Materials, methods and systems for cell redifferentiation and expansion

Abstract

Materials, methods, and systems are provided for cell redifferentiation and expansion of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) without the use of serum or additional cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application Nos. 63 / 387,541; 63 / 387,542; and 63 / 387,544, all filed on December 15, 2022, and 63 / 470,616, filed on June 2, 2023, the contents of each of which are incorporated herein in their entirety as if fully set forth herein. Technical Field

[0003] The present invention teaches cell production, including materials, methods and systems for, for example, cell growth, redifferentiation and amplification of hematopoietic stem cells (e.g., iHSC) derived from induced pluripotent stem cells (iPSCs), which are particularly useful for producing T cells (such as gamma-delta (γδ) T cells) derived from such iPSCs, including sustainable cell methods and strategies. The γδ T cells produced by these methods can be gene-edited. The materials, methods and systems of the present invention advantageously reduce or eliminate the use of expensive materials, energy, carbon raw materials and biomass, etc. (such as animal serum, additional cells (e.g., feeder cells or stromal cells)), human supervision, etc. in the manufacture and use of iHSCs. Background Art

[0004] It is reported that cell therapy (such as immune T cell immunotherapy, including autologous chimeric antigen receptor (CAR) T cell therapy) is an effective therapy for treating some diseases (such as some cancers, including some hematological malignancies). However, the application of cell therapy to treat a variety of cancer types has encountered many different challenges (see, for example, Advances and challenges of CAR T therapy and suitability of animal models (Review) (Advances and challenges of CAR T therapy and suitability of animal models (Review)), author: Xavier E. Ramos-Cardona Weichuan Luo Sulma I. Mohammed; published online on July 12, 2022). Summary of the Invention

[0005] Progress in T cell immunotherapy, such as autologous CAR T cell therapy, has been reported; however, the application of cell therapy to treat a variety of cancer types presents many unresolved challenges; in this context, the present invention encounters many challenges that hinder the availability of therapeutic cell products, such as, for example, prohibitively high manufacturing costs, excessive complexity, lack of consistent and scalable manufacturing processes, automation, and avoidance of scarce resources. For example, the inventors of the present invention use iPSCs, which can undergo, among other things, self-renewal, gene editing, multi-lineage differentiation, etc., in order to advance an off-the-shelf, allogeneic T cell therapy platform with a more commercially viable, automated, energy-efficient, and resource-saving manufacturing process. For example, according to the present invention, an iPSC-derived cell therapy platform is based on the generation of γδ T cells, which have inherent anti-tumor activity and whose cell tumor infiltration is associated with more favorable outcomes. Therefore, there is a need in the art for methods of efficiently redifferentiating iPSCs into iPSC-derived HSCs (iHSCs) as a source of sufficient numbers of robust allogeneic γδT cells for the production of the present invention. Such iHSCs have sufficient purity and multiple expansion, and have high T cell redifferentiation potential for use in T cell-based products, as well as γδT cells that have excellent increased purity and multiple expansion properties and are easier to prepare and use. The present invention relates to such improved methods, materials, and systems.

[0006] In addition, the present inventors have addressed the need in the art for a more sustainable method of generating iHSCs from iPSCs that does not use, for example, biological reagents, serum, and / or biomass, such as additional cells (e.g., feeder cells or stromal cells) and cell extracts, which produces iHSCs with high purity, viability, multiple expansion, and the potential to redifferentiate into immune effector cells, including lymphocytes, such as γδT cells, particularly iPSC-derived γδ (iγδ) T cells. The present disclosure addresses these and other needs.

[0007] The invention taught herein has multiple aspects. In one aspect, the present invention provides a method for generating hematopoietic stem cells (iHSCs) from scratch under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising: (i) inoculating iPSCs derived from human γδT cells in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; (ii) initiating redifferentiation of iPSCs to iHSCs by culturing iPSCs in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; (iii) culturing iPSC-derived cell intermediates for about 8 days; (iv) collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and (v) on about day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction.

[0008] In some embodiments, the method further comprises initiating redifferentiation of the iPSCs in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0009] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or antibiotics for about 2 days.

[0010] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic for about 2 days.

[0011] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic for about 2 days.

[0012] In some embodiments, the method further comprises culturing the redifferentiated iHSCs in a composition comprising L-glutamine and optionally an antibiotic for about 2 days, wherein the composition does not comprise cytokines or growth factors.

[0013] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0014] i) obtaining one or more human γδ T cell-derived induced pluripotent stem cells (iPSCs), and

[0015] The method further comprises one or more of the following steps:

[0016] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well is approximately 2.5×10 3 cells / well, preferably about 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0017] b) maintaining the iPSCs under culture conditions for about 5.5 days to about 6.5 days, preferably up to 6 days;

[0018] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, to obtain an iPSC-derived cell intermediate, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0019] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium (Advanced DMEM / F12 medium) and For Differentiation Medium differentiation medium), and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein SB431542 has a final concentration of about 1.5 μM to about 2.5 μM;

[0020] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0021] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, preferably about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0022] g) after about 7.8 to about 8.2 days of redifferentiation, preferably 8 days, collecting the non-adherent iPSC-derived cell intermediates in the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0023] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and PEG / ... For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0024] i) harvesting the non-adherent iHSCs in the culture by centrifugation, wherein the harvesting optionally does not include further culturing;

[0025] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0026] k) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0027] 1) not introducing serum in any of steps ak;

[0028] m) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a1; or

[0029] n) Obtaining de novo generated iHSCs.

[0030] In one aspect, the present invention includes a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 The invention relates to a method for the invention of the present invention to a method for the invention of the present invention to seed iPSCs at least 10 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs in the tissue culture treated dish for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) obtaining a suspension of the EBs and cells; and e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0031] In some embodiments, the present invention further comprises obtaining CD34+ and lineage marker negative iHSCs.

[0032] In some embodiments, the present invention further comprises obtaining iHSCs that are CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a cells.

[0033] In another aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0034] i) obtaining one or more human γδ T cell-derived induced pluripotent stem cells (iPSCs), and

[0035] The method further comprises one or more of the following steps:

[0036] a) iPSCs were cultured at a rate of approximately 0.55×10 6 cells / well is approximately 0.65×10 6 cells / well, preferably about 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0037] b) maintaining the iPSCs under culture conditions with agitation for about 44 hours to about 52 hours, preferably up to 48 hours, wherein embryoid bodies (EBs) are generated;

[0038] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0039] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein SB431542 has a final concentration of about 1.5 μM to about 2.5 μM;

[0040] e) culturing the suspension of EBs and cells in a third cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0041] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, preferably about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0042] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0043] h) resuspending the pelleted EB and cell suspension in a volume of about 18 mg / mL to about 20 mg / mL of type II collagenase and incubating the EB and cell suspension in type II collagenase;

[0044] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0045] j) mixing the single cell suspension;

[0046] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0047] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in a fifth cell culture medium to form a second cell suspension;

[0048] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0049] n) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0050] o) not introducing serum in any of steps a to b;

[0051] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a to b; or

[0052] q) Obtain de novo generated iHSCs.

[0053] In any of the embodiments herein, the cells are cultured under normoxic conditions.

[0054] In any of the embodiments herein, the method further comprises further purifying and / or isolating the iHSCs.

[0055] In any of the embodiments herein, the iHSCs obtained are isolated differentiated cells or are capable of being further purified and / or isolated.

[0056] In any of the embodiments herein, the step of determining the purity of the iHSCs comprises assaying one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or their equivalents.

[0057] In any of the embodiments herein, the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0058] In any of the embodiments herein, the step of resuspending the cells comprises gently dispersing the cells using a pipette or its equivalent.

[0059] In another aspect, the invention provides one or more cells obtained from any of the methods described herein.

[0060] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; initiating redifferentiation of the iPSCs to iHSCs by culturing the iPSCs in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; culturing the iPSC-derived cell intermediates for about 8 days; collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and on about day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction.

[0061] In some embodiments, the method of preparing cells further comprises initiating redifferentiation of the iPSCs in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0062] In some embodiments, the method of preparing cells further comprises culturing the iPSC-derived cell intermediate in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or antibiotics for about 2 days.

[0063] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic for about 2 days.

[0064] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic for about 2 days.

[0065] In some embodiments, the method further comprises culturing the redifferentiated iHSCs in a composition comprising L-glutamine and optionally an antibiotic for about 2 days, wherein the composition does not comprise cytokines or growth factors.

[0066] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδT cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, wherein the method comprises:

[0067] i) obtaining at least one human γδ T cell-derived iPSC, and

[0068] The method further comprises one or more of the following steps:

[0069] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well is approximately 2.5×10 3 cells / well, preferably about 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0070] b) maintaining the iPSCs in culture for up to 6 days;

[0071] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 2 days to obtain an iPSC-derived cell intermediate, wherein the first cell culture medium is Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0072] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein SB431542 has a final concentration of about 1.5 μM to about 2.5 μM;

[0073] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 2 days, wherein the third cell culture medium comprises Advanced DME M / F12 medium and ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0074] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0075] g) after about 8 days of redifferentiation, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0076] h) on day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions: a non-adherent cell fraction and an adherent cell fraction, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium comprising Advanced DMEM / F12 medium and 4:1 at a ratio of about 4:1. For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0077] i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing;

[0078] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0079] k) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0080] 1) not introducing serum in any of steps ak;

[0081] m) not introducing any additional cells, including feeder cells, in any of steps a1; or

[0082] n) Obtaining de novo generated iHSCs.

[0083] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the redifferentiation dish contains a suspension of EBs and cells; d) obtaining a suspension of the EBs and cells; and e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0084] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells, and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) obtaining the suspension of EBs and cells; e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension; and f) providing CD34+ and lineage marker negative iHSCs.

[0085] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) obtaining the suspension of EBs and cells; e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension; and f) obtaining CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- iHSCs.

[0086] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0087] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0088] The method further comprises one or more of the following steps:

[0089] a) iPSCs were cultured at a rate of approximately 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0090] b) maintaining the iPSCs in culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs);

[0091] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0092] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional approximately 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0093] e) culturing the suspension of EBs and cells in a third cell culture medium for about another 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0094] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0095] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0096] h) resuspending the pelleted EB and cell suspension in a volume of approximately 20 mg / mL of type II collagenase, and incubating the EB and cell suspension in type II collagenase;

[0097] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0098] j) mixing the single cell suspension;

[0099] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0100] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in a fifth cell culture medium to form a second cell suspension;

[0101] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0102] n) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0103] o) not introducing serum in any of steps a to b;

[0104] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a1; or

[0105] q) Obtain de novo generated iHSCs.

[0106] In any of the embodiments herein, the resulting iHSCs have high purity, viability, fold expansion, and the potential to redifferentiate into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

[0107] In another aspect, the invention provides a composition comprising a cell produced by any of the methods described herein.

[0108] In another aspect, the present invention provides the use of cells obtained by any of the methods described herein in preparing cells for treating lesions or diseases, in preparing lymphocytes, in bioreactors, in tissue engineering or in vitro drug screening for diseases.

[0109] In another aspect, the present invention provides a system wherein any of the methods described herein is performed manually or with automated robotic assistance, or a combination thereof.

[0110] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; initiating redifferentiation of the iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; culturing the iPSC-derived cell intermediates for about 8 days; collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and on about day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction.

[0111] In some embodiments, the method further comprises initiating redifferentiation of the iPSCs in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0112] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or antibiotics for about 2 days.

[0113] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic for about 2 days.

[0114] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic for about 2 days.

[0115] In some embodiments, the method further comprises culturing the redifferentiated iHSCs in a composition comprising L-glutamine and optionally an antibiotic for about 2 days, wherein the composition does not comprise cytokines or growth factors.

[0116] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising one or more components capable of performing the following method:

[0117] obtaining at least one human γδ T cell-derived iPSC, and

[0118] The method further comprises one or more of the following steps:

[0119] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well is approximately 2.5×10 3 cells / well, preferably about 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0120] b) maintaining the iPSCs as colonies in culture for up to 6 days;

[0121] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 2 days to obtain iPSC-derived cell intermediates, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0122] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DME M / F12 medium and dapoxetine at a ratio of about 4:1. ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0123] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 2 days, wherein the third cell culture medium comprises Advanced DME M / F12 medium and ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0124] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0125] g) after about 8 days of redifferentiation, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0126] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and PEG / T7 medium at a ratio of about 3.9:0.9 to about 4.1:1.1. ForDiffe rentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0127] i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing;

[0128] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0129] k) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0130] 1) not introducing serum in any of steps ak;

[0131] m) not introducing any additional cells, including feeder cells, in any of steps a1; or

[0132] n) Obtain de novo generated (iHSCs).

[0133] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells, and initiating redifferentiation of the EBs for about 10 days; d) obtaining a suspension of the EBs and cells; and e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0134] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells, and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) obtaining the suspension of EBs and cells; e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension; and f) providing CD34+ and lineage marker negative iHSCs.

[0135] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) obtaining the suspension of EBs and cells; e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension; and f) obtaining CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- iHSCs.

[0136] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδT cell-derived induced pluripotent stem cells (iPSCs) under serum-free, feeder-free culture conditions, the method comprising:

[0137] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0138] The method further comprises one or more of the following steps:

[0139] a) iPSCs were cultured at a rate of approximately 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0140] b) maintaining the iPSCs in culture for up to 48 hours to generate embryoid bodies (EBs);

[0141] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0142] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional approximately 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0143] e) culturing the suspension of EBs and cells in a third cell culture medium for about another 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0144] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0145] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0146] h) resuspending the pelleted EB and cell suspension in a volume of approximately 20 mg / mL of type II collagenase, and incubating the EB and cell suspension in type II collagenase;

[0147] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0148] j) mixing the single cell suspension;

[0149] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0150] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in a fifth cell culture medium to form a second cell suspension;

[0151] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0152] n) determining the purity of iHSCs in the single-cell suspension, defined as CD34+ and lineage marker negative;

[0153] o) not introducing serum in any of steps a to b;

[0154] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a to b; or

[0155] q) Obtain de novo generated iHSCs.

[0156] In some embodiments, the step of determining the purity of the iHSCs comprises assaying one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or its equivalent.

[0157] In some embodiments, lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0158] In some embodiments, the step of mixing the single cell suspension comprises gently dispersing the cells using a pipette or its equivalent.

[0159] In some embodiments, the system is automated or semi-automated.

[0160] In some embodiments, the system includes one or more software packages that operate and schedule the operation of the system.

[0161] In some embodiments, the software package is customized and / or customizable for a desired application, and is optionally menu driven.

[0162] In some embodiments, automation includes cell visualization, plate handling, plate spreading, inoculation, extraction, addition, cell feeding, incubation assays, and / or sampling.

[0163] In some embodiments, the system includes one or more in-line incubators.

[0164] In some embodiments, the system includes electronic humidity controls, a HEPA filter system, a turntable including a programmable step oscillation cycle, and / or a two-way communication interface, or any combination thereof.

[0165] In some embodiments, the addition or removal or supplementation of culture medium does not disturb or contaminate the cells.

[0166] In some embodiments, the system includes one or more computer-operated and controlled robotic arms. BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figures 1A to 1D A non-limiting, exemplary automated system format for performing any of the methods described herein is shown. Figure 1A shows a front view of the system, Figure 1B Shown Figure 1A A top view of the system, and Figure 1C (Exemplary vertically stackable views) and Figure 1D (Exemplary top-down view of six systems) shows a side view of the system with components stacked on top of each other ( Figure 1C ) or stacked side by side ( Figure 1D ), and each part is either fixed or moving.

[0168] Figure 2 A flow chart of a non-limiting exemplary SFFF 2D iHSC redifferentiation protocol is shown. On days 6 to 10, non-adherent cells harvested with media changes are added back to the culture.

[0169] Figure 3 A flow chart of a non-limiting exemplary SFFF 3D / 2DiHSC redifferentiation protocol is shown.

[0170] Figure 4 A schematic diagram of a non-limiting exemplary SFFF 2DiHSC redifferentiation protocol is shown.

[0171] Figures 5A to 5B A non-limiting exemplary SFFF 2D iHSC redifferentiation protocol according to one embodiment herein is shown ( Figure 5A ) and from a non-limiting exemplary SFFF 3D / 2D iHSC redifferentiation protocol ( Figure 5B ) obtained cells.

[0172] Figures 6A to 6C Shown are the effects of a non-limiting exemplary SFFF 2D iHSC redifferentiation protocol according to one embodiment herein. Figure 6A Shown is the time course of iHSC redifferentiation from three iPSC clones (top row, clone 1; middle row, clone 2; bottom row, clone 3) over a 10-day period. Figure 6B A non-limiting exemplary SFFF 2D iHSC redifferentiation protocol is shown for Figure 6A Effects of the purity (right panel) and fold expansion (left panel) of redifferentiated iHSCs obtained from the same three iPSC clones are shown, as well as the effects on adherent and non-adherent fractions. Figure 6C Shown are the effects of a non-limiting exemplary SFFF 2DiHSC redifferentiation protocol on the purity (right panel) and fold expansion (left panel) of iγδ T cells obtained from iHSCs derived from the same three iPSC clones, as well as on adherent and non-adherent fractions.

[0173] 7A to 7B Results are shown for a non-limiting exemplary 3D / 2D approach disclosed herein. Figure 7A Shown are the results of a non-limiting exemplary SFFF 3D / 2D iHSC redifferentiation protocol for iHSC purity (right panel) and fold expansion (left panel) of iPSC clone 1. Figure 7B Shown are the results of a non-limiting exemplary SFFF 3D / 2D iHSC redifferentiation protocol on iγδ T cell purity (right panel) and fold expansion (left panel) of clone 1.

[0174] Figures 8A to 8MM Shown are microscopic images of cells obtained at different stages of non-limiting exemplary 2D and 2D / 3D protocols disclosed herein. DETAILED DESCRIPTION

[0175] definition

[0176] Various terms related to various aspects of the specification are used throughout the specification and claims. Unless otherwise indicated, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

[0177] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and so forth.

