Hematopoietic lineage derived from pluripotent stem cells

Abstract

The present disclosure provides, in various aspects and embodiments, methods for generating hematopoietic lineage cells for hematopoietic cell therapy. Various hematopoietic lineages include T lymphocytes, including precursor T cells, natural killer cells, B lymphocytes, neutrophils, monocytes and / or macrophages, erythrocytes, megakaryocytes, and platelets. In various embodiments, the present invention provides an efficient ex vivo process for generating hematopoietic lineages, including but not limited to precursor T cells and T cell lineages, from human induced pluripotent stem cells (iPSCs). In various embodiments, the cells generated according to the present disclosure are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention provides, in some aspects, isolated cells and cell compositions produced by the methods disclosed herein, as well as methods of adoptive cell therapy.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 168,360, filed March 31, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The generation of hematopoietic cells from pluripotent cells ex vivo has attracted the interest of the scientific community for the possibility of allogeneic matched cell-based therapy. Induced pluripotent stem cells (iPSCs) could serve as a source for generating "off-the-shelf" therapeutic lymphocytes (Non-Patent Document 1). However, methods to generate clinically relevant numbers of hematopoietic cell lineages (e.g., immune cells, lymphocytes, etc.) and to generate cell lineages with clinically advantageous phenotypes remain a major obstacle. In various aspects and embodiments, the present invention achieves these objectives. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Nianias, A., & Themeli, M., Induced pluripotent stem cell (iPSC)-derived lymphocytes for adoptive cell immunotherapy: recent advances and challenges. Current Hematologic Malignancy Reports, 14(4), 261-268 (2019) Summary of the Invention

[0004] The present disclosure provides, in various aspects and embodiments, methods for generating hematopoietic lineages, including T lymphocytes (T cells, including precursor T cells), natural killer cells (NK cells), B lymphocytes (B cells) (including B cells engineered to produce specific antibodies), neutrophils, monocytes and / or macrophages, megakaryocytes, erythrocytes, and platelets. In various embodiments, the cells generated 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, in some aspects, provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods of cell therapy.

[0005] In one aspect, the disclosure provides a method of preparing a cell population of hematopoietic lineage. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population, differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34-enriched population. Endothelial hematopoietic transition (EHT) is induced in the CD34-enriched population, thereby preparing a hematopoietic stem cell (HSC) population, followed by differentiation into hematopoietic lineages. In various embodiments, the hematopoietic lineages are selected from T lymphocytes (i.e., T cells), progenitor T cells, natural killer cells (NK cells), B lymphocytes (i.e., B cells), monocytes and / or macrophages, megakaryocytes, and platelets. In accordance with aspects and embodiments of the disclosure, it has been discovered that induction of endothelial hematopoietic transition (EHT) of a CD34+ cell population derived from iPSC embryoid bodies can be used to generate superior hematopoietic lineages ex vivo.

[0006] In some aspects and embodiments, the disclosure provides methods of generating a CD7+ progenitor T cell population, or a derivative of this population. For example, the method includes generating a hematopoietic stem cell (HSC) population comprising human long-term hematopoietic stem cells (LT-HSC) from iPSCs (e.g., hiPSCs). The HSC population results from induction of endothelial hematopoietic transformation of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). The HSC population (or cells isolated therefrom) is cultured with partial or complete Notch ligands (including but not limited to DLL4, DLL1, SFIP, etc.), sonic hedgehog (SHH), TNF-α, retronectin (or other extracellular matrix components), and / or combinations thereof to generate a population comprising CD7+ progenitor T cells or a derivative cell population. The present disclosure provides HSC populations generated ex vivo from iPSCs and responsive to Notch ligands, Sonic Hedgehog (SHH), and / or extracellular matrix component(s) with robust ex vivo generation of T cell progenitors and T cell lineages.

[0007] In various embodiments, iPSCs are prepared by reprogramming somatic cells, such as, but not limited to, fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes, umbilical cord blood cells, PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs can be gene edited to aid HLA matching, such as by deletion of one or more HLA class I and / or class II alleles or their key regulators.

[0008] In some embodiments, hiPSCs are used to generate embryoid bodies (EBs) that can be used to generate (i.e., isolate or enrich) CD34+ cells. For example, EBs can be dissociated and CD34+ hematopoietic progenitors isolated or enriched. In some embodiments, processes according to aspects can include generating CD34-enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial hematopoietic differentiation.

[0009] In some embodiments, CD34 enrichment and EHT can be induced on days 8-14 of iPSC differentiation. EHT can be induced using any process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population including LT-HSC. In some embodiments, EHT generates HSC via endothelial cells or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological and / or genetic means. In some embodiments, the method includes increasing expression or activity of dnmt3b in PSCs, EBs, CD34 enriched cells, ECs, HECs or HSCs, which can be done by mechanical, genetic, biochemical or pharmacological means.

[0010] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezo1. Exemplary Piezo1 agonists include Yoda1, Jedil, and Jedi2. In some embodiments, the mechanosensitive receptor is Trpv4. Exemplary Trpv4 agonists are GSK1016790A. In certain embodiments, Piezo1 activation is applied to at least CD34+ cells isolated from EBs, thereby allowing for the generation of superior T cell progenitors compared to other methods of inducing EHT, according to various embodiments.

[0011] 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. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6.

[0012] Generally, at various stages, the cell population can be enriched for cells of a desired phenotype and / or depleted of cells of an undesired phenotype. 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 proliferation of CD34+ cells, thereby producing a population of expanded stem cells. Hematopoietic stem cells (HSCs), which give rise to the erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (designated Lin-).

[0013] In various embodiments, the HSC population or a fraction thereof (e.g., the CD34+ fraction) is differentiated into a hematopoietic lineage that can be selected from progenitor T cells, T cells and fractions thereof, B cells, NK cells, neutrophils, monocytes or macrophages, megakaryocytes, erythrocytes, and platelets.

[0014] In some embodiments, the cell population is cultured ex vivo with at least a partial or complete Notch ligand (including but not limited to DLL4, DLL1, SFIP, etc.), Sonic Hedgehog (SHH), TNF-α, retronectin (or other extracellular matrix components), and / or combinations thereof to convert HSCs into CD7 +The precursor T cells are optionally differentiated into a T cell lineage or other lineages (e.g., NK cells). In various embodiments, the Notch ligand comprises at least one of Delta-like 1 (DL1) and Delta-like 4 (DL4), SFIP, or functional portions thereof. In various embodiments, the Notch ligand is immobilized, functionalized, and / or embedded in the 2D or 3D culture system. The Notch ligand may be incorporated with a component of the extracellular matrix.

[0015] In another aspect, the invention provides a cell population produced by the methods described herein, or a pharma- ceutically acceptable composition thereof, and use in a method of treatment or therapy. In some embodiments, the cell population is a lymphocyte population (e.g., a T cell progenitor population) capable of engrafting in the thymus, spleen, or secondary lymphoid organs following administration to a subject in need thereof.

