemra), trastuzumab (Herceptin), secukinumab (Cosentyx), ranibizumab, abciximab, reslizumab, caplacizumab, infliximab, bevacizumab, dabigatran, idarucizumab, or ustekinumab (Stelara) or combinations thereof.In addition, the antibody can be selected from anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-EGFR antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis 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, 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-CD41 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-light chain lambda antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn antigen antibody.

[0154] If the administration times of the therapeutic agents are such that the pharmacological activities of the additional therapeutic agent and the active ingredient in the pharmaceutical composition overlap in time, thereby exerting a combined therapeutic effect, co-administration does not require simultaneous administration of the therapeutic agents. Generally, each agent will be administered at the dose determined for that agent and according to a schedule.

[0155] The pharmaceutical composition of the present invention can be administered at any suitable dose to achieve the desired result. In some embodiments, the desired result is to reduce the intensity, severity, frequency, and / or delay the onset of one or more symptoms of the infection. In some embodiments, the desired result is to inhibit or prevent the infection. The required dose varies from subject to subject and depends on the species, age, weight, and general condition of the subject, the severity of the infection to be prevented or treated, the specific composition used, and the mode of its administration.

[0156] In some embodiments, the pharmaceutical composition according to the present disclosure is administered in a single dose or multiple doses. In some embodiments, the pharmaceutical composition is administered in multiple doses administered on different dates.

[0157] As used herein, the term "about" means ±10% of the relevant value.

[0158] Certain aspects and embodiments of the present disclosure are further described with reference to the following examples.

[0159] Example

[0160] Example 1 – Overexpression of ETV2 increases the yield of hematopoietic endothelial cells and enhances the formation of CD34+ cells during iPSC differentiation, but does not affect pluripotency.

[0161] 方法

[0162] iPSCs were developed from hCD34+ cells by episomal reprogramming known in the art and were substantially as described by 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 body and hematopoietic endothelial differentiation were substantially as described in the following references: R. Sugimura, et al., Haematopoietic stem and progenitor cells from human pluripotent stem cells . Nature 545, 432 - 438, (2017); C. M. Sturgeon, et al., Wnt signaling controls the specificationofdefinitive 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 inducedpluripotent stem cells free of vector and transgene sequences . Science 324, 797 - 801, (2009).

[0163] Briefly, hiPSCs were dissociated and resuspended in a medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. Next, the cells were seeded in 10 cm culture 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 a medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with a medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with a medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. The cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs were dissociated on day 8, the cells were filtered through a 70 μm filter, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0164] result

[0165] An adenoviral vector containing the ETV2 and GFP sequences under the control of the EF1A promoter was used to transduce induced pluripotent stem cells (iPSCs). After transduction, approximately 45% of the iPSC culture was observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent characteristics of iPSCs, as shown by the expression of the stem cell marker TRA-1-60 ( Figure 1 ). Figure 1 FACS plots showing the transduction efficiency of iPSCs overexpressing the ETV2 and GFP sequences with an adenoviral vector.

[0166] Next, ETV2-OE-iPSCs (and control iPSCs transduced with a vector carrying the GFP sequence but not ETV2) were differentiated into embryoid bodies and subsequently into hematopoietic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 promoted the formation of hematopoietic endothelial cells, as demonstrated by the expression of CD235a - within the CD34 + and CD31 + markers ([[]] Figure 2 ). Specifically, Figure 2 Representative flow cytometric analysis of hematopoietic endothelial cells (defined here as CD235a-CD34+CD31+) is shown, and relative quantification indicates that ETV2-OE enhances the formation of hematopoietic endothelial cells compared to the control.

[0167] In addition, the results showed that ETV2-OE enhanced the formation of CD34 + cells ([ Figure 3 Figure 3 ). Figure 3 Representative flow cytometry analysis showing CD34+ cells, and relative quantification indicated that ETV2-OE enhanced the formation of CD34+ cells.

[0168] Overall, these data indicate that overexpression of ETV2 in iPSCs does not affect their pluripotency and promotes their ability to undergo hematopoietic endothelial and hematopoietic differentiation.

[0169] Example 2 - iPSC-derived HSCs generated using Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs.

[0170] method

[0171] To analyze EHT, EB-derived CD34+ cells were suspended in a 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 wells for approximately 4 to 18 hours (by visual inspection), Yoda1 was added to the cultures for some experiments. After 4 to 7 days, the cells were harvested for analysis.