[0178] As used herein, the term "about" when referring to a measurable value such as an amount, a time interval, etc., is meant to encompass variations of up to ±10% from the specified value, as such variations are suitable for performing the methods disclosed herein. Unless otherwise indicated, all numbers used in the specification and claims expressing amounts of ingredients, properties such as molecular weight, reaction conditions, etc. are to be understood as being modified by the term "about" in all instances. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the present invention. At a minimum, and without attempting to limit the application of the doctrine of equivalents to the scope of protection of the claims, each numerical parameter should at least be interpreted in light of the number of significant digits reported and by applying customary rounding techniques.

[0179] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0180] The term "treat" refers to any success or indication of success in alleviating or ameliorating an injury, condition, or disorder, including any objective or subjective parameter, such as symptom relief, remission, weakening, or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, reducing the degree of failure at a degenerative endpoint, improving the subject's physical or mental well-being, or prolonging survival. Treatment can be assessed by objective or subjective parameters, including the results of a physical examination, neurological examination, or psychiatric evaluation.

[0181] The term "subject" refers to humans and non-human animals, including all vertebrates, e.g., mammals and non-mammals such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians and reptiles. In many embodiments of the methods, the subject is a human.

[0182] The term "induced pluripotent stem cell" (hereinafter referred to as "iPSC") means a stem cell established by introducing reprogramming factors into a somatic cell, which has pluripotency that allows differentiation into many cell types present in a living organism, and also has proliferation (ie, self-renewal) ability. It encompasses any cell that can be redifferentiated into a hematopoietic stem cell (HSC) to be used in the present invention. iPSC is preferably derived from a primate (e.g., monkey, orangutan, chimpanzee, human), more preferably human.

[0183] The term "iHSC" refers to hematopoietic stem cells derived from iPSCs.

[0184] The term "multicellular body" includes, but is not limited to, embryoid bodies.

[0185] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary.

[0186] Methods for generating iHSCs

[0187] γδT cells were selected as the origin of iPSC cells because the epigenetic and transcriptional memory of the starting cells are known to affect and enhance the redifferentiation potential of iPSC to this cell type. An optimized two-step two-dimensional (2D) research-scale SFFF protocol was used to redifferentiate γδT-iPSC into iHSC, and then redifferentiate into iγδT cells. The purity and multiple expansion of each redifferentiation step were determined, and the cells were examined under a microscope to assess the morphological characteristics of the redifferentiation of iPSC to iHSC. Based on the results from the 2D redifferentiation protocol and from the 3D / 2D redifferentiation protocol, success criteria were set for the purity and multiple expansion of redifferentiated iHSC and iγδT cells. Optimizing the protocol for the redifferentiation of iPSC to iHSC resulted in an increase in its purity and multiple expansion, exceeding the success criteria set for the redifferentiation step. The protocol optimization of the iHSC redifferentiation step also increased the purity and multiple expansion of the iγδT cells generated in the second redifferentiation step. Notably, enhanced T cell redifferentiation potential was observed for three γδ T-iPSC clones (two of which were subclones), demonstrating the efficiency and effectiveness of the optimized protocol for iPSC to iHSC redifferentiation.

[0188] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs), which are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising one or more of the following steps, optionally all of the following steps: (i) maintaining iPSCs before initiating redifferentiation; (ii) redifferentiation of iPSCs into iHSCs; and (iii) redifferentiation of iHSCs into iγδT cells.

[0189] In another aspect, the present invention provides a method for de novo generation of hematopoietic stem cells under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: (i) seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; (ii) initiating redifferentiation of the iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; (iii) culturing the iPSC-derived cell intermediates for about 8 days; (iv) collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and (v) on about day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction.

[0190] In some embodiments, the method further comprises the step of initiating redifferentiation of the iPSCs in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0191] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or antibiotics for about 2 days.

[0192] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic for about 2 days.

[0193] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic for about 2 days.

[0194] In some embodiments, the method further comprises culturing the redifferentiated iHSCs in a composition comprising L-glutamine and optionally an antibiotic for about 2 days, wherein the composition does not comprise cytokines or growth factors.

[0195] In another aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0196] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0197] The method further comprises one or more of the following steps:

[0198] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well to about 2.5×10 3 cells / well, preferably about 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0199] b) maintaining the iPSCs under culture conditions for about 5.5 days to about 6.5 days, preferably up to 6 days;

[0200] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, to obtain an iPSC-derived cell intermediate, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0201] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the second cell culture medium comprises Advanced D MEM / F12 medium and PEG-177 at a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0202] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-100 at a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0203] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, preferably about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0204] g) after about 7.8 to about 8.2 days of redifferentiation, preferably 8 days, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0205] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and PEG / ... For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0206] i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing;

[0207] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0208] k) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0209] 1) not introducing serum in any of steps ak;

[0210] n) not introducing any additional cells, including feeder cells, in any of steps a to m; or

[0211] o) Obtaining de novo generated iHSCs.

[0212] In one aspect, the present invention includes a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells, and initiating redifferentiation of the EBs for about 10 days; d) obtaining a suspension of the EBs and cells; and e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0213] In some embodiments, the present invention further comprises obtaining CD34+ and lineage marker negative iHSCs.

[0214] In some embodiments, the present invention further comprises obtaining iHSCs that are CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- cells.

[0215] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0216] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0217] The method further comprises one or more of the following steps:

[0218] a) iPSCs were cultured at a rate of approximately 0.4×106 cells / well to about 1.2×10 6 cells / well, preferably about 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0219] b) maintaining the iPSCs in culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs);

[0220] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0221] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional approximately 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0222] e) culturing the suspension of EBs and cells in a third cell culture medium for about another 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0223] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0224] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0225] h) resuspending the pelleted EB and cell suspension in a volume of approximately 20 mg / mL of type II collagenase, and incubating the EB and cell suspension in type II collagenase;

[0226] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0227] j) mixing the single cell suspension;

[0228] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0229] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in a fifth cell culture medium to form a second cell suspension;

[0230] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0231] n) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0232] o) not introducing serum in any of steps a to b;

[0233] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a to b; or

[0234] q) Obtain de novo generated iHSCs.

[0235] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step for initiating redifferentiation of iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; (b) a step for culturing the iPSC-derived cell intermediates for about 8 days; (c) a step for collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and (d) a step for harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction - on about day 10 of culture.

[0236] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step for seeding the iPSCs; b) a step for culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step for transferring the EBs to a tissue culture-treated culture dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture-treated culture dish contains a suspension of EBs and cells; d) a step for obtaining the suspension of the EBs and cells; and e) optionally, a step for processing the suspension of the EBs and cells to obtain a single-cell suspension.

[0237] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step for seeding iPSCs; b) a step for culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step for transferring the EBs to a tissue culture-treated culture dish and initiating redifferentiation of the EBs for about 10 days, wherein the redifferentiation culture dish contains a suspension of EBs and cells; d) a step for culturing the suspension of EBs and cells for about 2 days to about 4 days; e) a step for obtaining a suspension of EBs and cells; f) a step for optionally processing the suspension of EBs and cells to obtain a single cell suspension; and g) a step for providing CD34+ and lineage marker-negative iHSCs.

[0238] In one aspect, the present invention provides a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step of seeding the iPSCs; b) a step of culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step of transferring the EBs to a tissue culture-treated dish and initiating redifferentiation of the EBs for about 10 day, wherein the redifferentiation culture dish contains a suspension of EBs and cells; d) a step for performing the following function, i.e., obtaining the suspension of the EBs and cells; e) optionally, a step for performing the following function, i.e., processing the suspension of the EBs and cells to obtain a single cell suspension; and f) a step for performing the following function, i.e., obtaining CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- iHSCs.

[0239] In some embodiments of any of the methods disclosed herein, the cells are cultured under normoxic conditions.

[0240] In some embodiments of any of the methods disclosed herein, the method further comprises further purifying and / or isolating the iHSCs.

[0241] In some embodiments of any of the methods disclosed herein, the iHSCs obtained are isolated differentiated cells or are capable of being further purified and / or isolated.

[0242] In some embodiments of any of the methods disclosed herein, the step of determining the purity of the iHSCs comprises assaying one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or its equivalent.

[0243] In some embodiments of any of the methods disclosed herein, the step of mixing the single-cell suspension comprises gently dispersing the cells using a pipette or its equivalent.

[0244] None of the methods described herein require the use of serum or additional cells (such as feeder cells or stromal cells) at any step. Each step is discussed in more detail below.

[0245] Biology, bioengineering in general, and biotechnology (including biomedicine) theoretically allow for fundamental biological process improvements that could potentially underpin research and development and advance industrial progress, including in areas such as medical therapeutics, diagnostics and immunological procedures, agriculture, food production, biofuel production, and environmental solutions. See, for example: Alejandro Barragán- et al., Biotechnology and Bioprocesses: Their Contribution to Sustainability (Biotechnology and Bioprocess: Contribution to Sustainability), Processes 2020, Vol. 8, p. 436; and KV Woodhoo et al., Bioprocess intensification: A route to efficient and sustainable biocatalytic transformations for the future (Bioprocess Intensification: A route to efficient and sustainable biocatalytic transformations for the future), Chemical Engineering & Processing: Process Intensification 172 (2022) 108793, each of which is incorporated herein by reference in its entirety as if fully set forth herein. Even so, within the framework of sustainability, only initial steps have identified the trajectory of scientific and technological bioprocesses. A study highlights the activities and confirms that the field is positioned as an emerging field of cognition aimed at, for example, improving economic development, environmental protection, and social welfare. With these factors in mind, the inventors of the present invention simplified and optimized materials and processes to, among other things, reduce and otherwise minimize the use of reagents and resources, reduce overall waste including waste of useful biomass, lower costs, develop systems for recycling and reusing materials, and reduce waste generation.

[0246] Maintaining iPSCs before initiating redifferentiation

[0247] iPSCs are cultured as colonies or multicellular bodies. As known in the art, iPSC colonies typically exhibit round colonies with densely packed cells, a high nucleus to cytoplasm ratio, well-defined edges, and distinct borders.

[0248] In such Figure 2 iPSCs are maintained in culture under non-limiting exemplary 2D conditions as shown in Figure 1 for about 5.5 days to about 6.5 days, preferably up to 6 days. The results of redifferentiating iPSCs into iHSCs and then redifferentiating iHSCs into iγδT cells as described herein are shown in Figure 1. Figures 6A to 6C middle.

[0249] To culture iPSCs into multicellular bodies (e.g., Figure 3The iPSCs can be cultured in low-attachment tissue culture dishes and can be cultured under agitation, as shown in the non-limiting exemplary 3D / 2D scheme. For example, the low-attachment tissue culture dishes can be incubated in a shaking incubator / multitron (full-function shaking incubator) at 37°C, 5% CO2, and 85% humidity at 100 RPM for about 44 hours to about 52 hours, preferably about 48 hours. As described herein, the results of redifferentiating iPSCs cultured as multicellular bodies into iHSCs, and then redifferentiating iHSCs into iγδT cells are shown in 7A to 7B middle.

[0250] Optionally, iPSCs are thawed from frozen stock solutions and the desired number of living cells can be used in the methods described herein. Cell count, size, and viability can be determined by any method known in the art, such as by using a NucleoCounter NC-200. Living cells with a preferred size (e.g., 10 μm to 25 μm) can be used in the steps described herein.

[0251] In a preferred embodiment, for the 2D protocol, dissociated iPSCs are plated at approximately 1.7 × 10 3 cells / well to about 2.5×10 3 cells / well, preferably about 2×10 3 The cells were seeded at a density of approximately 0.4 × 10 cells / well for the 3D / 2D protocol. 6 cells / well to about 1.2×10 6 cells / well, preferably about 0.6×10 6 One of ordinary skill in the art will be able to calculate the number of viable cells to be seeded given the size of the surface area (eg, the well).

[0252] After seeding, iPSCs are maintained in a suitable tissue or cell culture dish, such as, for example, but not limited to, a six-well tissue culture plate. The tissue or cell culture dish is optionally prepared to receive iPSCs by adding a matrix, such as laminin protein (e.g., iMatrix), and incubating at 37°C, 5% CO2. In a preferred embodiment of the 2D protocol, the iPSCs are grown in a 1% PBS solution at 0.25 μg / cm 2 The tissue or cell culture dish is prepared for use by adding iMatrix 511 solution at a concentration of 1:1 / well. Suitable coating methods are known in the art.

[0253] In a preferred embodiment of the 3D / 2D scheme, iPSC can be cultured in a low-attachment tissue culture dish, and can be cultured under agitation, for example, a low-attachment tissue culture dish can be in a shaking incubator / multitron (full-function shaking incubator) at 37°C, 5% CO2 and 85% humidity with 100RPM incubation for about 44 hours to about 52 hours, preferably about 48 hours. Such conditions result in the formation of embryoid bodies (EBs) or multicellular bodies. After incubation under agitation, EBs can be transferred from the low-attachment tissue culture dish to a tissue culture-treated tissue culture dish, and incubated without agitation.

[0254] iPSCs in the 2D protocol were cultured at 37°C and 5% CO2. The cells are grown in Basic 04 medium (which also contains basic fibroblast growth factor (bFGF) at a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF) for about 5.5 to about 6.5 days, preferably up to 6 days. For the first 24 hours, the medium contains 10 μM Y-27632 ROCK inhibitor. Basic 04 Cell Culture Medium (Ajinomoto) is an animal-free, defined medium for culturing human pluripotent stem cells. This medium is complete and does not require any additional supplements.

[0255] During the maintenance period, the cell morphology of iPSC-derived cell intermediates (2D protocol) or EBs and cell suspensions (3D / 2D protocol) can be examined at regular intervals (e.g., daily), as shown in FIG. Figures 8A to 8MM As shown. Cell morphology can be determined by microscopy (e.g., inverted phase contrast microscopy). Multicellular bodies (e.g., embryoid bodies) generated using the 3D / 2D scheme described herein should be visible in about 48 hours. Multicellular bodies can be collected by centrifugation once formed, and do not need to be incubated under agitation after formation. Once the cells become brighter and have obvious boundaries, the colonies can be redifferentiated into iHSCs as discussed herein. As known in the art, the culture medium can be replaced at regular intervals.

[0256] Redifferentiation of iPSC-derived cell intermediates or EB and cell suspensions into iHSC

[0257] In the 2D and 3D / 2D protocols, the iPSC-derived cell intermediates and / or EBs and cell suspensions can be cultured at 37° C., 5% CO 2 for about 1.8 days to about 2.2 days, preferably about two days, in a first culture medium comprising Basic 04 medium (which also contains basic fibroblast growth factor (bFGF) at a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF), and one or more of CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF). 3D / 2D protocols do not require agitation at this stage. The morphology of the suspension of iPSC-derived cell intermediates and / or EBs and cells can be determined by microscopy (e.g., as described in Example 1) at the beginning of the culture period and / or at regular intervals. Figures 8A to 8MM Culturing iPSC-derived cell intermediates and / or EBs and cell suspensions separately in this medium can induce mesoderm formation, a prerequisite for hematopoietic lineage specification and differentiation.

[0258] After the culture period for mesoderm induction in the 2D and 3D / 2D protocols, the iPSC-derived cell intermediates and / or EBs and suspensions of cells can be collected, respectively, and subsequently cultured in a second culture medium to initiate hematopoietic differentiation at 37° C. and 5% CO 2 for about 1.8 days to about 2.2 days, preferably about two days. At this stage, the 3D / 2D protocol does not require agitation. The second cell culture medium comprises Advanced DMEM / F12 medium (Thermo Fisher Scientific Catalog #12634) and PEG-1 in a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation Medium (Ajinomoto), and also contains L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF and / or stem cell factor (SCF), and optionally penicillin / streptomycin. SB431542 is diluted from a 5 mM stock concentration to a final concentration of approximately 1.5 μM to 2.5 μM, so that a minimal amount of dimethyl sulfoxide (DMSO) is used as the diluent. The morphology of the cells can be determined by microscopy (e.g., as described in the example of the DMSO dilution). Figures 8A to 8MM Advanced DMEM / F12 medium contains amino acids, vitamins, inorganic salts, proteins, reducing elements, trace elements, dextrose, ethanolamine, sodium hypoxanthine, linoleic acid, lipoic acid, phenol red, putrescine 2HCl, sodium pyruvate, and thymidine. For Differentiation Medium is a chemically defined and animal origin-free (CD-AOF) supplement for differentiating human embryonic stem (ES) cells and iPSCs.

[0259] After the culture period for initiating hematopoietic differentiation in the 2D and 3D / 2D protocols, the iPSC-derived cell intermediates and / or EBs and cell suspensions can be collected, respectively, and then cultured in a third culture medium to induce angiogenesis at 37° C. and 5% CO 2 for about 1.8 days to about 2.2 days, preferably about two days. At this stage, the 3D / 2D protocol does not require agitation. The third culture medium comprises Advanced DMEM / F12 medium and PEG-12 in a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3) and / or interleukin-6 (IL-6) One or more, and optional penicillin / streptomycin. EPO is prepared from a stock concentration of 500 IU / mL to a final concentration of 10 IU / mL (e.g., 240 μL of EPO stock solution is added to a six-well tissue culture plate). The morphology of the cells can be determined by microscopy (e.g., as described above) at the beginning of the culture period and / or at regular intervals. Figures 8A to 8MM The results were determined by inverted phase contrast microscopy (as shown).