[0016] Various other aspects and embodiments of the present disclosure will become apparent from the following detailed description. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 shows that ETV2 overexpression (OE) does not affect pluripotency. Figure 1 shows FACS plots showing the transduction efficiency of iPSCs with adenoviral vectors overexpressing ETV2 and GFP sequences. ETV2 overexpression does not affect iPSC stemness as shown by the expression of TRA-1-60 stemness marker. [Diagram 2] Figure 2 shows that ETV2 overexpression (OE) increases the yield of hemogenic endothelial cells. Representative flow cytometry analysis and relative quantification of hemogenic endothelial cells (designated CD235a-CD34+CD31+) demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells. [Diagram 3]1 shows that ETV2 overexpression (OE) enhances CD34+ cell formation during iPSC differentiation. Representative flow cytometry analysis and relative quantification of CD34+ cells demonstrates that ETV2-OE enhances CD34+ cell formation. [Figure 4A-4B] Figure 4 shows that iPSC-derived HSCs induced by Piezo1 activation undergo precursor T cell differentiation similar to bone marrow (BM)-HSCs. Figure 4A shows FACS plots of differentiation efficiency into CD34+CD7+ precursor T cells of bone marrow (BM)-HSCs and iPSC-HSCs induced by Piezo1 activation. Figure 4B shows quantification of CD34+CD7+ cells (%) induced by (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Figure 4B shows the average of three experiments. [Figure 5A-5B] Figure 5 shows that iPSC-derived HSCs generated by Piezo1 activation can undergo T cell differentiation and be activated with CD3 / CD28 beads similarly to BM-HSCs. Figure 5A is a FACS plot of activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs generated by Piezo1 activation. Figure 5B is a quantification of CD3+CD69+ cells (%) induced by (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Figure 5B shows the average of three experiments. [Figure 6] Figure 6 shows that iPSC-derived HSCs generated by Piezo1 activation can differentiate into functional T cells. IFNγ expression is a consequence of T cell activation following T cell receptor (TCR) stimulation via CD3 / CD28 beads. Expression of IFNγ in T cells differentiated from iPSC-derived HSCs generated by Piezo1 activation enhances the ability of HSCs to further differentiate into functional T cells. Figure 6 shows the average of three experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure provides, in various aspects and embodiments, methods for generating hematopoietic lineages for cell therapy. The various hematopoietic lineages include T lymphocytes (T cells, including precursor T cells), natural killer cells (NK cells), B lymphocytes (B cells) (including B cells engineered to produce specific antibodies), neutrophils, monocytes and / or macrophages, megakaryocytes, erythrocytes, and platelets. In various embodiments, the present invention provides an efficient ex vivo process for generating hematopoietic lineages, including but not limited to precursor T cells and T cell lineages, from human induced pluripotent stem cells (iPSCs). In various embodiments, the cells generated according to the present disclosure are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or lymphoid organs. The present invention provides, in some aspects, isolated cells and cell compositions produced by the methods disclosed herein, as well as methods of cell therapy.

[0019] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to produce essentially unlimited pluripotent stem cells (PSCs) is exploited to generate an unlimited source of hematopoietic cells, including but not limited to therapeutic human T lymphocytes ("T cells") or their progenitors. The use of primary T cells as therapeutic lymphocytes has been limited by limited availability, cell numbers, limited proliferation capacity, and histocompatibility issues. Furthermore, compared to primary cells, hiPSCs can be more easily subjected to genetic modification in vitro, thereby providing opportunities to improve cell targeting specificity, cell numbers, as well as circumventing HLA matching issues, for example. Furthermore, fully genetically engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). Moreover, unlike human embryonic stem cells (hESCs), hiPSCs are of non-embryonic origin and therefore are free of ethical concerns. Thus, the use of hiPSCs according to the present disclosure provides several advantages over primary cells to generate therapeutic hematopoietic lineages, such as T lymphocytes.

[0020] In one aspect, the present disclosure provides a method for preparing a cell population of hematopoietic lineage. The method includes preparing a pluripotent stem cell (PSC) population, such as an induced pluripotent stem cell (iPSC) population differentiated into embryoid bodies, and enriching for CD34+ cells, thereby preparing a CD34-enriched population. Endothelial hematopoietic transformation (EHT) is induced in the CD34-enriched population, thereby preparing a hematopoietic stem cell (HSC) population, optionally followed by further enrichment of CD34+ cells. The resulting HSC population (or a fraction thereof) is differentiated into a hematopoietic lineage. In various embodiments, the hematopoietic lineage is selected from T lymphocytes (i.e., T cells), progenitor T cells, natural killer cells (NK cells), B lymphocytes (i.e., B cells), monocytes and / or macrophages, megakaryocytes, and platelets.

[0021] Traditionally, hematopoietic lineages are prepared by differentiating iPSCs into embryoid bodies up to day 8 to harvest CD34+ cells. CD34 is commonly used as a marker for hemogenic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been discovered that induction of endothelial hematopoietic transition (EHT) of CD34+ cell populations that can be derived from iPSC embryoid bodies can be used to generate superior hematopoietic lineages ex vivo.

[0022] In some aspects and embodiments, the disclosure provides a method of generating a CD7+ progenitor T cell population, or a derivative of this population. For example, the method includes generating a hematopoietic stem cell (HSC) population comprising human long-term hematopoietic stem cells (LT-HSC) from iPSCs (e.g., hiPSCs). The HSC population is derived by inducing endothelial hematopoietic transformation of CD34+ cells (e.g., CD34+ cells derived from embryoid bodies). The HSC population (or cells isolated therefrom) is cultured with partial or complete Notch ligand, sonic hedgehog (SHH), retronectin (or other extracellular matrix component(s)), and / or combinations thereof to generate a population comprising CD7+ progenitor T cells or a derivative cell population.

[0023] The Notch signaling pathway regulates the formation, differentiation, and function of precursor T cells, pre-T cells, and / or mature T lymphocytes. In vivo, T cell development proceeds after lymphoid progenitor cells differentiate from bone marrow hematopoietic stem cells and migrate to the thymus. Specialized thymic epithelial cells guide T cell development along a regulated pathway. Notch signaling plays a key role during T cell lineage commitment in the thymus. As lymphoid progenitor cells enter the thymus, they encounter high density expression of Notch ligands on the thymic epithelium, promoting thymopoiesis. The present disclosure provides HSC populations generated ex vivo from iPSCs and respond to Notch ligands, SHH, and / or components of the extracellular matrix with robust ex vivo production of T progenitor cells and T cell lineages.

[0024] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cells" or "iPSCs" refers to cells derived from somatic cells, such as skin cells or blood cells, that have been reprogrammed 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., T cells, B cells, NK cells, etc.), cord blood cells (including CD3+ cells or CD8+ cells derived from cord blood), PBMCs, CD34+ cells, or other primary human tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) with respect to the recipient (subject in need of the treatment described herein). In various embodiments, iPSCs can be genetically edited to aid in HLA matching (e.g., deletion of one or more HLA class I and / or class II alleles or their key regulators, including but not limited to β2-microglobulin (B2M), CIITA, etc.) or to delete or express other functions. For example, iPSCs can be genetically edited to delete one or more of HLA-A, HLA-B, and HLA-C, or to delete one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one of the retained class I and class II loci. In some embodiments, iPSCs are derived from T cells, e.g., with known or unknown TCR specificity. In some embodiments, the T cells bear a TCR with specificity for a tumor-associated antigen.

[0025] Somatic cells can be reprogrammed by expression of reprogramming factors selected from Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, Nanog, Lin28, and klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c-Myc, and klf4. Methods for preparing iPSCs are described, for example, in U.S. Pat. No. 10,676,165, U.S. Pat. No. 9,580,689, and U.S. Pat. No. 9,376,664, which are incorporated by reference in their entirety. In various embodiments, the reprogramming factors are expressed using known viral vector systems, such as lentivirus, Sendai virus, or measles virus systems. Alternatively, the reprogramming factors can be expressed by introducing mRNA(s) encoding the reprogramming factors into the somatic cells. Furthermore, iPSCs may be generated by introducing non-integrated episomal plasmids expressing reprogramming factors, i.e., non-integrated episomal plasmids for generating transgene-free and virus-free iPSCs. Known episomal plasmids that have limited replicative capacity and are therefore lost within a few cell generations may also be used.