[0172] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells were harvested from the iPSC-derived embryoid bodies and cultured for an additional 5 to 7 days to induce the endothelial-to-hematopoietic cell (EHT) transition. Then, CD34+ cells were harvested from the EHT cultures between days 5 and 7 for further hematopoietic lineage differentiation.

[0173] CD34+ cells harvested between days 5 and 7 (or a total of days 13 to 21 from iPSC differentiation) from the EHT cultures were seeded into 48-well plates pre-coated with rhDL4 and recombinant human fibrin fragments. T lineage differentiation was induced in a medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1a, and SB203580.

[0174] On days 2 to 6, 80% of the medium was replaced every other day. On D7, the cells were transferred to a new coated plate and the presence of pro-T cells (CD34+CD7+CD5+ / -) was analyzed.

[0175] From day 8 to day 13, replace 80% of the medium every other day. At D14, transfer 100,000 cells / well to a new coated plate and analyze for the presence of pre-T cells (CD34-CD7+CD5+ / -) in the cells.

[0176] From day 15 to day 20, replace 80% of the medium every other day. Harvest the cells at D21 and analyze the expression of CD3, CD8, CD5, CD7, TCRab in the cells via FACS as a surrogate for T cells, and / or activate with CD3 / CD28 beads to evaluate their functional properties.

[0177] After 21 days of differentiation, collect the cells and re-seed approximately 80,000 cells into a new 96-well culture plate in RPMI 1640 (without L-glutamine; without phenol red) plus FBS, L-glutamine, IL-2, and then activate with 1:1 CD3 / CD28 beads. After activation with CD3 / CD28 beads for 72 hours, analyze the expression of CD3, CD69, CD25 in the cells by FACS and analyze IFN-γ expression using RT-qPCR. Analyze the supernatant by ELISA.

[0178] result

[0179] Figure 4A and Figure 4B showed that iPSC-derived HSCs activated with Piezo1 underwent pro-T cell differentiation similar to that of bone marrow (BM)-HSCs. In addition, Figure 5A and Figure 5B showed that iPSC-derived HSCs generated by activation with Piezo1 underwent T cell differentiation and could be activated with CD3 / CD28 beads similar to BM-HSCs. Figure 6 showed that iPSC-derived HSCs generated by activation with Piezo1 could differentiate into functional T cells, as demonstrated by INFγ expression after stimulation with CD3 / CD28 beads. Collectively, these results indicate that activation of Piezo1 during HSC formation enhances the ability of HSCs to further differentiate into progenitor T cells and functional T cells in vitro, as well as related lineages such as NK cells.

[0180] Figure 7 showed that HSCs differentiated from iPSCs (D8+7) differentiated into NK cells and were activated by pharmacological stimulation. Figure 7 showed that HSCs differentiated from iPSCs (D8+7, with Yoda1 or "Y") were pharmacologically activated (measured by the activity of the functional marker CD107a).

[0181] Fig. 8A and Figure 8BIt is shown that HSC-derived NK cells (D8+7) are superior to bone marrow-derived NK cells in killing tumor cells. Fig. 8A and 8B It is shown that HSC-derived NK cells have stronger killing ability against tumor cells compared with BM-derived NK cells. The ratio of NK cells to tumor cells is 5:1.

[0182] Example 3 – Evaluation of off-target editing in HLA-disrupted HSCs

[0183] HLA typing of HSC clones edited for HLA was performed to check for unwanted editing and to ensure that no major editing events such as deletions occurred in other regions of chromosome 6. Sequencing methods and analyses were performed to evaluate the extent of gRNA off-target activity, and gRNAs representing low risk of affecting off-target HLA genes were selected.

[0184] Sequencing was performed using in situ break labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to the ends of the 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). Subsequently, DNA sequencing was performed on the DNA library enriched for DNA-labeled fragments, removing all foreign, non-functional DNA. Since the library preparation did not contain PCR, each sequencing read obtained corresponded to a single labeled DSB end from the cell. This generated DNA break reads, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction and enabling the generation of a clear list of off-target mutations.

[0185] Table 1 below summarizes the results of the editing strategies of two representative HLA-edited clones relative to wild-type cells (gHSCs).

[0186] Table 1: Clonal HSC HLA knockouts.

[0187]

[0188]

[0189] Table 2 provides non-limiting examples of gRNAs used in the experiments, which can be used to knock out the expression of the designated HLA genes.