[0260] After the culture period for inducing angioblast formation in the 2D and 3D / 2D protocols, the iPSC-derived cell intermediates and / or EBs and cell suspensions can be collected, respectively, and then cultured in a fourth culture medium at 37° C. and 5% CO 2 for about 1.8 days to about 4.2 days, preferably about 2 days to about 4 days, for the transition stage from endothelial cells to hematopoietic cells. At this stage, the 3D / 2D protocol does not require agitation. The fourth culture medium comprises Advanced DMEM / F12 medium and PEG-12 at a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation culture medium, and also comprise one or more of L-glutamine, SCF, IL-6 and / or EPO, and optional penicillin / streptomycin. EPO is prepared from a stock solution concentration of 500 IU / mL to a final concentration of 10 IU / mL (e.g., 240 μL EPO stock solution is added to a six-well tissue culture plate). The morphology of the cells can be observed at the beginning of the culture period and / or at regular intervals (e.g., at 8 days of redifferentiation) by microscopy (e.g., as Figures 8A to 8MM The results were determined by inverted phase contrast microscopy (as shown in Figure 3).

[0261] In the 2D protocol, after about 7.8 to about 8.2 days of redifferentiation, preferably about 8 days, the non-adherent iHSCs in the culture medium can be collected, centrifuged, resuspended in a fourth cell culture medium, and then added back to the culture medium.

[0262] After about 10 days of redifferentiation in both the 2D and 3D / 2D protocols, the cells can be visualized by microscopy (e.g., Figures 8A to 8MM The appearance and morphology of iHSCs and / or EBs were monitored by inverted phase-contrast microscopy (as shown).

[0263] Redifferentiated iHSCs from the 2D protocol can be harvested into two fractions: non-adherent cells and adherent cells (cells obtained from the 2D protocol are shown in Figure 5A middle).

[0264] Non-adherent iHSCs from the 2D protocol can be harvested and added back to the culture medium. For example, non-adherent iHSCs from the 2D protocol can be harvested by centrifugation (e.g., at 300×g for 5 minutes) and resuspended in a fifth cell culture medium comprising Advanced DMEM / F12 medium and 4:1 in a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation culture medium, and also comprising L-glutamine and optional penicillin / streptomycin, but without cytokines or growth factors. iHSC can be counted as described herein. Optionally, the non-adherent cell fraction is not further cultured. Preferably, the non-adherent cell fraction is added back into the culture. Cell markers (e.g., CD34) and / or lineage markers (e.g., CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and / or CD235a) of the obtained cells can be measured.

[0265] Adhesion iHSC from 2D scheme can be dispersed gently using a pipette or its equivalent, and can optionally be merged and dispersed again, and then be resuspended in the fifth culture medium after dispersion. Dispersed cells can then be made to pass through two cell strainers, the first cell strainer having a sieve size of 70 μm, and the second cell strainer having a sieve size of 40 μm. Cell counting can be performed as described herein. Cell markers (e.g., CD34) and / or lineage markers (e.g., CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and / or CD235a) of the resulting cells can be measured.

[0266] In the fifth culture medium, after two iHSC fractions from the 2D scheme are harvested, the purity of the cell can be assessed, for example, flow cytometry or equivalent is used to assess, to analyze the presence or absence of certain markers. Purity can be defined by the absence of CD34 expression (CD34+) and one or more lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a. In one embodiment, each of lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a does not exist. In other words, the cells harvested are CD34+ and lineage marker negative. In an exemplary embodiment, the gating scheme determining the purity of iHSC includes gating lineage marker negative cells, and then gating CD34+. After purity analysis, the iHSC obtained can be cryopreserved. Exemplary success criteria include, for example, redifferentiation of γδT-iPSCs to iHSCs with a purity greater than 70% Lin-CD34+ cells and a fold expansion / input iPSC greater than 100 for 2D culture, and differentiation of iHSCs to iγδT cells with a purity of 20% to 40% CD3+TCRγδ+ cells and a fold expansion / input iCD34 greater than 5.

[0267] Redifferentiated iHSCs and / or EBs from the 3D / 2D protocol can be harvested by centrifugation, gently resuspended in about 1 mL of about 18 mg / mL to about 20 mg / mL type II collagenase and incubated therein for about 20 minutes, followed by the addition of TrypLE and incubation for about 20 minutes ( Figure 3 The cell suspension produced by incubation in type II collagenase and TrypLE can be gently dispersed by pipetting and mixing, followed by the addition of a fifth cell culture medium comprising Advanced DMEM / F12 medium and PEG / T12 medium in a ratio of about 3.9:0.9 to about 4.1:1.1, preferably in a ratio of about 4:1. In some embodiments, iHSC is expressed as a cell culture medium containing L-glutamine and optionally penicillin / streptomycin, but without cytokines or growth factors. The mixed cell suspension can then be centrifuged to produce a cell pellet, and resuspended in the fifth cell culture medium to form a second cell suspension. The second cell suspension can be passed through two cell strainers, the first cell strainer having a sieve size of 70 μm, and the second cell strainer having a sieve size of 40 μm. iHSC can be counted as described herein, and cell markers (e.g., CD34) and / or lineage markers (e.g., CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and / or CD235a) can be measured. For example, the purity of iHSC can be assessed, for example, using flow cytometry or equivalent to assess, to analyze the presence or absence of certain markers. Purity can be defined by the presence (CD34+) of CD34 expression and the absence of one or more lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a. In one embodiment, each of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a is absent. In other words, the harvested cells are CD34+ and lineage marker negative. In an exemplary embodiment, the gating scheme for determining the purity of iHSC includes gating lineage marker negative cells and then gating CD34+. After the purity analysis, the iHSC obtained can be cryopreserved. Exemplary success criteria include, for example, redifferentiation of γδT-iPSCs to iHSCs with a purity greater than 70% Lin-CD34+ cells and a fold expansion / iPSC greater than 0.5 for 3D / 2D cultures, and differentiation of iHSCs to iγδT cells with a purity of 20% to 40% CD3+TCRγδ+ cells and a fold expansion / iCD34 greater than 5.

[0268] Redifferentiation of iHSCs into iγδT cells

[0269] The following is an exemplary method for redifferentiating iHSCs obtained by the methods described herein into iγδ T cells.

[0270] Suitable planar surfaces, such as tissue or cell culture plates or dishes, can be prepared by coating the surface (e.g., wells) with one or more Notch ligand proteins and / or vascular cell adhesion molecule 1 (VCAM1). Preferred tissue culture dishes include 48-well tissue culture plates. Preferred Notch ligand proteins include delta-like canonical Notch ligand 1 (DLL1), DLL4, VCAM1, and Jagged 2, and particularly preferred proteins include a combination of VCAM1 and DLL4. Coating can be performed by any method known in the art.

[0271] Cryopreserved iHSCs can be stored in StemSpan TM The cells were thawed in Lymphoid Progenitor Expansion Medium (StemCell Technologies). The cells were counted and their viability determined as discussed herein.

[0272] 250 μL of StemSpan per well TM Lymphoid progenitor cell expansion medium can be seeded with approximately 1.2×10 4 Viable cells can be cultured and the cells can be incubated at 37° C., 5% CO 2 , 18% O 2 for further culture and differentiation. Alternatively, in one aspect, the cells are incubated under hypoxic conditions.

[0273] Additional StemSpan can be added during the culture process as is generally known in the art. TM Lymphocyte progenitor cell expansion medium. The cells can be analyzed for viability and / or T cell lineage marker expression by any method known in the art during the culture process. The culture medium can be replaced as is generally known in the art. For example, the culture medium can be replaced every three days, for example, on the 13th, 17th, and 20th day of the culture process. The cells can be harvested approximately 14 days after thawing.

[0274] Cells generated by the method

[0275] In another aspect, the present invention provides one or more cells obtained from any of the methods described herein. The resulting iHSCs have high purity, viability, multiple expansion, and the potential to redifferentiate into immune effector cells, such as Figures 5A to 5B 、 Figures 6A to 6C and 7A to 7B iHSCs generated by any of these methods can be further redifferentiated into lymphocytes, including iγδ T cells.

[0276] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPS Cs), the method comprising: seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; initiating redifferentiation of the iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; culturing the iPSC-derived cell intermediates for about 8 days; collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and on about day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction.

[0277] In some embodiments, the method of preparing cells further comprises the step of initiating redifferentiation of iPSCs in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0278] In some embodiments, the method of preparing cells further comprises culturing the iPSC-derived cell intermediate in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or antibiotics for about 2 days.

[0279] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic for about 2 days.

[0280] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic for about 2 days.

[0281] In some embodiments, the method further comprises culturing the redifferentiated iHSCs in a composition comprising L-glutamine and optionally an antibiotic for about 2 days, wherein the composition does not comprise cytokines or growth factors.

[0282] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδT cell-derived induced pluripotent stem cells (iPSCs) under serum-free and feeder-free culture conditions, wherein the method comprises:

[0283] i) obtaining at least one human γδ T cell-derived iPSC, and

[0284] The method further comprises one or more of the following steps:

[0285] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well to about 2.5×10 3 cells / well, preferably about 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0286] b) maintaining the iPSCs under culture conditions for about 5.5 days to about 6.5 days, preferably up to 6 days;

[0287] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, to obtain an iPSC-derived cell intermediate, wherein the first cell culture medium is Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0288] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the second cell culture medium comprises Advanced D MEM / F12 medium and PEG-177 at a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0289] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0290] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, preferably about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0291] g) after about 7.8 to about 8.2 days of redifferentiation, preferably 8 days, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0292] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and PEG / ... For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0293] i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing;

[0294] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0295] k) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0296] 1) not introducing serum in any of steps ak;

[0297] m) not introducing any additional cells, including feeder cells, in any of steps a1; or

[0298] n) Obtaining de novo generated iHSCs.

[0299] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the redifferentiation dish contains a suspension of EBs and cells; d) obtaining a suspension of the EBs and cells; and e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0300] In some embodiments, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method further comprising: obtaining CD34+ and lineage marker-negative iHSCs.

[0301] In some embodiments, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method further comprising: obtaining iHSCs that are CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- cells.

[0302] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0303] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0304] The method further comprises one or more of the following steps:

[0305] a) iPSCs were cultured at a rate of approximately 0.4×10 6 cells / well to about 1.2×10 6 cells / well, preferably about 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0306] b) maintaining the iPSCs in culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs);

[0307] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and initiating redifferentiation of the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0308] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional approximately 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0309] e) culturing the suspension of EBs and cells in a third cell culture medium for about another 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0310] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0311] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0312] h) resuspending the pelleted EBs and the suspension of cells in a volume of approximately 20 mg / mL of type II collagenase, and incubating the EBs and iHSCs in type II collagenase;

[0313] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0314] j) mixing the single cell suspension;

[0315] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0316] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in a fifth cell culture medium to form a second cell suspension;

[0317] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0318] n) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0319] o) not introducing serum in any of steps a to b;

[0320] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a1; or

[0321] q) Obtain de novo generated iHSCs.

[0322] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a device for seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; b) a device for initiating redifferentiation of the iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; c) a device for culturing the iPSC-derived cell intermediates for about 8 days; d) a device for collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and e) a device for harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction - on about day 10 of culture.

[0323] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6b) a device for seeding iPSCs with 100 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a device for culturing iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; and e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension.

[0324] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) a device for seeding iPSCs with 10 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a device for culturing iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture-treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the redifferentiation dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a device for providing CD34+ and lineage marker negative iHSCs.

[0325] In one aspect, the present invention provides one or more cells obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culture the iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6b) a device for seeding iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a device for obtaining CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- iHSCs.

[0326] In some embodiments, the one or more cells have high purity, viability, fold expansion, and the potential to redifferentiate into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

[0327] In some embodiments, the step of determining the purity of the iHSCs comprises assaying one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or its equivalent.

[0328] In some embodiments, lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0329] In some embodiments, the step of mixing the single cell suspension comprises gently dispersing the cells using a pipette or its equivalent.

[0330] In another aspect, the invention provides a composition comprising a cell produced by any of the methods described herein.

[0331] In another aspect, the present invention provides the use of cells obtained by any of the methods described herein in preparing cells for treating lesions or diseases, in preparing lymphocytes, in bioreactors, in tissue engineering or in vitro drug screening for diseases.

[0332] System for performing the method

[0333] In another aspect, the present invention provides a system wherein any of the methods described herein is performed manually or with automated robotic assistance or a combination thereof, such as Figures 1A to 1Dshown.

[0334] In one aspect, the present invention provides a system for performing a method of generating hematopoietic stem cells (iHSCs) from scratch under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising: seeding iPSCs derived from human γδT cells in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; initiating redifferentiation of the iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; culturing the iPSC-derived cell intermediates for about 8 days; collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and on about day 10 of culture, harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction. The system of the present invention is mobile or fixed, or a combination thereof.

[0335] In some embodiments, the method further comprises the step of initiating redifferentiation of the iPSCs in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0336] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or antibiotics for about 2 days.

[0337] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic for about 2 days.

[0338] In some embodiments, the method further comprises culturing the iPSC-derived cell intermediate in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic for about 2 days.

[0339] In some embodiments, the method further comprises culturing the redifferentiated iHSCs in a composition comprising L-glutamine and optionally an antibiotic for about 2 days, wherein the composition does not comprise cytokines or growth factors.

[0340] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising one or more components capable of performing the following method:

[0341] obtaining at least one human γδ T cell-derived iPSC, and

[0342] The method further comprises one or more of the following steps:

[0343] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well to about 2.5×10 3 cells / well, preferably about 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0344] b) maintaining the iPSCs as colonies under culture conditions for about 5.5 days to about 6.5 days, preferably up to 6 days;

[0345] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, to obtain an iPSC-derived cell intermediate, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, preferably about 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0346] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the second cell culture medium comprises Advanced D MEM / F12 medium and PEG-177 at a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0347] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 1.8 days to about 2.2 days, preferably about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-100 at a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0348] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, preferably about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0349] g) after about 7.8 to about 8.2 days of redifferentiation, preferably 8 days, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0350] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and PEG / ... For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0351] i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing;

[0352] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0353] k) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0354] 1) not introducing serum in any of steps ak;

[0355] m) not introducing any additional cells, including feeder cells, in any of steps a1; or

[0356] n) Obtain de novo generated (iHSCs).

[0357] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6 b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the redifferentiation dish contains a suspension of EBs and cells; d) obtaining a suspension of the EBs and cells; and e) optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0358] In some embodiments, the present invention provides a system for performing a method for generating de novo hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method further comprising obtaining CD34+ and lineage marker negative iHSCs.

[0359] In some embodiments, the present invention provides a system for performing a method for generating hematopoietic stem cells (iHSCs) de novo under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method further comprising: obtaining iHSCs that are CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a-iHSCs.

[0360] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδT cell-derived induced pluripotent stem cells (iPSCs) under serum-free, feeder-free culture conditions, the method comprising:

[0361] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0362] The method further comprises one or more of the following steps:

[0363] a) iPSCs were cultured at a rate of approximately 0.4×10 6 cells / well to about 1.2×10 6 cells / well, preferably about 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0364] b) maintaining the iPSCs in culture for up to 48 hours to generate embryoid bodies (EBs);

[0365] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0366] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional approximately 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0367] e) culturing the suspension of EBs and cells in a third cell culture medium for about another 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0368] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO, and optionally penicillin / streptomycin;

[0369] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0370] h) resuspending the pelleted EB and cell suspension in a volume of approximately 20 mg / mL of type II collagenase, and incubating the EB and cell suspension in type II collagenase;

[0371] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0372] j) mixing the single cell suspension;

[0373] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0374] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in a fifth cell culture medium to form a second cell suspension;

[0375] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0376] n) determining the purity of iHSCs, defined as CD34+ and lineage marker negative;

[0377] o) not introducing serum in any of steps a to b;

[0378] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a to b; or

[0379] q) Obtain de novo generated iHSCs.

[0380] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a device for seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; b) a device for initiating redifferentiation of iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; c) a device for culturing the iPSC-derived cell intermediates for about 8 days; d) a device for collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and e) a device for harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction - on about day 10 of culture.

[0381] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a method for growing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6b) a device for seeding iPSCs with 100 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a device for culturing iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; and e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension.

[0382] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a method for growing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6 b) a device for seeding iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a device for providing CD34+ and lineage marker negative iHSCs.

[0383] In one aspect, the present invention provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a method for growing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6b) a device for seeding iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a device for obtaining CD34+ and CD3-, and / or CD19-, and / or CD14-, and / or CD11b-, and / or CD11c-, and / or CD56-, and / or CD20-, and / or CD16-, and / or CD2-, and / or CD235a- iHSCs.

[0384] In some embodiments, the step of determining the purity of the iHSCs comprises assaying one or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or its equivalent.

[0385] In some embodiments, lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0386] In some embodiments, the step of mixing the single cell suspension comprises gently dispersing the cells using a pipette or its equivalent.

[0387] In some embodiments, the system is automated or semi-automated. Suitable systems are described in, for example, Maria Kempner and Robin Felder, A Review of Cell Culture Automation, JALA: Journal of the Association for Laboratory Automation, Volume 7, Issue 2, April 2002, Pages 56-62; and Miho Sasamata et al., Establishment of a Robust Platform for Induced Pluripotent Stem Cell Research Using Maholo Lab Droid, SLAS Technology 2021, Volume 26(5), Pages 441-453, each of which is incorporated herein by reference in its entirety as if set forth herein.

[0388] In some embodiments, the system includes one or more software packages that operate and schedule the operation of the system.

[0389] In some embodiments, the software package is customized and / or customizable for a desired application, and is optionally menu driven.

[0390] In some embodiments, automation includes cell visualization, plate handling, plate spreading, inoculation, extraction, addition, cell feeding, incubation assays, and / or sampling.

[0391] In some embodiments, the system includes one or more in-line incubators.

[0392] In some embodiments, the system includes electronic humidity controls, a HEPA filter system, a turntable including a programmable step oscillation cycle, and / or a two-way communication interface, or any combination thereof.

[0393] In some embodiments, the addition or removal or supplementation of culture medium does not disturb or contaminate the cells.

[0394] In some embodiments, the system includes one or more computer-operated and controlled robotic arms.