[0026] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are genetically edited. Genetic editing can include, but is not limited to, modification of 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 T cell receptor (TCR) genes, or addition of chimeric antigen receptor (CAR) genes. Exemplary CARs can target CD19, CD38, CD33, CD47, CD20, etc. For example, the iPSCs can be T cell receptor (TCR) transduced iPSCs. Such embodiments allow for large-scale production of regenerative T lymphocytes with desired antigen specificity. Alternatively, engineered iPSCs with one or more HLA knockouts and TCR knockouts can be placed in feeder- and serum-free differentiation bioreactors under GMP-grade conditions to generate fully functional, histocompatible T cells.

[0027] In some embodiments, the iPSCs are + T-iPSCs are prepared from cells or, in some embodiments, T lymphocytes (e.g., CTLs). For example, T lymphocytes can be isolated with a desired antigen specificity (e.g., using cell sorting with HLA-peptide ligands) and reprogrammed into T-iPSCs. These T-iPSCs are then redifferentiated into progenitor T cells, or their derivatives or T cell lineages, according to the present disclosure. When T-iPSCs are generated from antigen-specific T cells, they inherit rearranged T cell receptor (TCR) genes. In these embodiments, CTLs redifferentiated from T-iPSCs exhibit the same antigen specificity as the original CTLs.

[0028] In some embodiments, hiPSCs are used to generate embryoid bodies (EBs) that can be used to generate (i.e., isolate or enrich) CD34+ cells. For example, EBs can be dissociated and CD34+ hematopoietic progenitors isolated or enriched. In some embodiments, human iPSC aggregates are grown in a bioreactor, e.g., as described in Abecasis B. et al., Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches. J. of Biotechnol. 246 (2017) 81-93.

[0029] In some embodiments, the process according to each aspect can include generating CD34-enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial hematopoietic differentiation. HSCs, including relatively high frequencies of LT-HSCs, can be generated from cell populations using a variety of stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimuli, and factors such as extracellular matrix, niche factors, cell-extrinsic factors, induction of cell-intrinsic properties, and pharmacological and / or genetic means.

[0030] In some embodiments, the method includes preparing hematopoietic endothelial cells from pluripotent stem cells prior to induction of EHT. In some embodiments, combined overexpression of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can lead to the formation of hematopoietic endothelial cells from a PSC source. In some embodiments, the method includes overexpression of E26 transformation specific mutant 2 (ETV2) transcription factor in iPSCs. After CD34+ enrichment, HSCs are then generated from endothelial cells using mechanical, biochemical, pharmacological and / or genetic stimulation or modification. ETV2 can be expressed by constitutive or inducible expression of ETV2 and introduction of a coding non-integrated episomal plasmid for the production of transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. The mRNA can be introduced using any available method, including electroporation or lipofection. Differentiation of 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). Cells generated in this manner can be used to produce CD34+ cells and induce EHT according to embodiments of the present disclosure.

[0031] In some embodiments, CD34 enrichment and EHT can be induced on days 8-14 of iPSC differentiation, such as, for example, days 8, 9, 10, 11, 12, 13, or 14. Differentiation of iPSCs can be by known techniques. In some embodiments, iPSC differentiation involves factors such as, but not limited to, combinations of bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, hPSCs are differentiated using feeder-free, serum-free, and / or GMP-compliant materials. In some embodiments, hPSCs are co-cultured with mouse bone marrow-derived feeder cells, such as OP9 or MS5 cell lines, in serum-containing medium. Cultures can include growth factors and cytokines to support differentiation into embryoid bodies or monolayer systems. The OP9 co-culture system can be used to generate multipotent HSPCs that can be further differentiated into several hematopoietic lineages, including T lymphocytes, B lymphocytes, megakaryocytes, monocytes or macrophages, and erythrocytes. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells, Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using conditions defined by specific signals can be used. For example, expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs promotes direct differentiation into multipotent CD34+ / CD45+ progenitors. Furthermore, expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs.See Doulatov S. et al., Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors, Cell Stem Cell. 2013 Oct 3; 13(4).

[0032] Induction of EHT can be performed using any known process. In some embodiments, induction of EHT generates a hematopoietic stem cell (HSC) population including LT-HSC. In some embodiments, EHT generates HSC via endothelial cells or hemogenic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological and / or genetic means (e.g., via stimulation, inhibition and / or genetic modification). In some embodiments, EHT generates a stem cell population including one or more of long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic stem progenitor cells.

[0033] In some embodiments, the method comprises increasing expression or activity of dnmt3b in PSCs, embryoid bodies, CD34-enriched cells, ECs, HECs or HSCs, which can be done by mechanical, genetic, biochemical or pharmacological means. In some embodiments, the method comprises increasing activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO 2019 / 236943 and WO 2021 / 119061, which are incorporated by reference in their entireties. In some embodiments, the induction of EHT comprises increasing expression or activity of dnmt3b.

[0034] In some embodiments, the cells are contacted with an effective amount of an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezo1. An exemplary Piezo1 agonist is Yoda1. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed for the mechanosensitive ion channel Piezo1 (Syeda R, Chemical activation of the mechanotransduction channel Piezo1. eLife (2015)). Yoda1 has the following structure: [ka]

[0035] Derivatives of Yoda1 can be used in various embodiments. For example, derivatives containing a 2,6-dichlorophenyl core are used in some embodiments. Exemplary agonists are disclosed in Evans EL, et al., Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation, British J. of Pharmacology 175(1744-1759): 2018. Still other Piezo1 agonists include Jedi1, Jedi2, and their derivatives and analogues. 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. 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, about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM.

[0036] 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, hemogenic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineage(s). In certain embodiments, Piezo1 activation is applied to at least EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof, thereby enabling superior generation of T progenitor cells compared to other methods of inducing EHT according to various embodiments.

[0037] Alternatively or additionally, the activity or expression of Dnmt3b can be increased directly in cells, for example, CD34-enriched cells. For example, the mRNA expression of Dnmt3b can be increased by transgene-free methods, including but not limited to, delivering a transcript encoding Dnmt3b to cells, or introducing a transgene encoding Dnmt3b, or introducing a non-integrated episome into cells. In some embodiments, gene editing is used to introduce genetic modifications into the Dnmt3b expression element of cells, for example, but not limited to, to increase promoter strength, ribosome binding, RNA stability, and / or to affect RNA splicing.

[0038] In some embodiments, the method includes increasing the activity or expression of Gimap6 in a cell, alone or in combination with Dnmt3b and / or other genes that are up-regulated or down-regulated after repeat distortion or Piezo1 activation. To increase the activity or expression of Gimap6, an mRNA transcript encoding Gimap6 can be introduced into the cell, and a transgene-free approach, including but not limited to, introducing an episome into the cell, or alternatively a transgene encoding Gimap6 can be used. In some embodiments, gene editing is used to introduce genetic modifications (e.g., one or more modifications to increase promoter strength, ribosome binding, RNA stability, or affect RNA splicing) into a Gimap6 expression element of the cell.

[0039] In the embodiment of the present disclosure using mRNA delivery to cells, known chemical modifications can be used to avoid innate immune response in cells. For example, synthetic RNA containing only canonical nucleotides can bind to pattern recognition receptors and induce a strong immune response in cells. This response can cause translation block, secretion of inflammatory cytokines, and cell death. RNA containing certain non-canonical nucleotides can avoid detection by the innate immune system and can be translated into protein with high efficiency. See U.S. Patent No. 9,181,319 (incorporated herein by reference) for nucleotide modifications to avoid innate immune response in particular.