[0190] Table 2: Exemplary gRNA sequences

[0191]

[0192]

[0193] The results showed that the editing strategy successfully and selectively targeted the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0194] These results were confirmed by phenotypic analysis of HLA-edited clones by FACS and immunofluorescence. As Fig.9A and 9B shown, the overall expression of class I HLA molecules in HLA-edited cells tested positive compared to the overall expression of class I HLA molecules in wild-type cells. Specific expression of HLA-A by immunofluorescence confirmed that HLA-A was not expressed in HLA-edited cells, confirming the finding that the gene editing strategy only successfully deleted the HLA-A gene. Specifically, Fig.9A showed that HLA-edited cells were positive for class I HLA to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that, despite the HLA-A deletion, other class I molecules such as HLA-B and C were expressed and not affected by the gene editing strategy.

[0195] To confirm the HLA-A gene deletion, specific expression of HLA-A was analyzed by immunofluorescence. As Fig. 9B could be seen, HLA-A was not expressed in HLA-edited clones, indicating that the gene editing strategy was efficient only in specifically deleting the HLA-A gene. Such retention of all class I expression and HLA-A deletion would facilitate patient matching while avoiding NK cell-mediated rejection.

[0196] Example 4 - Evaluation of the pluripotency and immunocompatibility of HLA-edited HSCs

[0197] Evaluate the ability of HLA-edited cells to retain pluripotency. As Fig.11 shown, immunofluorescence evaluation of HLA-edited iPSC clones indicated that they maintained trilineage differentiation, with ectodermal differentiation indicated by nestin-488 and PAX6-594 staining, mesodermal differentiation indicated by GATA-488 staining, and endodermal differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0198] HLA class I molecules are expressed on the surface of all nucleated cells, and if there is a mismatch of HLA class I molecules between the donor and the recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatch may lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). Conversely, complete deletion of HLA-I molecules by B2M KO will make the cells targets of NK cell-mediated cytotoxicity. Retaining all class I expression and deleting HLA-A can facilitate patient matching while preventing NK cell-mediated rejection. Therefore, the immunocompatibility of HLA-edited HSCs was tested by co-culture with peripheral blood mononuclear cells (PBMCs) to evaluate whether immune cells would reject the grafts of HLA-edited and wild-type HSCs (gHSCs).

[0199] Wild-type (gHSCs) and HLA-edited HSCs were co-cultured with PBMCs that were matched for HLA-B and HLA-C markers but mismatched for HLA-A. B2M KO HSCs lacking HLA class I molecule expression and CIITA KO HSCs lacking HLA class II molecule expression were used as controls to compare the degrees of HLA-null and HLA-mismatched PBMC-mediated cytotoxicity, respectively. Fig.12 Results of PBMC-mediated cytotoxicity assays in co-cultures measured by Annexin V staining are shown. The results show that deletion of HLA-A in HLA-edited HSCs protects the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO are vulnerable to PBMC-mediated cytotoxicity. HSCs co-cultured with sorted CD8+ T cells from the same PBMC donor protect HLA-edited and B2M KO HSCs from CD8+ T cell cytotoxicity. Conversely, HSCs co-cultured with sorted NK cells protect only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0200] In summary, the immunocompatibility results show that CD8+ T cells present in the PBMC samples are responsible for killing cells with HLA molecule (WT and CIITA KO) mismatches, while NK cells present in the PBMCs are responsible for killing HLA-null cells (B2M KO). However, HLA-edited HSCs are protected from CD8+ T cell-mediated cytotoxicity (because the mismatched HLA-A has been knocked out) and from NK cell-mediated cytotoxicity (because HLA class I molecule expression is largely retained).

[0201] Example 5 - Evaluation of the in vivo engraftment potential of HLA-edited HSCs

[0202] To evaluate the transplantation potential of HLA-edited HSCs, the in vivo engraftment ability of the cells was assessed by competitive transplantation against WT HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs were mixed and transplanted into mice, where bone marrow (BM) and peripheral blood samples were recovered and evaluated by FACS to compare the relative amounts of each cell type present in the samples. As Fig.12 shown, both HLA-edited HSCs and WT HSCs contributed to roughly equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to WT HSCs in terms of their transplantation and reconstitution potential. Thus, the properties of WT (unedited, parental) HSCs are expected to be consistent with those of the HLA-edited HSCs of the present disclosure for generating T cell lineages.