[0395] Elements of a non-limiting exemplary automation system

[0396] Figures 1A to 1DA non-limiting exemplary automated system is shown in FIG. In several exemplary non-limiting embodiments, Figure 1A The elements shown are as follows: 1. Actuator; 2. Cooler; 3. Freezer; 4. Track; 5. Incubator (e.g., with and without inlet and outlet); 6. Transfer; 7. through 9. Contactors; 10. Vessel Holder; 11. Cell Lifter; 12. Tubular Holder; 13. Tubular Holder; 14. Tubular Holder; 15. Tubular Holder; 16. Incubator; 17. Transfer; 18. Mixer; 19. Reservoir; 20. Viewer; 21. Fluid Manipulator; 22. Separator; and 23. Phenotyper. One or more of these elements are connected to one another as known in the art, e.g., operatively connected. For example, one or more of the elements may be electrically and / or mechanically connected via a gas line, via a waste line, etc.

[0397] It will be appreciated by those skilled in the art that, according to common sense in the art, depending on the desired method for generating iHSC, one or more of these elements can be combined in different arrangements. For example, elements can be stacked vertically or can be stacked side by side, or can be a combination of such arrangements. One or more of these elements can be computer or software operated and / or controlled, individually or in operable combination with another element. One or more of these elements can be manually operated. Exemplary automation systems comprising one or more of the above elements are described in, for example, Maria Kempner and Robin Felder, A Review of Cell Culture Automation, JALA: Journal of the Association for Laboratory Automation, Vol. 7, No. 2, April 2002, pp. 56-62; and Miho Sasamata et al., Establishment of a Robust Platform for Induced Pluripotent Stem Cell Research Using Maholo LabDroid, SLAS Technology 2021, Vol. 26(5), pp. 441-453, each of which is incorporated herein by reference in its entirety as if set forth herein.

[0398] Method of the present invention can be carried out in the suitable system comprising one or more elements as described herein to obtain cell as described herein.One or more elements of the present invention taught herein are configured to be operably combined with another element.The system can be any size (for example, small-scale or large-scale) or type (for example, open, closed, in batches, fed-batch, perfusion, chemostat, continuously), as long as it can be used for cultured cells, for example mammalian cells or human cells.Cultivation unit can provide suitable culture conditions as described herein (for example, stirring, oxygen level and temperature), and can perform suitable action as described herein (for example, stirring, culture medium replacement, microscopy, cell sorting and / or flow cytometry).

[0399] Exemplary elements or components for use with the present invention include the following non-limiting sources and exemplary elements: RO1 (standardbots.com / ro1), UFACTORY and UFA CTORY 850 (ufactory.cc / ufactory-850 / ), and Interbotix X-series arms (trossenro botics.com / robotic-arms.aspx) from Standard Bots, including various arm types such as articulated arms, six-axis arms, collaborative robot arms, SCARA arms, Cartesian arms, cylindrical arms, spherical / polar arms, parallel / delta arms, anthropomorphic arms and dual-arm variants, and also see U.S. Patent Serial No. 4,806,066, U.S. Patent Serial No. 8,374,722, US20100158656, U.S. Patent Serial No. 6,826,977, US20120253513, etc.; cooler elements of various capacities and functions, available from VWR, Glo balIndustrial, American Biotech, Galaxy, Accucold, (k2sci.com / products / 2-cu-ft-combination-glass-door-refrigerator-10-cu-ft-solid-door-freezer); incubators, such as Powers Scientific, Micro Q, VWR, Thermo Scientific, Friocell, Benchmark, JeioTech, Stellar Scientific, Being Bit (see, e.g., labincubators.net / ); separators, including Beckman Coulter (beckman.com / landing / ppc / cent / benchtop / microcentrifuges), Thermo Fisher(thermofisher.com / us / en / home / life-science / lab-equipment / lab-centrifuges / benchtop-centrifuges / models.html), VWR(us.vwr.com / store / category / centrifuges), etc.; observers, including Leica(leica-microsystems.com / c / am / lsr-c / personal-confocal-promo), KEYENCE(keyence.com / landing / microscope / lp_all-in-one-microscope_01085523.jsp), ZEISS (.zeiss.com / metrology / products / systems / industrial-microscopy.html), etc.; and phenotyping devices, for example, include Nanocellect (nanocellect.com / products / wolf-g2-cell-sorter), Namocell (namocell.com), Beckman (beckman.com / landing / ppc / flow / cell-sorters), Sino Biologics (sinobiological.com / category / fcm-facs-facs), etc. Each reference is incorporated herein in its entirety. One of ordinary skill in the art will understand the exemplary embodiments and their usefulness with the present invention, as well as the process for evaluating and including other embodiments.

[0400] Example

[0401] The following examples are provided to further describe some of the embodiments disclosed herein.These examples are intended to illustrate, but not to limit, the disclosed embodiments.

[0402] Example 0: Serum-free, feeder-free, γδ T cell-derived iPSCs to iHSCs and then to iγδ T cells Two-dimensional redifferentiation .

[0403] The protocol for the first redifferentiation step (iPSC to iHSC) was optimized using two γδT cell-derived iPSC (γδT-iPSC) clones, clones 1 and 2. Clone 2 is a subclone of clone 1. Clone 3 was used to validate the optimized protocol. As shown herein, given that iPSC clones are heterogeneous in their T cell redifferentiation potential, it is important to use multiple iPSC clones to validate the optimized γδT-iPSC to iHSC redifferentiation protocol. To this end, the optimized protocol has been validated using other fully characterized clones. The data for clone 3 are shown in Figures 6A to 6C middle.

[0404] Material

[0405] Table 1. Reagents used for iPSC culture and maintenance .

[0406]

[0407] Table 2. Reagents required for hematopoietic redifferentiation (listed by redifferentiation day)

[0408]

[0409]

[0410] Table 3. Reagents used for iγδ T cell redifferentiation .

[0411] Reagents Product Number <![CDATA[StemSpan TM SAINTS]]> StemCell Technologies, catalog number 09655 Lymphocyte Progenitor Expansion Supplement (10X) StemCell Technologies, catalog number 09915 rh VCAM-1-Fc R&D, Cat. No. 862-VC-100 rh DLL4-Fc R&D, catalog number 10185-D4-050

[0412] Table 4. Supplies and consumables required for cell culture .

[0413]

[0414] Table 5. Flow cytometry antibody combinations .

[0415]

[0416]

[0417] Example Procedure

[0418] 1.1 Prepare supplemented StemFit Basic 04 complete medium as follows:

[0419] a. Provide StemFit Basic 04 Complete Medium in frozen form and store at -20°C or below until use. Use aseptic technique to prepare supplemented StemFit Basic 04 Complete Medium.

[0420] b. Thaw frozen StemFit Basic 04 Complete Medium at room temperature (15°C-25°C) or in the refrigerator (2°C-8°C) with occasional mixing.

[0421] c. Note: Do not thaw the culture medium at 37°C as this accelerates the degradation of the culture medium components.

[0422] d. Supplement StemFit Basic 04 complete medium with an additional 20 ng / mL of bFGF to achieve a final concentration of 100 ng / mL bFGF in the supplemented medium.

[0423] e. Store the supplemented medium at 4°C for up to two (2) weeks.

[0424] f. Bring the culture medium to room temperature (15°C-25°C) at least one (1) hour before use.

[0425] 1.2 Prepare 10 mM Y-27632 ROCK inhibitor stock solution as follows:

[0426] a. Receive Y-27632 ROCK inhibitor as a crystalline solid and store it at -20°C for up to 12 months from the date of receipt.

[0427] b. Prepare a 10 mM stock solution in 1X DPBS as follows:

[0428] i. Add 312 μL of 1X DPBS to 1 mg of Y-27632 and resuspend the solution completely.

[0429] ii. Aliquot 25 μL of the stock solution into Eppendorf tubes labeled Y-27632 Stock.

[0430] iii. Aliquots are stable at -20°C for up to 6 months.

[0431] iv. Aliquot the working volume to avoid repeated freeze-thaw cycles.

[0432] 1.3 Prepare a 6-well TC treatment plate coated with iMatrix 511 as follows:

[0433] a. Pipette 12 mL of 1X DPBS into a 15 mL conical tube.

[0434] b. Add 30 μL of iMatrix 511 to 1X DPBS and mix thoroughly immediately (coating concentration: 0.25 μg / cm 2 ).

[0435] c. Add 2 mL of iMatrix solution to each well of a 6-well plate.

[0436] d. Incubate the plate at 37°C, 5% CO2 for at least 60 minutes (the plate can be left at 37°C overnight).

[0437] e. Note: After the coating step, the plates can be stored at 4°C for one week. Before use, incubate the plates at 37°C for 1 hour.

[0438] 1.4 Prepare supplemented StemFit Basic 04 complete medium (from step 1.1) with 10 μM Y-27632 ROCK inhibitor as follows:

[0439] a. Thaw an aliquot of 10 mM Y-27632 ROCK inhibitor stock solution before use.

[0440] b. Aliquot 25 mL of supplemented StemFit Basic 04 complete medium (from step 1.1) into 50 mL conical tubes.

[0441] c. Add 25 μL of 10 mM Y-27632 ROCK inhibitor to the supplemented StemFit Basic 04 complete medium to reach a final concentration of medium with 10 μM Y-27632 ROCK inhibitor.

[0442] 1.5 Thaw a vial of γδT-iPSCs as follows:

[0443] a. Thaw a vial of frozen γδT-iPSCs in a 37°C water bath for 1 minute or until small clumps of frozen, floating cells are visible.

[0444] b. Slowly transfer the thawed γδT-iPSCs dropwise from the vial into a 15 mL conical tube containing 9 mL of room temperature supplemented StemFit 04 Basic complete medium with 10 μM Y-27632 ROCK inhibitor (from step 1.4).

[0445] c. Note: The total volume is 10 mL.

[0446] 1.6 Cell counting.

[0447] a. A NucleoCounter NC-200 instrument was used for cell counting purposes.

[0448] b. Remove a 150 μL aliquot from the cell suspension (step 1.5.b) into a 1.5 mL Eppendorf tube.

[0449] c. Collect cells from the Eppendorf tube into vial-box.

[0450] d. Insert the vial-box into the NC-200 cytometer and count the cells.

[0451] e. Note: Only use the total number of viable cells for cell seeding purposes

[0452] 1.7 Wash and plate thawed γδT-iPSCs as follows:

[0453] a. Centrifuge the 15 mL conical tube containing the thawed γδT-iPSCs (step 1.5b) at 300 × g for 3 minutes at room temperature.

[0454] b. Aspirate the supernatant without disturbing the cell pellet, leaving 0.5 mL of medium.

[0455] c. Gently tap the conical tube to loosen the cell pellet.

[0456] d. Add P1000 to 1 mL of supplemented StemFit Basic04 complete medium with 10 μM Y-27632 ROCK inhibitor (step 1.4) to resuspend the cells.

[0457] e. Add additional supplemented StemFit Basic 04 Basic complete medium (step 1.4) with 10 μM Y-27632 ROCK inhibitor to reach 1×10 4 The final cell concentration was 10 cells / mL.

[0458] f. Remove the iMatrix 511 solution from the 6-well plate by aspiration.

[0459] i. NOTE: Avoid allowing the plate to dry for an extended period of time (e.g., more than 10 minutes). Do not allow the wells to dry.

[0460] g. Mix the cells thoroughly and then plate them at 2 mL / well.

[0461] h. Distribute the cells evenly across the bottom of the wells by gently rocking the plate manually back and forth and side to side.

[0462] i. Incubate the plate at 37°C in 5% CO2 overnight.

[0463] 1.8 Day 1: Perform medium change as follows:

[0464] a. Move the supplemented StemFit Basic 04 complete medium (from step 1.1) from 4°C to room temperature.

[0465] b. Gently aspirate the culture medium from the 6-well plate.

[0466] c. Add 2 mL / well of freshly supplemented StemFit Basic 04 complete medium ( none ROCK inhibitor) (step 1.1) was added to the 6-well plate.

[0467] d. Plates were imaged using EVOS M7000 and images were recorded at 4x and 10x magnification.

[0468] e. Three images were taken from different areas of the plate to document the morphology and landscape of the culture. Single cells and two-cell colonies were present. The cells had a spiky morphology due to culture in the presence of ROCK inhibitor.

[0469] i. Note: As the cells divide, the boundaries become more distinct.

[0470] ii. Figure 8A An example view at 10x magnification is shown in .

[0471] 1.9 Continue culturing the cells until they reach typical morphology, exhibiting all iPSC characteristics, including round colonies with densely packed cells, a high nuclear to cytoplasm ratio, well-defined edges, and distinct borders. At this stage, the confluency of the iPSC colonies is approximately 70%-80%.

[0472] a. Note: It may take 7 days + / - 1 day for colonies to develop this morphology.

[0473] b. iPSC colonies have defined borders and dense cells.

[0474] i. Figure 8B An example view at 10x magnification is shown in .

[0475] 1.10 Passage cells and confirm pluripotency by flow cytometry before initiating redifferentiation :

[0476] a. Prepare as many iMatrix 511-coated 6-well plates as needed to passage iPSCs.

[0477] b. Passage iPSCs as follows:

[0478] i. Aspirate the culture medium from the cells plated in step 1.7.

[0479] ii. Rinse the wells with 1 mL of 1X DPBS.

[0480] iii. Aspirate 1X DPBS from the wells.

[0481] iv. Add 1 mL of TrypLE and incubate at 37°C for 6 to 8 minutes.

[0482] v. Check the hole under a microscope. Figure 8C As shown, cells appear bright with clear cell borders.

[0483] vi. Gently remove TrypLE.

[0484] vii. Add P1000 to 1 mL of supplemented StemFit Basic 04 complete medium with 10 μM Y-27632 ROCK inhibitor (from step 1.4), and then gently pipette to remove the cells from the plate. Avoid forming bubbles.

[0485] c. Use a microscope to ensure that 95% of the cells are removed from the wells.

[0486] i. If residual cells are still attached to the plate, add 1 mL of StemFit Basic 04 complete medium with 10 μM Y-27632 ROCK inhibitor (from step 1.4). Then scrape the well using a cell spatula and pipette three (3) times using a P1000 to ensure a uniform single cell suspension.

[0487] d. Collect the cells into a 15 mL conical tube.

[0488] e. Count cells as in step 1.6.

[0489] f. Remove 1×10 6 The cells were used for pluripotency flow cytometry analysis.

[0490] g. The pluripotency flow panel is shown in Table 5.

[0491] h. Meet acceptance criteria before proceeding to the redifferentiation step. Acceptance criteria for pluripotency are:

[0492] >85% Oct4+, Sox2+, SSEA3+, SSEA4+

[0493] <1% SSEA1+

[0494] <5% CD34+

[0495] i. Day 6: Redifferentiation of γδT cell-derived iPSCs to iγδT cells: 2×10 3 Cells / well were plated.

[0496] i. Note: Passage iPSCs at least 5 times before initiating redifferentiation. Continue maintaining iPSCs from step 1.10.ah.

[0497] ii. Based on the final cell count (step 1.10.e), add 10 μM Y-276 to 12 mL of

[0498] Prepare 1.2×10 4 cells.

[0499] iii. If there are any cells remaining after plating, cryopreserve them in Cryostor CS10.

[0500] a. Cryopreserve cells in CS-10 freezing medium as follows:

[0501] i. Centrifuge the cells at 300 × g for 5 minutes at room temperature.

[0502] ii. Carefully remove the supernatant.

[0503] iii. Cell pellet was collected at 1×10 6 -2×10 6 Cells / mL were suspended in CS-10 medium and aliquoted into 1 mL / freezing vial.

[0504] iv. Place the cryovials in a CoolCell freezing container and store at -80°C

[0505] Place in freezer overnight.

[0506] v. Within 3 days, transfer the cells to a liquid N2 tank for storage.

[0507] j. Day 5: Replace the existing culture medium (step 1.1) with fresh supplemented StemFit Basic 04 complete medium.

[0508] k. The cells were incubated and cultured at 37°C, 5% CO2, and the medium was changed every other day until day 0.

[0509] 1.11 Day 0: Beginning of hematopoietic redifferentiation - mesoderm induction phase

[0510] a. Preheat the StemFit Basic 04 complete medium (step 1.1) at room temperature, and

[0511] Prepare Step 1 Medium for Days 0 to 2:

[0512]

[0513] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0514] c. Images were recorded on day 0. Figure 8D An exemplary cell at 4x magnification is shown in FIG, and Figure 8E An exemplary cell at 10x magnification is shown in FIG.

[0515] d. Carefully aspirate the medium and add 2 mL / well of step 1 medium (step 1.11.a) to all wells.

[0516] e. The cells were cultured at 37°C and 5% CO2.

[0517] 1.12 Day 2: Hematopoietic redifferentiation begins - mesoderm to definitive hematopoietic lineage specification.

[0518] a. On the second day, pre-warm Advanced DMEM / F12 medium at room temperature and prepare

[0519] Prepare the culture medium for step 2 from day 2 to day 4:

[0520]

[0521] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0522] c. Record images on day 2 as in step 1.11.c. Figure 8F An exemplary cell at 4x magnification is shown in FIG, and Figure 8G An exemplary cell at 10x magnification is shown in FIG.

[0523] d. Carefully aspirate the medium and add 2 mL / well of step 2 medium (step 1.12.a) to all wells.

[0524] e. The cells were incubated and cultured at 37°C, 5% CO2.

[0525] 1.13 Day 4: Beginning of hematopoietic redifferentiation – angioblast induction phase

[0526] a. On day 4, pre-warm Advanced DMEM / F12 medium at room temperature and prepare the medium for step 3 from day 4 to day 6:

[0527]

[0528] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0529] c. Record images on day 4 as in step 1.11.c. Figure 8H An exemplary cell at 4x magnification is shown in FIG, and Figure 8I An exemplary cell at 10x magnification is shown in FIG.

[0530] d. Carefully aspirate the medium and add 2 mL / well of step 3 medium (step 1.13.a) to all wells without any washing or rinsing.

[0531] e. The cells were incubated and cultured at 37°C, 5% CO2.