[0040] In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into the cell that can direct the desired level of overexpression (by varying promoter strength or other selection of expression control elements). The transgene can be introduced using various viral vectors or transfection reagents (including lipid nanoparticles) known in the art. In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by transgene-free methods (e.g., episomal delivery). In some embodiments, the expression or activity of Dnmt3b and / or Gimap6 or other genes disclosed herein is increased using gene editing techniques to introduce one or more modifications to increase, for example, promoter strength, ribosome binding, or RNA stability.

[0041] A variety of editing techniques are known and can be applied in accordance with various embodiments of the present disclosure. Gene editing techniques include, but are not limited to, CRISPR-Cas (e.g., CRISPR-Cas9), zinc finger (ZF), and transcription activator-like effector (TALE). Fusion proteins comprising one or more of these DNA binding domains and the cleavage domain of Fok1 endonuclease can be used to generate double stranded breaks in desired regions of DNA within a cell (see, e.g., U.S. Patent Application Publication No. 2012 / 0064620, U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. 2011 / 0301073, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the contents of all of which are incorporated herein by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system (e.g., CRISPR-Cas9) known in the art. See, e.g., U.S. Patent No. 8,697,359, U.S. Patent No. 8,906,616, and U.S. Patent No. 8,999,641, which are incorporated herein by reference in their entireties.

[0042] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to the cells. In various embodiments, the cells subjected to cyclic 2D, 3D, or 4D stretch are selected from one or more of CD34 enriched cells, iPSCs, ECs, and HECs. For example, the cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretching, as described in WO 2017 / 096215, which is incorporated by reference in its entirety. The cyclic strain biomechanical stretching can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, a mechanical means applies a stretching force to the cells, or to a cell culture surface on which the cells (e.g., ECs or HECs) are cultured. For example, a computer-controlled vacuum pump system or other means of providing a stretching force (e.g., FlexCell™ Tension System, Cytostretcher System) attached to a flexible biocompatible and / or biomimetic surface can be used to apply cyclic 2D, 3D, or 4D stretch to cells ex vivo under defined, controlled, cyclic strain conditions. For example, the cyclic stretch applied can be about 1% to about 20% cyclic strain (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours.

[0043] Alternatively or additionally, EHT is stimulated by Trpv4 activation, which can be achieved by contacting cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, optionally selected from GSK1016790A, 4alpha-PDD, or analogs and / or derivatives thereof.

[0044] Generally, at various stages, the cell population can be enriched for cells of a desired phenotype and / or depleted of cells of an undesired phenotype. Such positive and negative selection methods are known in the art. For example, cells can be sorted based on cell surface antigens (including those described herein) using a fluorescence activated cell sorter or magnetic beads that bind cells to certain cell surface antigens. Negative selection columns can be used to remove cells expressing undesirable cell surface markers. In some embodiments, the cells are enriched (before and / or after undergoing EHT) for CD34+ cells. In some embodiments, the cell population is cultured under conditions that promote the proliferation of CD34+ cells, thereby producing a population of expanded stem cells.

[0045] In various embodiments, CD34+ cells (eg, suspension and / or adherent cells) are harvested from cultures undergoing endothelial-hematopoietic conversion between days 8 and 15 of iPSC differentiation.

[0046] In various embodiments, the HSCs or CD34 enriched cells are further expanded. For example, the HSCs or CD34 enriched cells can be expanded according to the methods disclosed in U.S. Patent No. 8,168,428, U.S. Patent No. 9,028,811, U.S. Patent No. 10,272,110, and U.S. Patent No. 10,278,990, which are incorporated herein by reference in their entirety. In some embodiments, the ex vivo expansion of the HSCs or CD34 enriched cells uses prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs comprise at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0047] Hematopoietic stem cells (HSCs), which give rise to the erythroid, myeloid, and lymphoid lineages, can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, a population of stem cells containing HSCs is enriched, for example, as described in U.S. Pat. No. 9,834,754, which is incorporated herein by reference in its entirety. For example, this process can include sorting the cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. CD34 + , CD90 + , CD38 - , and CD43 - One or more fractions of the CD34+ / CD48+ / CD39+ / CD4 ... + or at least about 90% CD34 + or at least about 95% CD34 + It is.

[0048] In some embodiments, the stem cell population, or CD34 enriched cell or fraction thereof, or derivative population is expanded as described in US Patent Publication No. 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells are expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55 and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells. Science 2010 Sep 10;329(5997):1345-1348.

[0049] In some embodiments, CD34 +Compounds that promote cell proliferation include, for example, pyrimidoindole derivatives including UM171 or UM729 (see U.S. Patent Application Publication No. 2020 / 0308540, incorporated herein by reference).

[0050] In some embodiments, the stem cell population or CD34 enriched cells are further enriched for cells expressing periostin and / or platelet derived growth factor receptor alpha (pdgfra) or cells modified to express periostin and / or pdgfra, as described in WO 2020 / 205969, which is incorporated by reference in its entirety. Such expression can be by transgene-free methods, including but not limited to delivering an encoding transcript to the cell, introducing an encoding transgene, or introducing a non-integrated episome into the cell. In some embodiments, gene editing is used to introduce genetic modifications into expression elements of the cell, for example to modify promoter activity or strength, ribosome binding, RNA stability, or to affect RNA splicing.

[0051] In yet another embodiment, the stem cell population or CD34-enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely deleted or inactivated, or temporarily suppressed, in the stem cell population. Inhibition of EZH1 can direct bone marrow progenitor cells (e.g., CD34+CD45+) to lymphoid lineages. See WO2018 / 048828, which is incorporated herein by reference in its entirety. In yet another embodiment, EZH1 is overexpressed in the stem cell population.

[0052] In various embodiments, the HSC population or fractions thereof are differentiated into hematopoietic lineages that can be selected from progenitor T cells, T cells and fractions thereof, B cells, B cells custom designed to produce certain antibodies, NK cells, neutrophils, monocytes or macrophages, megakaryocytes, erythrocytes, and platelets.

[0053] In some embodiments, the cell population is cultured ex vivo with a partial or complete Notch ligand, SHH, extracellular matrix component(s), and / or combinations thereof to convert HSCs into CD7 + The precursor T cells are optionally differentiated into T cell lineages or other lineages (e.g., NK cells). Furthermore, according to known processes, heterologous OP9-DL1 cells are often used for differentiation into T cells. The OP9-DL1 co-culture system uses a bone marrow stromal cell line (OP9) transduced with Notch ligand delta-like 1 (DLL1) to support T cell development from a stem cell source. The OP9-DL1 system limits the potential of the cells for clinical use. There is a need for a feeder cell-free system that can generate T lymphocytes from hiPSCs for clinical use, and in some embodiments, the present invention achieves this goal.

[0054] The term "Notch ligand" as used herein refers to a ligand capable of binding to a Notch receptor polypeptide present on the membrane of a hematopoietic stem cell or progenitor T cell. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate, and L-Lag2) containing 20-22 amino acids at the amino terminus and 3-8 EGF repeats on the extracellular surface. In various embodiments, the Notch ligand comprises at least one of Delta-like 1 (DLL1), Delta-like 4 (DLL4), SFIP3, or a functional portion thereof. A key signal transmitted by thymic stromal cells to incoming lymphoid progenitor cells in vivo is mediated by DL4 expressed by cortical thymic epithelial cells.