[0203] Example 6 - Evaluation of Degranulation and Cytotoxicity of Immunocompatible HSC-Derived NK Cells

[0204] Next, the ability of HSCs to differentiate into NK cells that retain their degranulation and cytotoxicity capabilities was evaluated. As Fig.13 shown, 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 demonstrated a differentiation ability at least comparable to that of 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 Fig.14A was performed. HSC-derived NK cells were co-cultured with K562 HLA-null cells for 3.5 hours. K562 HLA-null cells are a human erythroleukemia cell line derived from the pleural effusion of a patient with chronic myeloid leukemia. These cells express ligands for aNKR, and they lack HLA cell surface expression and also contribute to NK cell activation by eliminating negative signaling through iNKR. Thus, these HLA-null cell lines have the potential to induce different functional characteristics in NK cells and their subsets. After co-culture, FACS and annexin V staining as well as cytotoxicity assays were used to measure the extent of NK cell degranulation. As Fig. 14B shown, annexin V staining indicated that HSC-derived NK cells exhibited a higher degree of activation from HLA-null K562 cells compared to CD34+ BM and iPSC-EB CD34+ cells. This was confirmed by the results of the cytotoxicity assays, as Fig. 14C shown.

[0205] References

[0206] 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).

[0207] 2. Brauer, P.M., Singh, J., Xhiku, S. & J.C. T Cell Genesis: In Vitro Veritas Est? Trends Immunol 37, 889–901 (2016).

[0208] 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).

[0209] 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).

[0210] 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).

[0211] 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).

[0212] 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).

[0213] 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).

[0214] 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).

[0215] 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21–33 (2020).

[0216] 11. Nagano, S. et al. High Frequency Production of T Cell-Derived iPS Clones Capable of Generating Potent Cytotoxic T Cells. Molecular Therapy-Methods & Clinical Development 16, 126–135 (2020).

[0217] 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. A method for preparing a population of NK cells or their progenitor cells, the method comprising: Enriching CD34+ cells from a population of differentiated pluripotent stem cells (PSCs) to prepare a CD34+-enriched population; Inducing the CD34+-enriched cell population to undergo an endothelial-to-hematopoietic cell transition for at least two days but not more than 12 days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitors (HSPCs); and Differentiating the population comprising HSCs and / or HSPCs into a progenitor NK cell population or an NK cell population.

2. The method according to 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 tissues.

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

4. The method according to claim 2 or 3, wherein the iPSC is homozygous for one or more HLA class I and / or class II genes.

5. The method according to claim 4, wherein the iPSC is homozygous for HLA-DRB1.

6. The method according to claim 4, wherein the iPSC is homozygous for both HLA-B and HLA-C.

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

8. The method according to claim 7, wherein the iPSC comprises a deletion of HLA-A.

9. The method according to claim 7 or 8, wherein the iPSC contains a deletion of HLA-DPB1 and / or HLA-DQB1.

10. The method according to any one of claims 2 to 9, wherein the iPSC is HLA-A neg and homozygous for both HLA-B and HLA-C, HLA-DPB1 neg and HLA-DQB1 neg , and optionally further homozygous for HLA-DRB1.

11. The method according to claim 7, wherein the one or more genes that govern the expression or presentation ability of HLA or MHC are β2-microglobulin and / or CIITA.

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

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

14. The method according to claim 13, wherein the endothelial-to-hematopoietic cell transition generates an HSC population that comprises one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem cell progenitors.

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

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

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

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

19. The method according to claim 18, wherein the cyclic stretch is 2D, 3D or 4D cyclic stretch.

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

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

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

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

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

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

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

27. The method according to 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. The method according to any one of claims 24 to 27, wherein the Notch ligand is incorporated with an extracellular matrix component, the extracellular matrix component optionally selected from fibronectin, recombinant human fibrin fragment and laminin, derivatives or analogs thereof and / or combinations thereof.

29. The method according to claim 28, wherein the Notch ligand and / or extracellular matrix component is embedded in an inert material providing 3D culture conditions, and the inert material is optionally selected from cellulose, alginate, and combinations thereof.

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

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

32. The method according to any one of claims 24 to 31, wherein the HSC population or a fraction thereof is cultured in an artificial thymic organoid, which optionally comprises a Notch ligand selected from one or more of BMP2, delta-like 1 (DLL1), delta-like 4 (DLL4), SFIP3, and delta 最大 among others.

33. The method according to any one of claims 24 to 32, wherein the HSC population or 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 according to any one of claims 24 to 33, wherein the generated T cells or progenitor T cells are further cultured in the presence of IL-3 and / or IL-15.

35. The method according to claim 34, wherein, IL-15 is added to support NK cell differentiation, and optionally IL-3 is not included after early NK cell formation.

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

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

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

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

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

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

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

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

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

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