[0532] 1.14 Day 6: Beginning of hematopoietic redifferentiation – transition from endothelial cells to hematopoietic cells

[0533] a. On days 6 and 8, pre-warm Advanced DMEM / F12 medium at room temperature and prepare fresh medium from step 4:

[0534]

[0535] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0536] c. Record images on day 6 as in step 1.11.c. Figure 8J An exemplary cell at 4x magnification is shown in FIG, and Figure 8K An exemplary cell at 10x magnification is shown in FIG.

[0537] d. Carefully aspirate the medium and add 2 mL / well of step 4 medium (step 1.14.a) to all wells.

[0538] e. The cells were incubated and cultured at 37°C, 5% CO2.

[0539] f. On day 8, prepare 12 mL of Step 4 medium as outlined in the table above.

[0540] g. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0541] h. Record images on day 8 as in step 1.11.c. Figure 8L An exemplary cell at 4x magnification is shown in FIG, and Figure 8M An exemplary cell at 10x magnification is shown in FIG.

[0542] i. Collect the culture medium from all wells of the plate into 15 mL conical tubes.

[0543] i. The culture medium may contain non-adherent cells.

[0544] j. Add 1 mL of the medium from step 4 to each well.

[0545] k. Centrifuge the 15 mL conical tube containing the collected non-adherent cells at 300 x g for 5 minutes at room temperature.

[0546] 1. Discard the supernatant and resuspend the cell pellet in 6 mL of fresh step 4 medium and distribute 1 mL to each well for a final volume of 2 mL per well.

[0547] m. The cells were incubated and cultured for another two days at 37°C, 5% CO2.

[0548] 1.15 Day 10: Harvest hematopoietic stem cells (iCD34 / iHSC) and prepare cells for flow cytometry analysis and cryopreservation as follows:

[0549] a. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0550] b. Record images on day 10 as in step 1.11.c. Figure 8N An exemplary cell at 4x magnification is shown in FIG, and Figure 8O An exemplary cell at 10x magnification is shown in FIG.

[0551] c. Prepare 50 mL of the base medium from step 4 (step 1.14.a) without cytokines and growth factors.

[0552] d. Process the non-adherent cell fraction as follows: swirl the plate gently and collect the floating cells into a new 50 mL conical tube.

[0553] i. Centrifuge the cells at 300 × g for 5 minutes at room temperature.

[0554] ii. Discard the supernatant and resuspend the cells in step 4 basal medium (step 1.15.c) without added cytokines or growth factors.

[0555] iii. Perform cell counting using NC-200 as in step 1.6.

[0556] e. The adherent cell fraction was treated as follows Using a P1000, detach adherent cells from the plate by gently pipetting up and down 4-6 times in the base medium from step 4 (step 1.15.c) without added cytokines and growth factors.

[0557] i. Collect adherent cells into a new 50 mL conical tube and gently resuspend using a 10 mL pipette. Then pass the cells through a 70 μm cell strainer and then through a 40

[0558] μm cell strainer.

[0559] f. Note: Cells from the non-adherent and adherent fractions remain separate.

[0560] g. Perform cell counting using NC-200 as in step 1.6.

[0561] h. 0.5×10 6 to 1.0×10 6 Cells were aliquoted and used for flow cytometric analysis using the iHSC group.

[0562] i.iHSC flow groups are shown in Table 5.

[0563] j. Success criteria for the iHSC redifferentiation step: purity of each input iPSC >70% lineage-CD34+ cells and fold expansion >100.

[0564] k. As known in the art, iHSC cells can be redifferentiated into iγδT cells.

[0565] Alternatively, iHSCs / iCD34 cells can be cryopreserved in CS-10 freezing medium as in step 1.10.i.iii.a.

[0566] i. Note: Cryopreserved iHSC / iCD34 cells can be thawed and redifferentiated at a later date.

[0567] Example 1: Serum-free, feeder-free regeneration of γδT cells from induced pluripotent stem cells derived from γδT cells change

[0568] method

[0569] Induced pluripotent stem cells (iPSCs): Peripheral blood Vγ9Vδ2 T cells from healthy donors were selectively expanded and then reprogrammed into iPSCs using Yamanaka factors delivered by Sendai virus according to the method of Watanabe et al.

[0570] iPSC characterization: Stored γδT-iPSCs were tested for pluripotency, genomic stability, sterility, morphology, doubling time, HLA genotype, and TCR monoclonality. A two-step 2D research-scale SFFF redifferentiation protocol was used to determine the T cell redifferentiation potential of ΓδT-iPSCs that met the defined acceptance criteria (see Table 6).

[0571] Table 6. Assays used to characterize iγδT-iPSC clones

[0572]

[0573] SFFF iHSC Redifferentiation Protocol: Based on the results of the previously established two-step 2D research-scale SFFF redifferentiation protocol, success criteria were set for the purity and fold expansion of differentiated iHSCs and iγδT cells. Exemplary success criteria include, for example, γδT-iPSC redifferentiation to iHSCs with a purity greater than 70% Lin-CD34+ cells and a fold expansion / input iPSC greater than 100 for 2D culture and greater than 0.5 for 3D / 2D culture, and iHSC differentiation to iγδT cells with a purity of 20% to 40% CD3+TCRγδ+ cells and a fold expansion / input iCD34 greater than 5. The protocol for the first redifferentiation step (iPSC to iHSC) was optimized using two γδT-iPSC clones, clone 1 and 2. Clone 2 is a subclone of clone 1. Clone 3 was used to validate the optimized protocol. Figure 2 A flow chart of the 2D SFFF redifferentiation protocol is shown in FIG. On days 6 to 10, non-adherent cells harvested with media changes were added back to the culture. Figures 6A to 6B As shown, flow cytometry was used to measure the purity and viability of iHSCs (defined as Lin-CD34+ cells).

[0574] SFFF iγδT cell redifferentiation protocol: A previously established 2D protocol for the second redifferentiation step (iHSC to iγδT cells) was used to measure the T cell redifferentiation potential of iHSCs generated in the first redifferentiation step of the optimized protocol. The culture conditions for the iHSC to iγδT cell redifferentiation protocol were as follows: iHSCs were cultured in a 2D culture medium containing StemSpan TM The cells were cultured in a lymphocyte progenitor expansion medium (containing SCF, TPO, Flt3L, and IL-7). The medium was changed on days 13, 17, and 20. iHSCs were redifferentiated on tissue culture dishes coated with VCAM1+DLL4. In addition, redifferentiation of iHSCs into iγδT cells was performed under normoxic conditions (18% O2). Flow cytometry was used to measure the purity and viability of iγδT cells (defined as CD3+Vγ9+Vδ2+ cells). Figure 6C shown.

[0575] result

[0576] γδ T cells were chosen as the origin of iPSCs because the epigenetic and transcriptional memory of these starting cells is known to influence and enhance the redifferentiation potential of iPSCs into this cell type. 4 γδT-iPSCs were characterized in detail using assays that assessed pluripotency, genomic stability, morphology, doubling time, sterility, identity, and TCR monoclonality. Table 6 lists the assays used to characterize iPSC clones and the acceptance criteria for each assay.

[0577] A previously established protocol for the first redifferentiation step (iPSC to iHSC) was optimized using two γδ T-iPSC clones, clones 1 and 2, with the goal of increasing iHSC purity, fold expansion, and T cell redifferentiation potential. Figures 6A to 6C The results of this optimization are shown. Figure 6A Figure 4 shows the morphological changes of iPSC colonies during hematopoietic redifferentiation. Figure 6B The purity and fold expansion of iHSCs in adherent and non-adherent fractions from two to three independent experiments are summarized in . Notably, the purity and fold expansion of iHSCs redifferentiated using the optimized protocol exceeded the success criteria defined for this redifferentiation step by an unexpected and surprising magnitude.

[0578] To determine whether the optimized SFFF iHSC redifferentiation protocol also increased the T cell redifferentiation potential of redifferentiated iHSCs, a previously established SFFF iHSC redifferentiation protocol was used to redifferentiate iHSCs from the adherent fraction into iγδ T cells. Figure 6C As shown, the purity and fold expansion of redifferentiated iγδ T cells unexpectedly and surprisingly exceeded the success criteria defined for this redifferentiation step. Taken together, these data demonstrate that modifications to the SFFF iHSC redifferentiation protocol not only increased iHSC purity and fold expansion but also increased their T cell redifferentiation potential.

[0579] Given the potential of iPSC clones for T cell redifferentiation 5 Since γδT-iPSCs are heterogeneous in terms of differentiation, it is important to use multiple iPSC clones to validate the optimized γδT-iPSC to iHSC redifferentiation protocol. To this end, the optimized protocol has been validated using other fully characterized clones. The data for clone 3 are shown in Figures 6A to 6C In the three clones, similar morphological changes were observed during hematopoietic redifferentiation ( Figure 6A Furthermore, even though the purity of iHSCs from clone 3 was close to the 70% definition standard, its iHSC fold expansion reached or exceeded the 100-fold definition standard ( Figure 6B Importantly, when the T cell redifferentiation potential of clone 3-derived iHSCs from the adherent fraction was assessed, the purity and fold expansion of iγδ T cells were found to unexpectedly and surprisingly exceed the defined criteria ( Figure 6C ). Thus, unexpectedly excellent enhanced T cell redifferentiation potential was observed for two γδ T-iPSC subclones and two individual γδ T-iPSC clones, demonstrating the efficiency and effectiveness of the optimized iHSC redifferentiation protocol.

[0580] Example 2: Redifferentiation protocol of γδT-iPSC clones into iγδT cells

[0581] Material

[0582] Table 7. Reagents and supplies used for cell culture and differentiation

[0583]

[0584] Example Procedure

[0585] 1.2 Prepare supplemented StemFit Basic 04 medium as follows:

[0586] a. StemFit Basic 04 Medium is supplied frozen. StemFit Medium should be stored below -20°C until use. Use aseptic technique to prepare supplemented StemFit Medium.

[0587] b. Thaw frozen StemFit Basic 04 medium at room temperature (15°C-25°C) or in the refrigerator (2°C-8°C) with occasional mixing.

[0588] c. Culture medium should not be thawed at 37°C as this accelerates the degradation of culture medium components.

[0589] d. Supplement StemFit Basic 04 medium with an additional 20 ng / mL of bFGF to achieve a final concentration of 100 ng / mL bFGF in the complete medium.

[0590] 1.3 Prepare a 6-well tissue culture plate with iMatrix 511 as follows:

[0591] a. Pipette 12 mL of 1X PBS into a 15 mL conical tube.

[0592] b. Add 30 μL of iMatrix 511 to 1X PBS and mix thoroughly immediately (coating concentration: 0.25 μg / cm 2 ).

[0593] c. Add 2 mL of iMatrix solution to each well.

[0594] d. Incubate the plate at 37°C, 5% CO2 for at least 60 minutes (the plate can be left at 37°C overnight).

[0595] e. Note: Plates can be stored at 4°C for one week.

[0596] 1.4 Prepare supplemented with 10 μM Y-27632 ROCK inhibitor as follows Basic 04 Medium:

[0597] a. Thaw the culture medium before use.

[0598] b. Aliquot 25 mL of supplemented Stemfit Basic 04 100 ng / mL bFGF into 50 mL conical tubes.

[0599] c. Add 10 μM Y-27632 ROCK inhibitor.

[0600] 1.5 Thaw a vial of γδT-iPSCs as follows:

[0601] a. Thaw a frozen vial of γδT-iPSCs in a 37°C water bath for 1 minute or until small pieces of frozen, floating cells are visible.

[0602] b. Slowly transfer the thawed γδT-iPSCs dropwise from the vial into a 15 mL conical tube containing 9 mL of room temperature supplemented StemFit 04 Basic medium with 10 μM Y-27632 ROCK inhibitor (step 1.4).

[0603] c. Note: The total volume can be 10 mL.

[0604] 1.6 Count the cells as follows:

[0605] a. NucleoCounter NC-200 was used for cell counting purposes.

[0606] b. Pipette a 150 μL aliquot from the cell suspension (step 1.5.b) into a 1.5 mL Eppendorf tube.

[0607] c. Insert vial 1-cassette into the cell suspension from the Eppendorf tube and collect the cells in the cassette.

[0608] d. Insert vial 1-box into the NC-200 cell counter and count the cells.

[0609] e. Only the total number of viable cells was considered for cell seeding purposes.

[0610] 1.7 Remove freezing medium as follows:

[0611] a. Centrifuge the conical tube at 300X g for 3 minutes at room temperature.

[0612] b. Aspirate the supernatant without disturbing the cell pellet, leaving 0.5 mL of supplemented StemFit Basic 04 medium.

[0613] c. Gently tap the conical tube to loosen the pellet.

[0614] d. Add P1000 to 1 mL of supplemented StemFit Basic 04 medium (step 1.4).

[0615] e. Add additional supplemented StemFit Basic 04 medium to obtain 1×10 4 The final cell concentration was 10 cells / mL.

[0616] f. Remove the iMatrix 511 solution from the 6-well plate by aspiration.

[0617] i. NOTE: Avoid allowing the plate to dry for extended periods of time (e.g., more than 10 minutes). The wells should not dry out.

[0618] g. Mix the cells thoroughly and plate at 2 mL / well.

[0619] h. Evenly distribute the cells by rocking the plate back and forth and side to side.

[0620] i. Incubate the plate at 37°C and 5% CO2 overnight.

[0621] 1.8 Day 1: Change the culture medium as follows:

[0622] a. Place Basic 04 medium containing 100 ng / mL bFGF at room temperature.

[0623] b. Aspirate the culture medium from the 6-well plate.

[0624] c. Add 2 mL / well of fresh culture medium (without ROCK inhibitor) to the 6-well plate.

[0625] d. Plates were imaged using EVOS7000 and recorded at 4x and 10x magnification.

[0626] e. Take three images to document the morphology and landscape of the culture. Figure 8P An exemplary cell at 10x magnification is shown in FIG.

[0627] Continue culturing the cells until they reach typical iPSC morphology, displaying all iPSC characteristics, including round colonies with densely packed cells, a higher nuclear to cytoplasm ratio, well-defined edges, and distinct borders. At this stage, iPSC colonies are approximately 70%-80% confluent.

[0628] a. Note that this may take 7 days + / - 1 day.

[0629] b.iPSCs have defined borders and dense cells, such as Figure 8Q Shown, magnified 10 times.

[0630] 1.10 Passage cells and characterize pluripotency by flow cytometry before initiating redifferentiation:

[0631] a. Prepare iMatrix 6-well plates as in step 1.3.

[0632] b. Aspirate the culture medium from the wells.

[0633] c. Rinse the wells with 1 mL of 1X PBS.

[0634] d. Aspirate PBS from the wells.

[0635] e. Add 1 mL / well of TrypLE.

[0636] f. Incubate the plate at 37°C for 5 to 7 minutes.

[0637] g. Inspect the holes under a microscope.

[0638] i. The cells become brighter and cell boundaries appear, e.g. Figure 8R shown.

[0639] h. Gently remove TrypLE.

[0640] i. Add 1 mL of 20 ng / mL bFGF and P1000 to each well of StemFitBasic 04 medium supplemented with 10 μM Y-27632 ROCK inhibitor and pipette to remove cells. Avoid air bubbles.

[0641] j. Use a microscope to ensure that 95% of the cells are removed from the wells.

[0642] If residual cells are still adherent, add 1 mL of StemFit Basic 04 medium supplemented with 20 ng / mL bFGF and 10 μM Y-27632 ROCK inhibitor. Scrape the well using a cell spatula and pipette three (3) times with a P1000 to ensure a uniform population.

[0643] k. Collect the wells into a 15 mL conical tube.

[0644] l. Perform cell count as in step 1.6g.

[0645] m. Collect 1×10 6 The cells were used for pluripotency flow cytometry analysis.

[0646] n. Using Stemfit medium (step 1.4) containing 100 ng / mL bFGF and 10 μM ROCK inhibitor, 2 × 10 3 Cells / well were plated.

[0647] i. Depending on the cell number, prepare enough volume to expand the cells into the desired number of 6-well plates.

[0648] ii. If there is an excess of cells, freeze the remaining cells using Cryostor CS10.

[0649] 1.11 After 24 hours, replace the existing culture medium with StemFit Basic 04 medium containing 100 ng / mL bFGF.

[0650] 1.12 Continue to incubate and culture the cells at 37°C, 5% CO2, and change the medium every other day until day 0.

[0651] Day 0: Beginning of hematopoietic differentiation – mesoderm induction phase

[0652] 2.1 Prepare step 1 culture medium for day 0 to day 2 as follows:

[0653]

[0654] 2.2 Remove the plate from the incubator and observe the cell growth and morphology under an inverted phase contrast microscope.

[0655] a. Images were recorded on day 0. Figure 8S An exemplary cell at 4x magnification is shown in FIG, and Figure 8T An exemplary cell at 10x magnification is shown in FIG.

[0656] 2.3 Carefully aspirate the culture medium and replace 2 mL / well of the culture medium from step 1 in all wells without any washing or rinsing.

[0657] 2.4 The cells were continued to be incubated and cultured at 37°C and 5% CO2.

[0658] Day 2: Hematopoietic differentiation begins – mesoderm to definitive hematopoietic lineage specification

[0659] 2.5 On Day 2, prepare Step 2 medium for Days 2 to 4 as follows:

[0660]

[0661] 2.6 Remove the plate from the incubator and observe the cell growth and morphology under an inverted phase contrast microscope.

[0662] a. Record images on day 2 as in step 2.2a. Figure 8U An exemplary cell at 4x magnification is shown in FIG, and Figure 8V An exemplary cell at 10x magnification is shown in FIG.

[0663] 2.7 Carefully aspirate the culture medium and replace 2 mL / well of step 2 culture medium in all wells without any washing or rinsing.

[0664] 2.8 Continue to incubate and culture the cells at 37°C, 5% CO2.