[0055] The earliest intrathymic progenitor cells express high levels of CD34 and CD7, do not express CD1a, and are triple negative (TN) for the mature T cell markers CD4, CD8, and CD3. Commitment to the T cell lineage is associated with expression of CD1a by CD7-expressing prothymocytes. Thus, the immature stage of T cell development is typically characterized by the expression of CD34 + CD1a - Cells (most immature) and CD34 + CD1a + Depicted as cells. CD34 by early thymocytes + CD7 + CD1a - From CD34 + CD7 + CD1a + Transition to CD34 is associated with T cell commitment. + CD7 + CD1a + The cells are likely to be T cell lineage restricted. After this stage, thymocytes progress to a CD4 immature single positive stage, at which point they express CD4 in the absence of CD8. A subset of cells then expresses CD4 + CD8 + Finally, after TCRα reconstitution, TCRαβ-expressing DP thymocytes undergo positive and negative selection and differentiate into CD4 + CD8 - Single positive (SP) T cells and CD4 - CD8 + Generate single positive (SP) T cells.

[0056] In some embodiments, the precursor T cells are isolated by enrichment for CD7 expression. In some embodiments, the precursor T cells are expanded as described in US Patent Publication No. 2020 / 0308540, which is incorporated herein by reference in its entirety. For example, the cells can be expanded by exposing them to an aryl hydrocarbon receptor antagonist, including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55. In some embodiments, compounds that promote proliferation include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US Patent Publication No. 2020 / 0308540, which is incorporated herein by reference).

[0057] Differentiation into progenitor T cells can, in some embodiments, further include the presence of stem cell factor (SCF), Flt3L, and interleukin (IL)-7. In various embodiments, the generated CD7+ progenitor T cells express CD1a. The CD7+ progenitor T cells do not express CD34 or express reduced levels 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 is 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 + It is.

[0058] In some embodiments, the progenitor T cells exhibit reduced levels of CD34 expression, minimal CD34 expression (relative to the HSC population), or no CD34 expression, hi some embodiments, CD34 expression is reduced by at least about 50%, or at least about 75%, in the population relative to the HSC population.

[0059] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody capable of binding and participating in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including a human or humanized antibody), a single chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule capable of activating the Notch signaling pathway.

[0060] In some embodiments, the Notch ligand is a Notch ligand of the Delta family. In some embodiments, the Delta family ligand is Delta-1 (Genbank Accession No. AF003522, Homo sapiens), Delta-like 1 (DLL1, Genbank Accession Nos. NM_005618 and NP_005609, Homo sapiens, Genbank Accession Nos. X80903, 148324, Mus musculus (M. Notch ligands are commercially available or can be produced, for example, by recombinant DNA techniques.

[0061] In some embodiments, the Notch ligand comprises an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical (e.g., about 100% identical) to a human DLL1 or DLL4 Notch ligand. A functional derivative of a Notch ligand (including a fragment or a portion thereof) can bind to and activate a Notch receptor. Binding to a Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0062] In various embodiments, the Notch ligand is soluble and optionally immobilized on a microparticle or nanoparticle, which is optionally paramagnetic to allow for a magnetic enrichment or concentration process. In yet other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally with other adhesion molecules such as VCAM-1. See U.S. Patent Application Publication No. 2020 / 0399599, which is incorporated herein by reference in its entirety. In other embodiments, the beads or particles are composed of biological materials, such as particles that are polymers (e.g., polystyrene or PLGA), gold, iron dextran, or formed from lipids and / or proteins. In various embodiments, the particles have a diameter or maximum dimension of about 0.01 μm (10 nm) to about 500 μm (e.g., 1 μm to about 7 μm). In still other embodiments, polymer scaffolds with conjugated ligands can be used, as described in WO 2020 / 131582, which is incorporated herein by reference in its entirety. For example, the scaffold can be comprised of polylactic acid, polyglycolic acid, PLGA, alginic acid or alginic acid derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-ε-caprolactone, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic® polyols, polyoxamers, poly(uronic acids), poly(anhydrides), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold comprises pores having diameters of about 1 pm to 100 pm.

[0063] In some embodiments, the C-terminus of the Notch ligand is conjugated to a selected support. In some embodiments, this may include adding a sequence to the C-terminus of the Notch ligand that can be enzymatically conjugated to the support, for example, via a biotin molecule. In another embodiment, the Notch ligand-Fc fusion is prepared such that the Fc segment can be immobilized by binding to Protein A or Protein G that is conjugated to the support. Of course, any of the known protein conjugation methods can be used.

[0064] Thus, in various embodiments, the Notch ligand is immobilized, functionalized, and / or embedded in the 2D or 3D culture system. The Notch ligand may be incorporated with one or more extracellular matrix components, for example, selected from fibronectin, retronectin, and laminin. In some embodiments, the Notch ligand and / or the extracellular matrix components 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, the extracellular matrix components, or combinations thereof are in contact with culture conditions that provide a topographical pattern and / or texture (e.g., roughness) to cells that promotes differentiation and / or proliferation.

[0065] In some embodiments, HSCs are differentiated into precursor T cells by culturing in medium containing TNF-α and / or an antagonist of aryl hydrocarbon / dioxin receptor (SR1) in the presence of a Notch ligand. See U.S. Patent Application Publication Nos. 2020 / 0390817, 2021 / 0169934, and 2021 / 0169935, which are incorporated by reference in their entireties. In some embodiments, HSCs are cultured in medium containing TNF-α, IL-7, thrombopoietin (TPO), Flt3L, stem cell factor (SCF), and optionally SR1 in the presence of immobilized Delta-like 4 ligand and fibronectin fragments. In some embodiments, cells are cultured with Retronectin, a recombinant human fibronectin that contains three functional domains: human fibronectin cell binding domain (C domain), heparin binding domain (H domain), and CS-1 sequence domain. In some embodiments, cells are cultured in the presence of immobilized Delta-like 4 ligand and Retronectin. In some embodiments, cells are cultured in the presence of immobilized Delta-like 4 ligand, TNF-α, and Retronectin. In some embodiments, cells are cultured in the presence of immobilized Delta-like 1 ligand and Retronectin. In some embodiments, cells are cultured in the presence of SFIP3 and Retronectin. In some embodiments, cells are cultured in the presence of immobilized Delta-like 4 ligand and SHH molecule and / or functional derivatives thereof. Exemplary fibronectin fragments include one or more of RGDS, CS-1, and heparin binding motifs. Fibronectin fragments may be free in solution or immobilized on a culture surface or particle. In some embodiments, the cells are cultured for 5 to 7 days to prepare CD7+ precursor T cells.

[0066] In various embodiments, the method produces progenitor T cells, or T cell lineages, by culturing a population of HSCs with a Notch ligand (including any of the embodiments described above), with or without a component(s) of the extracellular matrix, and optionally adding TNF-α to the culture at certain stages of differentiation. Thus, in some embodiments, the cells produced are progenitor or precursor cells committed to a T cell lineage ("progenitor T cells"). In some embodiments, the cells are CD7 + In some embodiments, the cells are progenitor T cells. + Immature T cells, or cells committed to the CD4 or CD8 lineage. In some embodiments, the cells are CD4 + CD8 + Double positive (DP), CD4 - CD8 + , or CD4 + CD8 - In some embodiments, the cells are CD4 - CD8 + or CD4 + CD8 - and TCR hi In some embodiments, the cells are single positive (SP) cells, which are TCRαβ. + and / or TCRγΔ + In various embodiments, the cells are CD3 + It is.

[0067] Adoptive transfer of precursor T cells is a strategy to promote T cell reconstitution. Precursor T cells are developmentally immature and undergo positive and negative selection in the host thymus. Thus, they become restricted to the recipient's major histocompatibility complex (MHC), generating host-tolerant T cells that can circumvent the clinical challenges associated with graft-versus-host disease (GVHD). Importantly, engraftment with precursor T cells restores thymic architecture and improves subsequent thymic seeding with HSC-derived precursor cells. In addition to their inherent regenerative medicine properties, precursor T cells can also be genetically engineered (either via gene or mRNA delivery) with T cell receptors (TCRs) and chimeric antigen receptors (CARs) to confer specificity for tumor-associated antigens.

[0068] In various embodiments, the precursor T cells are further cultured under suitable conditions to generate cells of the desired T cell lineage, including one or more Notch ligands. For example, the cells can be cultured in the presence of one or more Notch ligands as described for a sufficient time to form cells of the T cell lineage. In some embodiments, the stem cells or precursor T cells are cultured in suspension with soluble Notch ligand, or Notch ligand conjugated to particles or other supports, or Notch ligand-expressing cells. In some embodiments, the precursor T cells or stem cells are cultured in suspension or in adherent form with soluble or conjugated Notch ligand in suspension in a bioreactor, optionally a closed or closed automated bioreactor. One or more cytokines, extracellular matrix component(s), and thymic niche factor(s) that promote commitment and differentiation to the desired T cell lineage can also be added to the culture or reactor. Such cytokines or factors are known in the art. In various embodiments, the HSC population is cultured with a Notch ligand for about 4 to about 21 days, or about 6 to about 18 days, or about 7 to about 14 days to generate progenitor T cells. In some embodiments, the stem cell population or derivatives thereof are cultured for at least about 21 days, or at least about 28 days to generate mature T cell lineages or NK cells.

[0069] In various embodiments, the HSC population is cultured in artificial thymic organoids (ATO). See Hagen, M. et al. (2019). ATO includes the culture of HSCs (or aggregates of HSCs) with a Notch ligand-expressing stromal cell line in serum-free conditions. Artificial thymic organoids are a 3D system that cultures hematopoietic precursors, naive CD3 + CD8 + and CD3 + CD4 + Induces differentiation into T cells.

[0070] In various embodiments, the method includes generating a derivative of the precursor T cells or generating a T cell lineage from the precursor T cells. In certain embodiments, the derivative of the precursor T cells or the T cell lineage expresses CD3 and a T cell receptor. In some embodiments, the T cell lineage expresses CD8 + and / or CD4 + For example, the T cell lineage includes CD8 + CD4 - , CD8 - CD4 + , CD8 + CD4 + , and CD8 - CD4 - In some embodiments, the iPSCs, CD34+ cells, or derivatives thereof are engineered to express a chimeric antigen receptor (CAR) at the progenitor T cell, T cell, and / or NK cell level.

[0071] In some embodiments, the T cell lineage is a regulatory T cell. Regulatory T cells (or Tregs) are CD4 + CD25 + Tregs are defined as immune responses to self and foreign antigens and help prevent autoimmune diseases. Differentiation of progenitor T cells into Tregs in some embodiments includes culturing progenitor T cells or Treg precursors with TGFβ and optionally IL-2 and / or IL-10.

[0072] In some embodiments, the HSC population or fraction thereof is differentiated into B lymphocytes ("B cells"). For example, CD34+ or CD34+CD43+ cells can be cultured with MS5 stromal cells or S17 stromal cells (e.g., for 15-25 days, or about 21 days) to generate a B lymphoid identity with expression of CD19, CD45, and CD10. See Carpenter L. et al., Human induced pluripotent stem cells are capable of B-cell lymphopoiesis, Blood 117(15):4008-4011; Dubois F. et al., Toward a better definition of hematopoietic progenitors suitable for B cell differentiation, Plos One Dec. 15, 2020. In various embodiments, B cells produced according to the present disclosure express surface IgM (sIgM) and undergo VDJ rearrangement. In various embodiments, B cells produced in accordance with the present disclosure engraft in the spleen and secondary lymphoid tissues of a subject for maturation.

[0073] In some embodiments, the HSC population or fraction thereof is differentiated into monocytes, macrophages, or neutrophils. For example, erythromyeloid precursors (EMPs) (CD43+CD45+) can be generated by culture with IL-6, IL-3, thyroid peroxidase (TPO), SCF, FGF2, and VEGF, followed by differentiation into monocytes. Differentiation into monocytes using culture with M-CSF, IL-3, and IL-6. See Cao X et al., Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives, Stem Cell Reports. 2019 Jun 11; 12(6): 1282-1297. Monocyte and macrophage lineages prepared according to the present disclosure are CD14+ and exhibit endocytosis and phagocytic functions. In some embodiments, macrophages are polarized ex vivo towards M1 (pro-inflammatory) or M2 (immunosuppressive) phenotypes. In some embodiments, differentiation of iPSC-derived hCD34+ cells generates CD45+ hematopoietic cells with phagocyte markers such as CD33 and CD11b, and optionally subsequently cells with neutrophil-specific markers such as CD66b, CD16b, GPI-80, etc. These processes can use differentiation media containing a mixture of cytokines and growth factors, including but not limited to SCF, IL3, FLT3, IL6, GM-CSF, G-CSF, EPO, TPO, and / or combinations thereof.In some embodiments, neutrophils and their precursors can be derived from neutrophils or their precursors as described in Saeki L., et al., A Feeder-Free and Efficient Production of Functional Neutrophils from Human Embryonic Stem Cells, Stem Cells Vol. 27, Issue 1, 2009, Pages 59-67; Morishima T. et al., Neutrophil differentiation from human-induced pluripotent stem cells. J. Cell. Physiol. 226: 1283-1291, 2011; Yokoyama Y. et al., Derivation of functional mature neutrophils from human embryonic stem cells. Blood 2009 Jun 25;113(26):6584-92; and Sweeney CL et al., Generation of functionally mature neutrophils from induced pluripotent stem cells. Methods Mol Biol 2014; 1124:189-206.

[0074] In some embodiments, the HSC population or fraction thereof is differentiated into megakaryocytes or platelets. For example, megakaryocytes (as a renewable source of platelets) can be prepared from HSC or fractions thereof by culture with SCF, IL-11, and TPO for several days (e.g., about 5 days). Alternatively, other cytokines and growth factors such as IL-3, IL-6, SDF-1, and FGF-4 can be used. Megakaryocytes will be CD42b+CD61+. See Liu L., Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents, Stem Cells Transl Med. 2015 Apr; 4(4): 309-319. Platelets can be further generated from megakaryocytes by culture in serum-free medium containing IL-11. CD41+CD42a+ platelet-like particles are collected from the medium.

[0075] In some embodiments, the derivative of the progenitor T cells is a natural killer (NK) cell. In some embodiments, the NK cell is generated from a progenitor T cell as described in U.S. Patent No. 10,266,805, which is incorporated herein by reference in its entirety. For example, the progenitor T cell can give rise to a NK cell when cultured with IL-15. In some embodiments, the NK cell expresses a CAR based on gene editing of iPSC, embryonic bodies, hCD34+ cells, or NK cells, or via mRNA expression in NK cells.

[0076] In some embodiments, the HSC population or fraction thereof is differentiated into red blood cells or derivatives thereof. Red blood cells produced according to the present disclosure can be administered or used, for example, in therapy for inherited or acquired red blood cell disorders, bone marrow failure disorders, high altitude related physiological and pathological conditions, conditions associated with chemical or radiation exposure, and / or for treatment of subjects undergoing HSC transplantation. In further embodiments, red blood cells prepared according to the present disclosure are provided as pharma- ceutically acceptable compositions that deliver or encapsulate drugs (including but not limited to enzymes), oxygen carriers, or other suitable materials to treat human diseases or physiological or pathological conditions.