[0665] Day 4: Hematopoietic differentiation begins—angioblast induction period

[0666] 2.9 On Day 4, prepare the culture medium for Step 3 from Day 4 to Day 6 as follows:

[0667]

[0668] 2.10 Remove the plate from the incubator and observe the cell growth and morphology under an inverted phase contrast microscope.

[0669] a. Record images on day 4 as in step 2.2a. Figure 8X An exemplary cell at 4x magnification is shown in FIG, and Figure 8Y An exemplary cell at 10x magnification is shown in FIG.

[0670] 2.11 Carefully aspirate the culture medium and replace 2 mL / well of the culture medium from step 3 in all wells without any washing or rinsing.

[0671] 2.12 The cells were continued to be incubated and cultured at 37°C and 5% CO2.

[0672] Day 6: Hematopoietic differentiation begins—transition from endothelial cells to hematopoietic cells

[0673] 2.13 On Day 6, prepare the culture medium for Step 4 for Days 6 to 10 as follows:

[0674]

[0675]

[0676] 2.14 Remove the plate from the incubator and observe the cell growth and morphology under an inverted phase contrast microscope.

[0677] a. Record images on day 6 as in 2.2a. Figure 8Z An exemplary cell at 4x magnification is shown in FIG, and Figure 8AA An exemplary cell at 10x magnification is shown in FIG.

[0678] 2.15 Carefully aspirate the culture medium and replace 2 mL / well of the culture medium from step 4 in all wells without any washing or rinsing.

[0679] 2.16 The cells were continued to be incubated and cultured at 37°C and 5% CO2.

[0680] 2.17 On day 8, prepare the culture medium in step 4 as shown in the table above.

[0681] 2.18 Remove the plate from the incubator and observe the cell growth and morphology under an inverted phase contrast microscope.

[0682] a. Record images on day 8 as in step 2.2a. Figure 8BB An exemplary cell at 4x magnification is shown in FIG, and Figure 8CC An exemplary cell at 10x magnification is shown in FIG.

[0683] 2.19 Collect the culture medium from all wells of the plate into 15 mL conical tubes.

[0684] 2.20 Replace 1 mL of the medium from step 4 in each well.

[0685] 2.21 Centrifuge the 15 mL conical tube containing cells and culture medium at 300×g for 5 minutes at room temperature.

[0686] 2.22 Discard the supernatant and resuspend the cell pellet in 6 mL of fresh step 4 medium and distribute 1 mL to each well so that the final volume is 2 mL per well.

[0687] 2.23 The cells were continued to be incubated and cultured at 37°C and 5% CO2.

[0688] Day 10: Harvest hematopoietic cells (iCD34 / iHSC) and prepare samples for flow cytometry characterization and Freezing of iCD34 / iHSCs

[0689] 2.24 Remove the plate from the incubator and observe the cell growth and morphology under an inverted phase contrast microscope.

[0690] a. Record images on day 10 as in step 2.2a. Figure 8DD An exemplary cell at 4x magnification is shown in FIG, and Figure 8EE An exemplary cell at 10x magnification is shown in FIG.

[0691] 2.25 Process the non-adherent fraction as follows: Collect the floating cells into a new 50 mL conical tube by gently swirling the plate.

[0692] 2.26 Centrifuge the conical tube at 300X g for 5 minutes at room temperature.

[0693] 2.27 Discard the supernatant and resuspend the cells in step 4 medium without cytokines or growth factors and count the cells.

[0694] 2.28 Count cells using NC-200.

[0695] 2.29 Treat the adherent cell fraction as follows: Resuspend the adherent cell fraction using the medium from step 4 without cytokines and growth factors to allow adherent cells to detach from the plate (mix 4-6 times using a P1000).

[0696] 2.30 Collect the cells into a new 50 mL conical tube and gently resuspend with a 10 mL pipette and pass through a 70 μm cell strainer and then through a 40 μm cell strainer.

[0697] 2.31 Using the iHSC group, an appropriate number of cells were used for flow cytometry analysis.

[0698] 2.32 Cryopreserve iHSC / iCD34 cells using CS-10 freezing medium.

[0699] 2.33 Cryopreservation method: Centrifuge the cells at 300X g for 5 minutes.

[0700] 2.34 Remove the supernatant.

[0701] a. Cell pellet was prepared at 1×10 6 -2×10 6 Cells / mL were resuspended in CS-10 and frozen into cryovials.

[0702] b. Place the cryovial in a CoolCell and store the container in a -80°C freezer.

[0703] c. The next day, transfer the cells to a liquid N2 tank for storage.

[0704] Day 10: Differentiation of iCD34 / iHSCs into iγδT cells

[0705] 3.1 Preparation of VCAM1 / DLL1 / 4 / Jagged 2-coated plates.

[0706] 3.2 Prepare 10 μg / mL of DLL4 or DLL1 or Jagged 2 or all three Notch ligand proteins + 5 μg / mL of VCAM1 (without Ca) in PBS. +2 or Mg +2 ).

[0707] 3.3 Add 100 μL / well to a tissue culture treated 48-well plate and shake it well to cover the surface of the wells.

[0708] 3.4 Incubate the plate at 4°C overnight or at 37°C for 1 hour (add PBS or water to the wells surrounding the coated wells to avoid evaporation).

[0709] 3.5 Resuspend the harvested iCD34 / iHSCs in lymphoid progenitor cell expansion medium (StemCell Technologies).

[0710] 3.6 Perform cell counting.

[0711] 3.7 Remove VCAM1 / DLL1 / 4 / Jagged 2 / PBS from the pre-coated plate and replace the plate with PBS (without Ca +2 or Mg +2 )Wash once.

[0712] 3.8 will be 1.2×10 4 viable cells / 250 μL of lymphoid progenitor expansion medium were seeded per well of a pre-coated 48-well plate.

[0713] 3.9 Incubate at 37°C under 5% CO2 and 18% O2 for further culture and differentiation.

[0714] 3.10 Day 13: Add 250 μL of lymphocyte progenitor cell expansion medium.

[0715] 3.11 Day 17: Remove 250 μL of culture medium and add 270 μL of *Lymphoid Progenitor Expansion Medium.

[0716] (*Aim for a half medium change, but add slightly more volume than removed to account for evaporation).

[0717] 3.12 Day 20: Remove 250 μL of culture medium and add 270 μL of *Lymphoid Progenitor Expansion Medium without disturbing the cells on the plate until day 24.

[0718] 3.13 Day 24: Harvest cells.

[0719] 3.14 Count the cells and prepare samples for flow cytometry analysis to assess the purity of iγδ T cells.

[0720] Example 3: Serum-free, feeder-free, γδ T cell-derived iPSCs to iHSCs and then to iγδ T cells 3D / 2D redifferentiation .

[0721] Material

[0722] Table 8. Reagents used for iPSC culture and maintenance .

[0723]

[0724] Table 9. Reagents required for hematopoietic differentiation (listed by redifferentiation day) .

[0725]

[0726] Table 10. Reagents for iγδ T cell redifferentiation .

[0727] Reagents Product Number <![CDATA[StemSpan TM SAINTS]]> StemCell Technologies, catalog number 09655 Lymphocyte Progenitor Expansion Supplement (10X) StemCell Technologies, catalog number 09915 rh VCAM-1-Fc R&D, Cat. No. 862-VC-100 rh DLL4-Fc R&D, catalog number 10185-D4-050

[0728] Table 11. Supplies and consumables required for cell culture .

[0729]

[0730] Table 12. Flow cytometry antibody combinations .

[0731]

[0732]

[0733] Example Procedure

[0734] 1.1 Prepare supplemented StemFit Basic 04 complete medium as follows:

[0735] a. Provide StemFit Basic 04 Complete Medium in frozen form and store at -20°C or below until use. Use aseptic technique to prepare supplemented StemFit Basic 04 Complete Medium.

[0736] b. Thaw frozen StemFit Basic 04 Complete Medium at room temperature (15°C-25°C) or in the refrigerator (2°C-8°C) with occasional mixing.

[0737] c. Note: Do not thaw the culture medium at 37°C as this accelerates the degradation of the culture medium components.

[0738] d. Supplement StemFit Basic 04 complete medium with an additional 20 ng / mL of bFGF to achieve 100 ng / mL bFGF in the supplemented medium.

[0739] e. Store the supplemented medium at 4°C for up to two (2) weeks.

[0740] f. Bring the culture medium to room temperature (15°C-25°C) at least one (1) hour before use.

[0741] 1.2 Prepare 10 mM Y-27632 ROCK inhibitor stock solution as follows:

[0742] a. Y-27632 ROCK inhibitor was received as a crystalline solid and stored at -20°C for up to 12 months from the date of receipt.

[0743] b. Prepare a 10 mM stock solution in 1X DPBS as follows:

[0744] i. Add 312 μL of 1X DPBS to 1 mg of Y-27632 and resuspend the solution completely.

[0745] ii. Aliquot 25 μL of the stock solution into Eppendorf tubes labeled Y-27632 Stock.

[0746] iii. Aliquots are stable at -20°C for up to 6 months.

[0747] iv. Aliquot the working volume to avoid repeated freeze-thaw cycles.

[0748] 1.3 Prepare a 6-well tissue culture plate coated with iMatrix 511 as follows:

[0749] a. Pipette 12 mL of 1X DPBS into a 15 mL conical tube.

[0750] b. Add 30 μL of iMatrix 511 to 1X DPBS and mix thoroughly immediately (coating concentration: 0.25 μg / cm 2 ).

[0751] c. Add 2 mL of iMatrix solution to each well.

[0752] d. Incubate the plate at 37°C, 5% CO2 for at least 60 minutes (the plate can be left at 37°C overnight).

[0753] e. Note: After the coating step, the plates can be stored at 4°C for one week. Before use, incubate the plates at 37°C for 1 hour.

[0754] 1.4 Prepare complete StemFit Basic 04 medium supplemented with 10 μM Y-27632 ROCK inhibitor (step 1.1) as follows:

[0755] a. Thaw an aliquot of 10 mM Y-27632 ROCK inhibitor stock solution before use.

[0756] b. Aliquot 25 mL of supplemented StemFit Basic 04 complete medium (step 1.1) into 50 mL conical tubes.

[0757] c. Add 25 μL of 10 mM Y-27632 ROCK inhibitor to the supplemented StemFit Basic 04 complete medium to reach a final concentration of medium with 10 μM Y-27632 ROCK inhibitor.

[0758] 1.5 Thaw a vial of γδT-iPSCs as follows:

[0759] a. Thaw a vial of frozen γδT-iPSCs in a 37°C water bath for 1 minute or until small clumps of frozen, floating cells are visible.

[0760] b. Slowly transfer the thawed γδT-iPSCs dropwise from the vial into a 15 mL conical tube containing 9 mL of room temperature supplemented StemFit 04 Basic complete medium with 10 μM Y-27632 ROCK inhibitor (from step 1.4).

[0761] c. Note: The total volume is 10 mL.

[0762] 1.6 Perform cell counting as follows:

[0763] a. A NucleoCounter NC-200 instrument was used for cell counting purposes.

[0764] b. Remove a 150 μL aliquot from the cell suspension (step 1.5.b) into a 1.5 mL Eppendorf tube.

[0765] c. Collect cells from the Eppendorf tube into vial-box.

[0766] d. Insert the vial-box into the NC-200 cytometer and count the cells.

[0767] e. Note: Only use the total number of viable cells for cell seeding purposes

[0768] 1.7 Wash and plate thawed γδT-iPSCs as follows:

[0769] a. Centrifuge the 15 mL conical tube containing the thawed γδT-iPSCs (step 1.5b) at 300 × g for 3 minutes at room temperature.

[0770] b. Aspirate the supernatant without disturbing the cell pellet, leaving 0.5 mL of culture medium.

[0771] c. Gently tap the conical tube to loosen the cell pellet.

[0772] d. Add P1000 to 1 mL of supplemented StemFit Basic04 complete medium with 10 μM Y-27632 ROCK inhibitor (step 1.4) to resuspend the cells.

[0773] e. Add additional supplemented StemFit Basic 04 complete medium (step 1.4) with 10 μM Y-27632 ROCK inhibitor to reach 1×10 4 The final cell concentration was 10 cells / mL.

[0774] f. Remove the iMatrix 511 solution from the 6-well plate by aspiration.

[0775] i. NOTE: Do not allow the plate to dry for an extended period of time (e.g., more than 10 minutes). Do not allow the wells to dry.

[0776] g. Mix the cells thoroughly and then plate them at 2 mL / well.

[0777] h. Distribute the cells evenly across the bottom of the wells by gently rocking the plate manually back and forth and side to side.

[0778] i. Incubate the plate at 37°C in 5% CO2 overnight.

[0779] 1.8 Day 1: Perform medium change as follows:

[0780] a. Transfer the supplemented StemFit Basic 04 complete medium (step 1.1) from 4°C to room temperature.

[0781] b. Gently aspirate the culture medium from the 6-well plate.

[0782] c. Add 2 mL / well of fresh StemFit Basic 04 complete medium (without ROCK inhibitor) (step 1.1) to the 6-well plate.

[0783] d. Plates were imaged using EVOS M7000 and recorded at 4X and 10X magnification.

[0784] e. Three images were taken from different areas of the plate to document the morphology and landscape of the culture. Single cells and two-cell colonies were present. The cells had a spiky morphology due to the ROCK inhibitor.

[0785] i. Note: As the cells divide, the boundaries become more distinct.

[0786] ii. Figure 8FF An example view at 10x magnification is shown in .

[0787] 1.9 Continue culturing the cells until they reach typical morphology, exhibiting all iPSC characteristics, including round colonies with densely packed cells, a high nuclear to cytoplasm ratio, well-defined edges, and distinct borders. At this stage, the confluency of the iPSC colonies is approximately 70%-80%.

[0788] a. Note: It may take 7 days + / - 1 day for colonies to develop this morphology.

[0789] b. iPSC colonies have defined borders and dense cells.

[0790] i. Figure 8GG An example view at 10x magnification is shown in .

[0791] 1.10 Passage cells and confirm pluripotency by flow cytometry before initiating redifferentiation

[0792] a. Passage iPSCs as follows:

[0793] i. Aspirate the culture medium from the cells plated in step 1.7.

[0794] ii. Rinse the wells with 1 mL of 1X DPBS.

[0795] iii. Aspirate 1X DPBS from the wells.

[0796] iv. Add 1 mL of TrypLE and incubate at 37°C for 6 to 8 minutes.

[0797] v. Check the hole under a microscope. Figure 8HH As shown, cells appear bright with clear cell borders.

[0798] vi. Gently remove TrypLE.

[0799] vii. Add P1000 to 1 mL of supplemented StemFit Basic 04 complete medium with 10 μM Y-27632 ROCK inhibitor (from step 1.4), and then gently pipette to remove the cells from the plate. Avoid forming bubbles.

[0800] b. Inspect the wells microscopically to ensure that 95% of the cells are removed from the wells.

[0801] c. If residual cells are still attached to the plate, add 1 mL of StemFit Basic 04 complete medium with 10 μM Y-27632 ROCK inhibitor (step 1.4). Then scrape the well using a cell spatula and pipette three (3) times using a P1000 to ensure a uniform single cell suspension.

[0802] d. Collect the cells into a 15 mL conical tube.

[0803] e. Count cells as in step 1.6.

[0804] f. Remove 1×10 6 The cells were used for pluripotency flow cytometry analysis.

[0805] g. The multipotency flow groups are shown in Table 12.

[0806] h. Meet acceptance criteria before proceeding to the redifferentiation step. Acceptance criteria for pluripotency are:

[0807] >85%Oct4+, Sox2+, SSEA3+, SSEA4+

[0808] <1% SSEA1+

[0809] <5% CD34+

[0810] i. Day 2: Redifferentiation of γδT cell-derived iPSCs to iγδT cells: Based on cell counts (step 1.10.e), 400 cells were plated in a low-attachment 6-well plate using supplemented StemFit Basic04 complete medium with 10 μM Y-27632 ROCK inhibitor (step 1.

[0811] 4), with 0.6×10 6 Cells / well were plated.

[0812] i. Note: iMatrix-511 coating is not required.

[0813] ii. Note: iPSCs should be passaged at least 5 times before initiating redifferentiation. Continue to maintain iPSCs from step 1.10.ah.

[0814] iii. Based on the cell count (step 1.10.e), prepare 3.6×10 cells / mL in 24 mL of StemFit Basic 04 complete medium supplemented with 10 μM Y-27632 ROCK inhibitor (step 1.4). 6 cells and distributed 4 mL / well in a low attachment 6-well plate.

[0815] iv. If there are any cells remaining after plating, cryopreserve them in Cryostor CS-10.

[0816] a. Cryopreserve cells in CS-10 freezing medium as follows:

[0817] i. Centrifuge the cells at 300 × g for 5 minutes at room temperature.

[0818] ii. Carefully remove the supernatant.

[0819] iii. Cell pellet was collected at 1×10 6 -2×10 6 Cells / mL were suspended in CS-10 medium and aliquoted into 1 mL / freezing vial.

[0820] iv. Place the cryovials in a CoolCell freezing container and store at -80°C

[0821] Place in freezer overnight.

[0822] v. Within 3 days, transfer the cells to a liquid N2 tank for storage.

[0823] j. Inspect the low attachment plate under a microscope to ensure that the cells are evenly distributed in the wells.

[0824] k. The plate was incubated in a shaking incubator / multitron (full-function shaking incubator) and the cells were cultured at 37°C, 5% CO2 and 85% humidity for 48 hours. The plate was agitated at 100 RPM. Uniform embryoid bodies (EBs) were observed within 48 hours.

[0825] 1.11 Day 0: Beginning of hematopoietic differentiation - mesoderm induction phase

[0826] a. Pre-warm StemFit Basic 04 Complete Medium (step 1.1) at room temperature and prepare the medium for step 1 from day 0 to day 2:

[0827]

[0828] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0829] c. Images were recorded on day 0. Figure 8II An exemplary cell at 4x magnification is shown in FIG.