[0077] In another aspect, the present invention provides a cell population produced by the methods described herein, or a pharma- ceutically acceptable composition thereof. In some embodiments, the cell population is a lymphocyte population that can engraft in the thymus, spleen, or secondary lymphoid organs after administration to a subject in need. In various embodiments, a composition for cell therapy is prepared that includes the desired cell population and a pharma- ceutically acceptable vehicle. The pharmaceutical composition is a composition that contains 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 For example, in some embodiments, a pharmaceutical composition containing about 100,000 to about 400,000 cells per kg of recipient body weight (e.g., about 200,000 cells / kg) is administered.

[0078] The cell compositions of the present disclosure may further comprise a pharma- ceutically acceptable carrier or vehicle suitable for intravenous infusion or other routes of administration, and the compositions may include a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). The cell compositions may be provided in unit vials or bags and stored frozen until use. In certain embodiments, the volume of the composition is about 1 fluid ounce to 1 pint.

[0079] In some embodiments, the present disclosure provides CD7+ precursor T cells, or a pharma- ceutically acceptable composition thereof, produced by the methods disclosed herein. In various embodiments, the precursor T cells can engraft in the thymus or spleen of the recipient. The precursor T cells have the potential to reduce the risk of recurrence of leukemia or other types of cancer in bone marrow transplant patients, and to reduce the number of post-transplant infections that cause significant patient morbidity and mortality. In another aspect, the present disclosure provides a derivative or T cell lineage of the precursor T cells, or a pharma-ceutically acceptable composition thereof, produced by the methods disclosed herein.

[0080] In some embodiments, the cell population is a T cell population (or a precursor T cell population) or a NK cell population, and is useful for adoptive cell therapy for human subjects suffering from a condition selected from, for example, lymphopenia, cancer, immunodeficiency, viral infection, autoimmune disease (particularly when the T cell population comprises Tregs), skeletal dysplasia, bone marrow failure syndrome, or genetic disorders that impair T cell development or function. Exemplary genetic disorders affect the immune system and may be manifested as immunocompromised conditions, or as autoimmune or proinflammatory conditions. In some embodiments, the subject is suffering from cancer, optionally a hematological malignancy or solid tumor. In some embodiments, the T cells are CAR-T cells.

[0081] In some embodiments, the cell population is a B lymphocyte population and can engraft in the spleen or secondary lymphoid tissues of the subject. The B cell population according to the present disclosure has the potential to partially reconstitute humoral immunity in immunocompromised patients, providing protection against or treatment of infectious diseases, including, for example, viral, bacterial, fungal, or parasitic infections. In various embodiments, the B cells according to the present disclosure can be differentiated into plasma cells to produce antigen-specific antibodies in vivo. In other embodiments, the B cells produced according to the present disclosure can be used for cancer immunotherapy. In some embodiments, chimeric antigen B cells (CAR B cells) are prepared by genetic modification at the iPSC, embryonic bodies, hCD34+ cells, hematopoietic progenitor cells, or B cell level. The CAR B cells express a surface BCR and / or secrete recombinant monoclonal antibodies that recognize a target antigen, such as a cancer antigen or an infectious disease antigen. In yet other embodiments, the B cells produced according to the present disclosure are used for ex vivo production of antibodies (e.g., vaccine antibodies that provide protection against infectious pathogens).

[0082] In some embodiments, the cell population is a monocyte or macrophage cell population, and the cell population is capable of engrafting and maturing in various tissues of a subject, including tumors. In various embodiments, the monocyte or macrophage cell population is capable of forming tissue-resident macrophages in the subject. In various embodiments, the macrophages are predominantly of the M1 (pro-inflammatory) or M2 (immunosuppressive) phenotype. In various embodiments, the subject in need of treatment is suffering from cancer of any of a variety of tissues or organs, inflammatory disease of the liver or kidney, or bacterial infection (e.g., sepsis or infection or colonization of an indwelling medical device).

[0083] In some embodiments, the cell population is a megakaryocyte population or platelets developed therefrom, which are useful, for example, in the treatment of inherited platelet disorders that affect the coagulation pathway.

[0084] In some embodiments, the cell population is an erythrocyte population.

[0085] In some embodiments, the cell population (or platelets) are derived from autologous cells, or universally compatible donor cells, or HLA modified or HLA null cells (e.g., as described herein), i.e., the cell population is generated from iPSCs prepared from the recipient subject's cells, or prepared from donor cells (e.g., universal donor cells, HLA compatible cells, HLA modified cells, or HLA null cells).

[0086] In another aspect, the invention provides a method of cell therapy comprising administering a cell population described herein, or a pharma- ceutically acceptable composition thereof, to a human subject in need thereof. In various embodiments, the methods described herein are used to treat hematological disorders (malignant and non-malignant), bone marrow disorders, and immune disorders. In various embodiments, the human subject suffers from a condition comprising one or more of lymphopenia, cancer, immunodeficiency, autoimmune disease, skeletal dysplasia, hemoglobinopathies, anemia, bone marrow failure syndromes, and genetic disorders (e.g., genetic disorders affecting the immune system). In some embodiments, the subject suffers from cancer, such as a hematological malignancy or a solid tumor.

[0087] In some embodiments, the subject is suffering from a condition selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorder, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, inborn errors of metabolism, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and leukocyte adhesion deficiency.

[0088] The cell lines generated using the methods described herein are administered to a subject, for example, by intravenous infusion. In some embodiments, the methods can be performed following myeloablative, non-myeloablative, or immunotoxin-based (e.g., anti-c-Kit, anti-CD45, etc.) conditioning regimes.

[0089] As used herein, the term "about" means plus or minus 10% of the relevant numerical value.

[0090] Certain aspects and embodiments of the present disclosure are further illustrated with reference to the following examples. EXAMPLES

[0091] Example 1 - ETV2 overexpression increases the yield of hemogenic endothelial cells and enhances CD34+ cell formation during iPSC differentiation but does not affect pluripotency method iPSCs were generated from hCD34+ cells by episomal reprogramming as known in the art, essentially as described in Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells, Science 318, 1917-1920, (2007), and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009). Embryoid body and hemogenic endothelial differentiation was performed essentially as described in 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 specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801, (2009).

[0092] Briefly, hiPSCs were dissociated and resuspended in medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holotransferrin, monothioglycerol, BMP4, and Y-27632. The cells were then seeded onto 10 cm dishes (EZSPHERE or low attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. 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, cells were filtered through a 70 μm strainer, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0093] result Adenoviral vectors containing both ETV2 and GFP sequences under the control of the EF1A promoter were used to transduce induced pluripotent stem cells (iPSCs). After transduction, approximately 45% of the iPSC cultures were observed to be GFP positive, confirming ETV2 overexpression (ETV2-OE). It was further observed that ETV2-OE in iPSC cells retained the pluripotent properties of iPSCs, as indicated by the stemness marker expression TRA-1-60 (Figure 1). Figure 1 shows a FACS plot depicting the transduction efficiency of iPSCs with adenoviral vectors overexpressing ETV2 and GFP sequences.

[0094] Next, ETV2-OE-iPSCs (along with control iPSCs transduced with a vector carrying the GFP sequence but without ETV2) were differentiated into embryoid bodies and subsequently into hemogenic endothelial cells (Strugeon et al., 2014). - CD34 in the population + and CD31 +As demonstrated by marker expression, it is suggested that overexpression of ETV2 enhances the formation of hemogenic endothelial cells (Figure 2). Specifically, Figure 2 shows a representative flow cytometry analysis of hemogenic endothelial cells (defined here as CD235a-CD34+CD31+), and the relative quantification demonstrates that ETV2-OE enhances the formation of hemogenic endothelial cells compared to the control.

[0095] Furthermore, the results showed that ETV2-OE inhibits CD34 + These results suggest that ETV2-OE enhances CD34+ cell formation (FIG. 3). FIG. 3 shows a representative flow cytometry analysis of CD34+ cells, and the relative quantification demonstrates that ETV2-OE enhances CD34+ cell formation.

[0096] Taken together, these data indicate that ETV2 overexpression in iPSCs promotes their ability to undergo hemogenic endothelial and hematopoietic differentiation without affecting their pluripotent properties.

[0097] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs method To analyze EHT, EB-derived CD34+ cells were suspended in medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. Yoda1 was added to the cultures after the cells had adhered to the bottom of the wells for approximately 4-18 hours (by visual inspection). After 4-7 days, cells were harvested for analysis.

[0098] 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 5-7 days to induce endothelial-hematopoietic transition (EHT). CD34+ cells were then harvested from the EHT cultures between days 5-7 for further hematopoietic lineage differentiation.

[0099] CD34+ cells harvested from EHT cultures between days 5 and 7 (or total days 13 to 21 of differentiation from iPSCs) were seeded onto 48-well plates precoated with rhDL4 and retronectin. T cell lineage differentiation was induced with medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1a, and SB203580.

[0100] 80% of the medium was replaced every other day between days 2 and 6. On day 7, cells were transferred to new coated plates and analyzed for the presence of precursor T cells (CD34+CD7+CD5+ / -).

[0101] 80% of the medium was changed every other day between days 8 and 13. On day 14, 100,000 cells / well were transferred to new coated plates and cells were analyzed for the presence of pre-T cells (CD34-CD7+CD5+ / -).

[0102] 80% of the medium was changed every other day between days 15 and 20. Cells were harvested on day 21 and analyzed for expression of CD3, CD8, CD5, CD7, TCRab as a surrogate for T cells via FACS and / or activated using CD3 / CD28 beads to assess functional properties.

[0103] After 21 days of differentiation, cells were harvested and reseeded at approximately 80,000 cells in new 96-well culture plates in RPMI 1640 (without L-glutamine, without phenol red) supplemented with FBS, L-glutamine, IL-2, and then activated with CD3 / CD28 beads 1:1. After 72 hours of activation with CD3 / CD28 beads, cells were analyzed for expression of CD3, CD69, CD25 by FACS and IFN-γ using RT-qPCR. Supernatants were analyzed by ELISA.

[0104] result Figures 4A and 4B show that iPSC-derived HSCs induced by Piezo1 activation undergo precursor T cell differentiation similar to bone marrow (BM)-HSCs. Furthermore, Figures 5A and 5B show that iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation and can be activated with CD3 / CD28 beads similar to BM-HSCs. Figure 6 shows that iPSC-derived HSCs generated by Piezo1 activation can differentiate into functional T cells, as indicated by IFNγ expression after stimulation with CD3 / CD28 beads. Taken together, these results indicate that Piezo1 activation during HSC formation enhances the ability of HSCs to further differentiate into precursor T cells and functional T cells ex vivo.

[0105] References 1. Nianias, A. & Themeli, M. Induced Pluripotent Stem Cell (iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep 14, 261-268 (2019). 2. Brauer, PM, Singh, J., Xhiku, S. & Zuniga-Pfluecker, JC T Cell Genesis: In Vitro Veritas Est? Trends Immunol 37, 889-901 (2016). 3. Kennedy, M. et al. T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures. Cell Reports 2, 1722-1735 (2012). 4. Sturgeon, C. M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells. Nat Biotechnol 32, 554-561 (2014). 5. Chang, C.-W., Lai, Y.-S., Lamb, L. S. & Townes, T. M. Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells. PLoS One 9, (2014). 6. Nishimura, T. et al. Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation. Cell Stem Cell 12, 114-126 (2013). 7. Themeli, M. et al. Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 31, 928-933 (2013). 8. Vizcardo, R. et al. Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells. Cell Stem Cell 12, 31-36 (2013). 9. Montel-Hagen, A. et al. Organoid-induced differentiation of conventional T cells from human pluripotent stem cells. Cell Stem Cell 24, 376-389.e8 (2019). 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21-33 (2020). 11. Nagano, S. et al. High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells. Molecular Therapy - Methods & Clinical Development 16, 126-135 (2020). 12. Iriguchi, S. et al. A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nature Communications 12, 430 (2021).

Claims

1. 1. A method for preparing a cell population of hematopoietic lineage, comprising: Preparing a pluripotent stem cell (PSC) population; enriching for CD34+ cells to prepare a CD34 enriched population; inducing endothelial-hematopoietic conversion of said CD34 enriched population to prepare a population comprising hematopoietic stem cells (HSCs), and optionally harvesting cells from the CD34 enriched population that undergo endothelial-hematopoietic conversion; Differentiating the HSC population into a hematopoietic lineage; A method comprising:

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

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

4. the iPSCs have been gene-edited to delete one or more HLA class I genes, to delete one or more class II genes, and / or to delete one or more genes that govern HLA or MHC expression or presentation capabilities; The one or more genes that control the expression or presentation ability of HLA or MHC are β2-microglobulin and / or CIITA; The method of claim 2.

5. The method of claim 1, wherein CD34 enrichment and endothelial-hematopoietic conversion are induced from day 8 to day 15 of iPSC differentiation.

6. The method of claim 5, wherein the endothelial hematopoietic conversion generates a HSC population comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem progenitor cells.

7. The method of claim 5, wherein CD34+ cells are harvested from a culture undergoing endothelial-hematopoietic conversion, including harvesting of CD34+ floating and / or adherent cells.

8. The method of claim 6, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSCs).

9. The method of claim 5, wherein said inducing endothelial hematopoietic transition comprises increasing expression or activity of dnmt3b.

10. The method of claim 5 , wherein the induction of endothelial-hematopoietic conversion comprises Piezo1 activation.

11. The method of claim 10, wherein the Piezo1 activation is by contacting the CD34-enriched cells or a fraction thereof with one or more Piezo1 agonists, optionally selected from Yoda1, Jedi1, Jedi2, or analogs or derivatives thereof.

12. 2. The method of claim 1, wherein the hematopoietic lineage is selected from progenitor T cells, T lymphocytes, B lymphocytes, natural killer cells, neutrophils, monocytes, macrophages, erythrocytes, megakaryocytes, and platelets.

13. 13. The method of claim 12, wherein the HSC population or a fraction thereof is differentiated ex vivo into progenitor T cells, T cells, NK cells, and / or fractions or analogs thereof.

14. 14. The method of claim 13, wherein the HSC population or a fraction thereof is cultured with a partial or complete Notch ligand to produce a population comprising CD7+ progenitor T cells or a derivative cell population.

15. The method of claim 14, wherein the CD7+ progenitor T cells express CD1a.

16. The method of claim 15, wherein the CD7+ progenitor T cells do not express CD34 or express reduced levels of CD34 compared to the HSC population.

17. The method of claim 16, wherein the CD7+ progenitor T cells express CD5.

18. 15. The method of claim 14, wherein the Notch ligand comprises at least one of DLL1, DLL4, SFIP3, or a functional portion thereof.

19. The method of claim 18, wherein the Notch ligand is immobilized, functionalized and / or embedded in a 2D or 3D culture system, or optionally incorporated together with a component of the extracellular matrix selected from fibronectin, retronectin and laminin, derivatives or analogues thereof, and / or combinations thereof.

20. generating a population of HSCs comprising human long-term hematopoietic stem cells (LT-HSCs) from human pluripotent stem cells; wherein the HSC population is derived by endothelial-hematopoietic transformation of CD34+ cells; Culturing said HSC population or cells isolated therefrom with partial or complete Notch ligand and / or components of the extracellular matrix to produce a population comprising CD7+ precursor T cells or a derivative cell population; A method comprising:

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