[0830] d. Collect EBs containing medium from all wells of the low attachment plate and place them into 50 mL conical tubes.

[0831] e. Centrifuge the 50 mL conical tube at 500 x g for 3 minutes at room temperature.

[0832] f. Carefully aspirate the supernatant and gently resuspend the pelleted EBs in 12 mL of fresh Step 1 medium (Step 1.11.a) and distribute 2 mL into each well of a 6-well TC-treated plate.

[0833] g. Continue to incubate and culture the cells at 37°C, 5% CO2. No agitation is required at this stage.

[0834] 1.12 Day 2: Beginning of hematopoietic differentiation - mesoderm to definitive hematopoietic lineage specification

[0835] a. On day 2, pre-warm Advanced DMEM / F12 medium at room temperature and prepare the medium for step 2 from day 2 to day 4:

[0836]

[0837] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0838] c. Record images on day 2 as in step 1.11.c. Figure 8JJ An exemplary cell at 4x magnification is shown in FIG.

[0839] d. Collect EBs and cells in culture medium from all wells of a 6-well TC-treated plate and place them in 15 mL conical tubes.

[0840] e. Add 1 mL of Step 2 medium (Step 1.12.a) to each well.

[0841] f. Centrifuge the 15 mL conical tube containing EBs, cells, and culture medium at 500 x g for 3 minutes at room temperature.

[0842] g. Carefully aspirate the supernatant and gently resuspend the pelleted EBs and cells in 6 mL of fresh Step 2 medium (Step 1.12.a) and distribute 1 mL to each well, so that the final volume per well of the 6-well TC-treated plate is 2 mL.

[0843] h. Continue to incubate and culture the cells at 37°C, 5% CO2. No agitation is required at this stage.

[0844] 1.13 Day 4: Beginning of hematopoietic differentiation – angioblast induction phase

[0845] a. On day 4, pre-warm Advanced DMEM / F12 medium at room temperature and prepare the medium for step 3 from day 4 to day 6:

[0846]

[0847] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0848] c. Record images on day 4 as in step 1.11.c. Figure 8KK An exemplary cell at 4x magnification is shown in FIG.

[0849] d. Collect EBs and cells in culture medium from all wells of the TC-treated plate into 15 mL conical tubes.

[0850] e. Add 1 mL of step 3 medium (1.13.a) to each well.

[0851] f. Centrifuge the 15 mL conical tube containing EBs, cells, and culture medium at 500 x g for 3 minutes at room temperature.

[0852] g. Carefully aspirate the supernatant and gently resuspend the pelleted EBs and cells in 6 mL of fresh step 3 medium (1.13.a) and distribute 1 mL to each well, so that the final volume per well of the 6-well TC-treated plate is 2 mL.

[0853] h. Continue to incubate and culture the cells at 37°C, 5% CO2. No agitation is required at this stage.

[0854] 1.14 Day 6: Beginning of hematopoietic differentiation – transition from endothelial cells to hematopoietic cells

[0855] a. On days 6 and 8, pre-warm Advanced DMEM / F12 medium at room temperature and prepare fresh medium from step 4:

[0856]

[0857] b. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0858] c. Record images on day 6 as in step 1.11.c. Figure 8LL An exemplary cell at 4x magnification is shown in FIG.

[0859] d. Collect EBs and cells in culture medium from all wells of the TC-treated plate into 15 mL conical tubes.

[0860] e. Add 1 mL of step 4 culture medium (step 1.14.a) to each well.

[0861] f. Centrifuge the 15 mL conical tube containing EBs, cells, and culture medium at 500 X g for 3 minutes at room temperature.

[0862] g. Carefully aspirate the supernatant and gently resuspend the pelleted EBs and cells in 6 mL of fresh step 4 medium (step 1.14.a) and distribute 1 mL to each well, so that the final volume per well of the 6-well TC-treated plate is 2 mL.

[0863] h. Continue to incubate and culture the cells at 37°C, 5% CO2. No agitation is required at this stage.

[0864] i. On day 8, prepare step 4 medium (step 1.14.a).

[0865] j. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0866] k. Record images on day 8 as in 1.11.c. Figure 8MM An exemplary cell at 4x magnification is shown in FIG.

[0867] 1. Collect EBs and cells in culture medium from all wells of the TC plate into a 15 mL conical tube.

[0868] m. Add 1 mL of step 4 medium (step 1.14.a) to each well.

[0869] n. Centrifuge the 15 mL conical tube containing EBs, cells, and culture medium at 500 × g for 3 minutes at room temperature.

[0870] Carefully aspirate the supernatant and gently resuspend the pelleted EBs and cells in 6 mL of fresh Step 4 medium (Step 1.14.a) and distribute 1 mL so that the final volume per well of a 6-well TC-treated plate is 2 mL.

[0871] p. Continue to incubate and culture the cells at 37°C, 5% CO2. No agitation is required at this stage.

[0872] 1.15 Day 10: Harvest hematopoietic stem cells (iCD34 / iHSC) and prepare samples for flow cytometry analysis and cryopreservation as follows:

[0873] a. Remove the plate from the incubator and place it on an inverted phase contrast microscope to observe cell growth and morphology.

[0874] b. Record images on day 10 as in step 1.11.c. Figure 8W Exemplary cells at 4x magnification are shown.

[0875] c. Prepare 50 mL of the base medium from step 4 (step 1.14.a) without cytokines and growth factors.

[0876] d. Gently pipette the EBs and cells in the wells up and down to ensure that all EBs are in suspension. Transfer the suspended EBs and cells from all wells to a 15 mL conical tube.

[0877] e. Centrifuge the EB and cell suspension at 300 × g for 5 minutes at room temperature.

[0878] f. Carefully aspirate the supernatant and add 1 mL of collagenase II (20 mg / mL or 2500 U / mL) to the pellet. Gently pipette the supernatant up and down to mix. Incubate the EBs and cells at 37°C, 5% CO2 for 20 minutes.

[0879] g. Add 3 mL of TryPLE Select to the EB and cell suspension and mix by gently pipetting up and down. Incubate the EB and cell suspension at 37°C, 5% CO2 for 20 minutes.

[0880] h. Remove the suspension from the incubator and mix gently to break up any remaining clumps.

[0881] i. Add 6 mL of basal medium from step 4 (step 1.15.c) without cytokines and growth factors.

[0882] j. Centrifuge the suspension at 300 x g for 5 minutes at room temperature.

[0883] k. Carefully aspirate the supernatant and gently resuspend the pellet in 10 mL of step 4 basal medium (step 1.15.c) without cytokines and growth factors.

[0884] 1. Gently mix the cell suspension and then pass it through a 70 μm cell strainer, followed by a 40 μm cell strainer.

[0885] m. Perform cell counting using NC-200 (step 1.6).

[0886] n.0.5×10 6 to 1.0×10 6 Cells were aliquoted and used for flow cytometric analysis using the iHSC group.

[0887] The o.iHSC flow groups are shown in Table 12.

[0888] p. Success criteria for the iHSC redifferentiation step: purity of each input iPSC >70% lineage marker negative CD34+ cells and fold expansion >0.5.

[0889] q.iHSC / iCD34 cells were either cryopreserved in CS-10 freezing medium as in step 1.10.i.iv.a. or redifferentiated into iγδ T cells.

[0890] i. Note: Cryopreserved iHSC / iCD34 cells can be thawed and redifferentiated at a later date.

[0891] Implementation Plan List The following is a non-exhaustive list of contemplated embodiments of the present invention.

[0892] 1. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0893] seeding human γδ T cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days;

[0894] Initiating redifferentiation of the iPSCs into iHSCs by culturing in the tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates;

[0895] culturing the iPSC-derived cell intermediate for about 8 days;

[0896] collecting non-adherent iPSC-derived cell intermediates and adding them back into the culture; and

[0897] At approximately day 10 of culture, redifferentiated iHSCs were harvested as two different cell fractions—a non-adherent cell fraction and an adherent cell fraction.

[0898] 2. The method according to item 1, further comprising:

[0899] The step of redifferentiation of the iPSCs is initiated in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

[0900] 3. The method according to item 1, further comprising:

[0901] culturing the iPSC-derived cell intermediate for about 2 days in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or an antibiotic; and / or

[0902] Culturing the iPSC-derived cell intermediate for about 2 days in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic; and / or

[0903] The iPSC-derived cell intermediates are cultured for about 2 days in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally antibiotics; and / or the redifferentiated iHSCs are cultured for about 2 days in a composition comprising L-glutamine and optionally antibiotics, wherein the composition does not comprise cytokines or growth factors.

[0904] 4. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0905] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0906] The method further comprises one or more of the following steps:

[0907] a) iPSCs were cultured at a rate of approximately 1.7 × 10 3 cells / well is approximately 2.5×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0908] b) maintaining the iPSCs under culture conditions for about 5.5 days to about 6.5 days;

[0909] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 1.8 days to about 2.2 days to obtain an iPSC-derived cell intermediate, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0910] d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 1.8 days to about 2.2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEG-177 at a ratio of about 3.9:0.9 to about 4.1:1.1. For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0911] e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 1.8 days to about 2.2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 3.9:0.9 to about 4.1:1.1. For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0912] f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEG-170 at a ratio of about 3.9:0.9 to about 4.1:1.1. For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0913] g) after about 7.8 days to about 8.2 days of redifferentiation, collecting non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0914] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and PEG / T7 medium at a ratio of about 3.9:0.9 to about 4.1:1.1. For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0915] i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing;

[0916] j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size;

[0917] k) determining the purity of the iHSCs, defined as CD34+ and lineage marker negative;

[0918] 1) not introducing serum in any of steps ak;

[0919] n) not introducing any additional cells, including feeder cells, in any of steps a to m; or

[0920] o) Obtaining de novo generated iHSCs.

[0921] 5. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0922] a) The iPSCs were cultured at a rate of approximately 0.4×10 6 cells / well to about 1.2×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0923] b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs);

[0924] c) transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells, and initiating redifferentiation of the EBs for about 10 days;

[0925] d) obtaining a suspension of the EBs and cells;

[0926] e) Optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

[0927] 6. The method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions according to item 5, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method further comprising:

[0928] Provides CD34+ and lineage marker negative iHSCs.

[0929] 7. The method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions according to item 6, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method further comprising:

[0930] Obtain CD34+ and CD3 - , and / or CD19 - , and / or CD14 - , and / or CD11b - , and / or CD11c-, and / or CD56 - , and / or CD20 - , and / or CD16 - , and / or CD2 - , and / or CD235a - iHSC.

[0931] 8. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0932] i) obtaining one or more human γδ T cell-derived iPSCs, and

[0933] The method further comprises one or more of the following steps:

[0934] a) The iPSCs were cultured at a rate of approximately 0.55×10 6 cells / well is approximately 0.65×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0935] b) maintaining the iPSCs under culture conditions with agitation for about 44 hours to about 52 hours to generate embryoid bodies (EBs);

[0936] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 1.8 days to about 2.2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 90 ng / mL to about 110 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0937] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional about 1.8 to about 2.2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0938] e) culturing the suspension of EBs and cells in a third cell culture medium for an additional about 1.8 days to about 2.2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0939] f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 1.8 days to about 4.2 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0940] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0941] h) resuspending the pelleted EB and cell suspension in a volume of about 18 mg / mL to about 20 mg / mL of type II collagenase, and incubating the EB and cell suspension in the type II collagenase;

[0942] i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension;

[0943] j) mixing the single cell suspension;

[0944] k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DM EM / F12 medium and ForDifferentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors;

[0945] 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in the fifth cell culture medium to form a second cell suspension;

[0946] m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size;

[0947] n) determining the purity of the iHSCs, defined as CD34+ and lineage marker negative;

[0948] o) not introducing serum in any of steps a to b;

[0949] p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a to b; or

[0950] q) Obtain de novo generated iHSCs.

[0951] 9. The method according to item 4 or item 8, wherein the cells are cultured under normoxic conditions.

[0952] 10. The method according to any one of items 4 and 8-9, further comprising further purifying and / or isolating the iHSC.

[0953] 11. The method according to any one of items 4 and 8-10, wherein the iHSCs obtained are isolated differentiated cells or are capable of being further purified and / or isolated.

[0954] 12. The method according to any one of items 4 and 8-11, wherein the step of determining the purity of the iHSC comprises: measuring all of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a via flow cytometry or its equivalent.

[0955] 13. The method of any one of items 4 and 8-12, wherein the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0956] 14. One or more cells, said one or more cells being obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising the steps according to any one of items 1-13.

[0957] 15. The one or more cells according to item 14, wherein the step of determining the purity of the iHSC comprises: measuring all of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a via flow cytometry or its equivalent.

[0958] 16. One or more cells according to any one of items 14 or 15, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a.

[0959] 17. One or more cells according to any one of items 14-16, wherein the one or more cells have high purity, viability, multiplication expansion and potential to redifferentiate into immune effector cells, wherein the immune effector cells include iPSC-derived γδ (iγδ) T cells.

[0960] 18. A composition comprising one or more cells according to any one of items 14-17.

[0961] 19. Use of one or more cells according to any one of items 14 to 17 in the preparation of cells for treating lesions or diseases, in the preparation of lymphocytes, in bioreactors, in tissue engineering or in vitro drug screening for diseases.

[0962] 20. A system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps according to claim 1 or 5.

[0963] 21. The system according to item 20, wherein the process is performed manually or with the assistance of automatic robots or a combination thereof.

[0964] 22. The system according to any one of items 20-21, wherein the process is automated or semi-automated.

[0965] 23. A system according to any one of items 20-22, comprising one or more software packages that operate and schedule the operation of the system.

[0966] 24. A system according to item 23, wherein the software package is customized and / or customizable for a desired application and is optionally menu driven.

[0967] 25. The system of claim 22, wherein the automation comprises cell visualization, plate handling, plate spreading, inoculation, extraction, addition, cell feeding, incubation assays and / or sampling.

[0968] 26. The system according to any one of items 20-25, comprising one or more online incubators.

[0969] 27. A system according to any one of items 20-26, wherein the system includes electronic humidity control, a HEPA filter system, a turntable including a programmable step oscillation cycle and / or a two-way communication interface or any combination thereof.

[0970] 28. The system according to any one of items 20-27, wherein the addition or removal or supplementation of culture medium does not disturb or contaminate the cells.

[0971] 29. A system according to any one of items 20 to 28, comprising one or more computer operated and controlled robotic arms.

[0972] 30. The method according to any one of items 4 and 8-13, wherein the method further comprises one or more of the following steps:

[0973] a) The iPSCs were cultured at a rate of approximately 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0974] b) maintaining the iPSCs under culture conditions for up to 6 days;

[0975] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 2 days to obtain iPSC-derived cell intermediates, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0976] d) culturing the iPSC-derived cell intermediate in the second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEG-1 at a ratio of about 4:1. ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0977] e) culturing the iPSC-derived cell intermediate in the third cell culture medium for an additional about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0978] f) culturing the iPSC-derived cell intermediate in the fourth cell culture medium for an additional about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and For Diffusion Medium, and further comprising L-glutamine, SCF, IL-6, EPO, and optionally penicillin / streptomycin; or

[0979] g) after about 8 days of redifferentiation, collecting non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium;

[0980] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in the fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and 4:1 at a ratio of about 4:1. For Differentiation medium, and further comprises L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors.

[0981] 31. The method according to any one of items 8 to 13, further comprising:

[0982] a) The iPSCs were cultured at a rate of approximately 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[0983] b) maintaining the iPSCs under culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs);

[0984] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0985] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0986] e) culturing the suspension of EBs and cells in the third cell culture medium for another about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and Glucocorticoids (GlcNAc) in a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0987] f) culturing the suspension of EBs and cells in the fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0988] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[0989] h) resuspending the pelleted EB and cell suspension in a volume of about 20 mg / mL of type II collagenase and incubating the EB and cell suspension in the type II collagenase; or

[0990] k) adding the fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprises L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors.

[0991] 32. The one or more cells according to item 14, wherein the method of generating the one or more cells further comprises:

[0992] a) The iPSCs were cultured at a rate of approximately 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[0993] b) maintaining the iPSCs under culture conditions for up to 6 days;

[0994] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 2 days to obtain iPSC-derived cell intermediates, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[0995] d) culturing the iPSC-derived cell intermediate in the second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEG-1 at a ratio of about 4:1. ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[0996] e) culturing the iPSC-derived cell intermediate in the third cell culture medium for an additional about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[0997] f) culturing the iPSC-derived cell intermediate in the fourth cell culture medium for an additional about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and For Diffe rentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[0998] g) after about 8 days of redifferentiation, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium; or

[0999] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in the fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and 4:1 at a ratio of about 4:1. For Differentiation medium, and further comprises L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors.

[1000] 52. The one or more cells according to item 14, wherein the method of generating the cells further comprises:

[1001] a) The iPSCs were cultured at a rate of approximately 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[1002] b) maintaining the iPSCs under culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs);

[1003] c) transferring the suspension of EBs and cells from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in the first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of about 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021

[1004] (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[1005] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[1006] e) culturing the suspension of EBs and cells in the third cell culture medium for another about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-177 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[1007] f) culturing the suspension of EBs and cells in the fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[1008] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[1009] h) resuspending the pelleted EB and cell suspension in a volume of about 20 mg / mL of type II collagenase and incubating the EB and cell suspension in the type II collagenase; or

[1010] k) adding the fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprises L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors.

[1011] 53. The system according to item 20,

[1012] The method further comprises one or more of the following steps:

[1013] a) The iPSCs were cultured at a rate of approximately 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent;

[1014] b) maintaining the iPSCs under culture conditions for up to 6 days;

[1015] c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 2 days to obtain iPSC-derived cell intermediates, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[1016] d) culturing the iPSC-derived cell intermediate in the second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEG-1 at a ratio of about 4:1. ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[1017] e) culturing the iPSC-derived cell intermediate in the third cell culture medium for an additional about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[1018] f) culturing the iPSC-derived cell intermediate in the fourth cell culture medium for an additional about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and For Diffe rentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[1019] g) after about 8 days of redifferentiation, collecting the non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium; or

[1020] h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in the fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and 4:1 at a ratio of about 4:1. For Differentiation medium, and further comprises L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors.

[1021] 54. The system according to item 20, wherein the method further comprises:

[1022] a) The iPSCs were cultured at a rate of approximately 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent;

[1023] b) maintaining the iPSCs under culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs);

[1024] c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in the first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF);

[1025] d) culturing the suspension of EBs and cells in a second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein the SB431542 has a final concentration of about 1.5 μM to about 2.5 μM, and optionally wherein the SB431542 is diluted from a stock concentration of about 5 mM;

[1026] e) culturing the suspension of EBs and cells in the third cell culture medium for another about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-177 at a ratio of about 4:1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin;

[1027] f) culturing the suspension of EBs and cells in the fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin;

[1028] g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells;

[1029] h) resuspending the pelleted EB and cell suspension in a volume of about 20 mg / mL of type II collagenase and incubating the EB and cell suspension in the type II collagenase; or

[1030] k) adding the fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprises L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors.

[1031] 55. A method for generating hematopoietic stem cells (iHSCs) de novo under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising: (a) a step for initiating redifferentiation of the iPSCs to iHSCs by culturing in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; (b) a step for culturing the iPSC-derived cell intermediates for about 8 days; (c) a step for collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and (d) a step for harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction - on about the 10th day of culture.

[1032] 56. A method for generating hematopoietic stem cells (iHSCs) de novo under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising: a) a step for inoculating the iPSCs; b) a step for culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step for transferring the EBs to a tissue culture-treated culture dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture-treated culture dish contains a suspension of EBs and cells; d) a step for obtaining the suspension of the EBs and cells; and e) optionally, a step for processing the suspension of the EBs and cells to obtain a single-cell suspension.

[1033] 57. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step of seeding the iPSCs; b) a step of culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs);

[1034] c) a step of transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a step of obtaining a suspension of the EBs and cells; e) a step of optionally processing the suspension of EBs and cells to obtain a single cell suspension; and f) a step of providing CD34+ and lineage marker negative iHSCs.

[1035] 58. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising: a) a step of seeding the iPSCs; b) a step of culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step of transferring the EBs to a tissue culture-treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture-treated dish contains a suspension of EBs and cells; d) a step of obtaining the suspension of EBs and cells; e) optionally, a step of processing the suspension of EBs and cells to obtain a single cell suspension; and f) a step of obtaining a CD34+ and CD3 - , and / or CD19 - , and / or CD14 - , and / or CD11b - , and / or CD11c-, and / or CD56 - , and / or CD20 - , and / or CD16 - , and / or CD2 - , and / or CD235a - iHSC.

[1036] 59. An iHSC generated according to the method of any one of items 55-58.

[1037] 60. One or more cells, wherein the one or more cells are obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method comprising: a) a device for seeding human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; b) a device for initiating redifferentiation of the iPSCs into iHSCs by culturing in the tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; c) a device for culturing the iPSC-derived cell intermediates for about 8 days; d) a device for collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and e) a device for harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction - on about day 10 of culture.

[1038] 61. One or more cells, said one or more cells being obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising: a) culture said iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) a device for seeding the iPSCs with 100 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a device for culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; and e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension.

[1039] 62. One or more cells, said one or more cells being obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising: a) culture said iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6b) a means for seeding the iPSCs with 10 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a means for culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a means for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a means for obtaining the suspension of EBs and cells; e) optionally, a means for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a means for providing CD34+ and lineage marker negative iHSCs.

[1040] 63. One or more cells, said one or more cells being obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising: a) culture said iPSCs at a concentration of about 0.4×10 6 cells / well to about 1.2×10 6 b) a device for culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells and initiating redifferentiation of the EBs for about 10 days; d) a device for obtaining the suspension of EBs and cells; e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a device for obtaining CD34+ and CD3 - , and / or CD19 - , and / or CD14 - , and / or CD11b - , and / or CD11c-, and / or CD56 - , and / or CD20 - , and / or CD16 - , and / or CD2 - , and / or CD235a - iHSC device.

[1041] 64. A system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the system comprising: a) a device for inoculating human γδT cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; b) a device for initiating redifferentiation of the iPSCs into iHSCs by culturing in the tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; c) a device for culturing the iPSC-derived cell intermediates for about 8 days; d) a device for collecting non-adherent iPSC-derived cell intermediates and adding them back to the culture; and e) a device for harvesting the redifferentiated iHSCs into two different cell fractions - a non-adherent cell fraction and an adherent cell fraction - on about day 10 of culture.

[1042] 65. A system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a method for culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6 b) a device for seeding the iPSCs with 100 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a device for culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a device for obtaining the suspension of EBs and cells; and e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension.

[1043] 66. A system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a method for culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6b) a means for seeding the iPSCs with 10 cells / well in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) a means for culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a means for transferring the EBs to a tissue culture treated dish and initiating redifferentiation of the EBs for about 10 days, wherein the tissue culture treated dish contains a suspension of EBs and cells; d) a means for obtaining the suspension of EBs and cells; e) optionally, a means for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a means for providing CD34+ and lineage marker negative iHSCs.

[1044] 67. A system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the system comprising: a) a method for culturing the iPSCs at a rate of about 0.4×10 6 cells / well to about 1.2×10 6 b) a device for culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a device for transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells and initiating redifferentiation of the EBs for about 10 days; d) a device for obtaining the suspension of EBs and cells; e) optionally, a device for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a device for obtaining CD34+ and CD3 - , and / or CD19

[1045] - , and / or CD14 - , and / or CD11b - , and / or CD11c-, and / or CD56 - , and / or CD20 - , and / or CD16 - , and / or CD2 - , and / or CD235a - iHSC device.

[1046] References

[1047] 1. Montel-Hagen A, et al. From pluripotent stem cells to T cells. Exp Hematol. 2019;71:24-31.

[1048] 2. Gentles AJ et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med. 2015;21:938-945

[1049] 3. Watanabe D, et al. The generation of human γδT cell-derived induced pluripotent stem cells from whole peripheral blood mononuclear cell culture. Stem Cells Transl Med. 2018;7:34-44.

[1050] 4. Williams RL, et al. Retention of epigenetic and transcriptional memory among reprogrammed T cells (T-iPSCs) imparts a selective advantage for T cell re-differentiation. J Immunol. 2022;208:107.

[1051] 5. Cahan P et al. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat Rev Mol Cell Biol. 2013;14:357-368. ***

[1052] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[1053] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

1. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; Initiating redifferentiation of the iPSCs into iHSCs by culturing in the tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; culturing the iPSC-derived cell intermediate for about 8 days; collecting non-adherent iPSC-derived cell intermediates and adding them back into the culture; as well as At approximately day 10 of culture, redifferentiated iHSCs were harvested as two different cell fractions—a non-adherent cell fraction and an adherent cell fraction.

2. The method according to claim 1, further comprising: The step of redifferentiation of the iPSCs is initiated in a composition comprising basic fibroblast growth factor (bFGF), a glycogen synthase kinase inhibitor, bone morphogenetic protein (BMP4), and vascular endothelial growth factor (VEGF).

3. The method according to claim 1, further comprising: culturing the iPSC-derived cell intermediate for about 2 days in a composition comprising an effective amount of L-glutamine, an inhibitor of the activin / BMP / TGFβ pathway, VEGF, bFGF, and optionally stem cell factor (SCF) and / or an antibiotic; and / or Culturing the iPSC-derived cell intermediate for about 2 days in a composition comprising L-glutamine, SCF, VEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally, an antibiotic; and / or culturing the iPSC-derived cell intermediate for about 2 days in a composition comprising L-glutamine, SCF, IL-6, EPO, and optionally, an antibiotic; and / or The redifferentiated iHSCs are cultured for about 2 days in a composition comprising L-glutamine and optionally antibiotics, wherein the composition does not comprise cytokines or growth factors.

4. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: i) obtaining one or more human γδ T cell-derived iPSCs, and The method also Include one or more of the following steps: a) iPSCs were cultured at a rate of approximately 2×10 3 Cells / well were seeded in a six-well tissue culture dish or its equivalent; b) maintaining the iPSCs under culture conditions for up to 6 days; c) initiating redifferentiation of the iPSCs into iHSCs by culturing the iPSCs in a first cell culture medium for about 2 days to obtain iPSC-derived cell intermediates, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF); d) culturing the iPSC-derived cell intermediate in a second cell culture medium for an additional about 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiatio medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein SB 431542 has a final concentration of about 1.5 μM to about 2.5 μM; e) culturing the iPSC-derived cell intermediate in a third cell culture medium for an additional about 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin; f) culturing the iPSC-derived cell intermediate in a fourth cell culture medium for an additional about 2 days to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and optionally penicillin / streptomycin; g) after about 8 days of redifferentiation, collecting non-adherent iPSC-derived cell intermediates from the fourth cell culture medium and adding them back to the culture in the fourth cell culture medium; h) harvesting the redifferentiated iHSCs into two different cell fractions, a non-adherent cell fraction and an adherent cell fraction, on day 10 of culture, wherein the redifferentiated iHSCs are harvested in a fifth cell culture medium, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and 4:1 at a ratio of about 4:

1. For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors; i) harvesting non-adherent iHSCs by centrifugation, wherein said harvesting optionally does not include further culturing; j) harvesting adherent iHSCs by dispersing the cells, optionally resuspending the cells obtained from the dispersion, and subsequently passing them through a first cell strainer and a second cell strainer, wherein the first cell strainer has a 70 μm mesh size and the second cell strainer has a 40 μm mesh size; k) determining the purity of the iHSCs, defined as CD34+ and lineage marker negative; 1) not introducing serum in any of steps ak; n) not introducing any additional cells, including feeder cells, in any of steps a to m; or o) Obtaining de novo generated iHSCs.

5. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) The iPSCs were cultured at a rate of approximately 0.4×10 6 cells / well to about 1.2×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) culturing the iPSCs with agitation for up to about 48 hours to generate embryoid bodies (EBs); c) transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells, and initiating redifferentiation of the EBs for about 10 days; d) obtaining a suspension of said EBs and cells; and e) Optionally, processing the suspension of EBs and cells to obtain a single cell suspension.

6. The method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions according to claim 5, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method further comprising: Provides CD34+ and lineage marker negative iHSCs.

7. The method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions according to claim 6, wherein the hematopoietic stem cells (iHSCs) are derived from induced pluripotent stem cells (iPSCs) derived from human γδT cells, the method further comprising: Obtain CD34+ and CD3 - , and / or CD19 - , and / or CD14 - , and / or CD11b - , and / or CD11c-, and / or CD56 - , and / or CD20 - , and / or CD16 - , and / or CD2 - , and / or CD235a - iHSC.

8. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: i) obtaining one or more human γδ T cell-derived iPSCs, and The method also Include one or more of the following steps: a) The iPSCs were cultured at a rate of approximately 0.6×10 6 Cells / well were seeded in a six-well ultra-low adhesion tissue culture dish or its equivalent; b) maintaining the iPSCs under culture conditions with agitation for up to 48 hours to generate embryoid bodies (EBs); c) transferring the EBs from the ultra-low adhesion tissue culture dish to a tissue culture treated tissue culture dish, and redifferentiating the EBs by culturing the EBs in a first cell culture medium for about 2 days, wherein the first cell culture medium comprises Basic 04 medium supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF, wherein the first cell culture medium further comprises CHIR99021 (a glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF); d) culturing the suspension of EBs and cells in a second cell culture medium for an additional approximately 2 days, wherein the second cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... For Differentiation medium, and further comprising L-glutamine, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, stem cell factor (SCF), and optionally penicillin / streptomycin, wherein SB431542 has a final concentration of about 1.5 μM to about 2.5 μM; e) culturing the suspension of EBs and cells in a third cell culture medium for about another 2 days, wherein the third cell culture medium comprises Advanced DMEM / F12 medium and PEG-177 at a ratio of about 4:

1. ForDifferentiation medium, and further comprising L-glutamine, SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and optionally penicillin / streptomycin; f) culturing the suspension of EBs and cells in a fourth cell culture medium for an additional about 2 to about 4 days, wherein the fourth cell culture medium comprises Advanced DMEM / F12 medium and PEGylated ... ForDifferentiation medium, and also contains L-glutamine, SCF, IL-6, EPO and penicillin / streptomycin; g) after about 10 days of redifferentiation, harvesting the suspension of EBs and cells by centrifugation, thereby forming a suspension of pelleted EBs and cells; h) resuspending the pelleted EB and cell suspension in a volume of about 20 mg / mL of type II collagenase, and incubating the EB and cell suspension in the type II collagenase; i) adding TrypLE to the suspension of EBs and cells, and incubating the suspension of EBs and cells to obtain a single cell suspension; j) mixing the single cell suspension; k) adding a fifth cell culture medium to the cell suspension, wherein the fifth cell culture medium comprises Advanced DMEM / F12 medium and For Differentiation medium, and further comprising L-glutamine and optionally penicillin / streptomycin, wherein the fifth cell culture medium does not comprise cytokines or growth factors; 1) centrifuging the cell suspension to form a cell pellet, and resuspending the cell pellet in the fifth cell culture medium to form a second cell suspension; m) passing the second cell suspension through a first cell strainer and a second cell strainer to obtain iHSCs, wherein the first cell strainer has a 70 μm pore size and the second cell strainer has a 40 μm pore size; n) determining the purity of the iHSCs, defined as CD34+ and lineage marker negative; o) not introducing serum in any of steps a to b; p) not introducing any additional cells, including feeder cells or stromal cells, in any of steps a to b; or q) Obtain de novo generated iHSCs.

9. The method of claim 4 or claim 8, wherein the cells are cultured under normoxic conditions.

10. The method according to any one of claims 4 and 8-9, further comprising further purifying and / or isolating the iHSCs.

11. The method according to any one of claims 4 and 8-10, wherein the iHSCs obtained are isolated differentiated cells or are capable of being further purified and / or isolated.

12. The method according to any one of claims 4 and 8-11, wherein the step of determining the purity of the iHSCs comprises: One or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a are assayed via flow cytometry or its equivalent.

13. The method of any one of claims 4 and 8-12, wherein the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

14. One or more cells, said one or more cells being obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising the steps according to any one of claims 1 to 13.

15. The one or more cells of claim 14, wherein the one or more cells have high purity, viability, fold expansion, and the potential to redifferentiate into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

16. The one or more cells of claim 14 or 15, wherein the step of determining the purity of the iHSCs comprises: One or more, optionally all, of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a are assayed via flow cytometry or its equivalent.

17. The one or more cells of any one of claims 14-16, wherein the lineage markers comprise one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

18. A composition comprising one or more cells according to any one of claims 14-17.

19. Use of one or more cells according to any one of claims 14 to 17 in the preparation of cells for treating lesions or diseases, in the preparation of lymphocytes, in bioreactors, in tissue engineering or in vitro drug screening for diseases.

20. A system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps according to claim 1 or 5.

21. The system according to claim 20, wherein the method according to claim 1 or 5 is performed manually or with the assistance of automatic robots or a combination thereof.

22. The system of claim 20 or 21, wherein the system is automated or semi-automated.

23. The system of any one of claims 20-22, comprising one or more software packages that operate and schedule the operation of the system.

24. A system according to claim 23, wherein the software package is customised and / or customisable for a desired application and is optionally menu driven.

25. The system of claim 22, wherein the automation comprises cell visualization, plate handling, plate spreading, inoculation, extraction, addition, cell feeding, incubation assays, and / or sampling.

26. The system of any one of claims 20-25, comprising one or more in-line incubators.

27. The system of any one of claims 20-26, wherein the system comprises an electronic humidity control, a HEPA filter system, a turntable comprising a programmable step oscillation cycle and / or a bidirectional communication interface, or any combination thereof.

28. The system of any one of claims 20-27, wherein addition or removal or supplementation of culture medium does not disturb or contaminate the cells.

29. The system of any one of claims 20-28, comprising one or more computer operated and controlled robotic arms.

30. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free, feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: A step for initiating redifferentiation of the iPSCs into iHSCs in a tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; a step for culturing the iPSC-derived cell intermediate for about 8 days; a step for collecting non-adherent iPSC-derived cell intermediates and adding them back into the culture; and A procedure is provided for harvesting the redifferentiated iHSCs into two distinct cell fractions, a non-adherent cell fraction and an adherent cell fraction, at approximately day 10 of culture.

31. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step for performing the following function, namely seeding the iPSCs; b) culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step of transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells and initiating redifferentiation of the EBs for about 10 days; d) a step for obtaining a suspension of said EBs and cells; and e) Optionally, a step for processing the suspension of EBs and cells to obtain a single cell suspension.

32. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step for performing the following function, namely seeding the iPSCs; b) culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step of transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells and initiating redifferentiation of the EBs for about 10 days; d) a step for obtaining a suspension of EBs and cells; e) a step for optionally processing the suspension of EBs and cells to obtain a single cell suspension; and f) Steps for providing CD34+ and lineage marker negative iHSCs.

33. A method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, wherein the hematopoietic stem cells (iHSCs) are derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising: a) a step for performing the following function, namely seeding the iPSCs; b) culturing the iPSCs under agitation for up to about 48 hours to generate embryoid bodies (EBs); c) a step of transferring the EBs to a tissue culture treated dish containing a suspension of EBs and cells and initiating redifferentiation of the EBs for about 10 days; d) a step for obtaining a suspension of EBs and cells; e) optionally, a step for processing the suspension of EBs and cells to obtain a single cell suspension; and f) a step for performing the following functions, namely obtaining CD34+ and CD3 - , and / or CD19 - , and / or CD14 - , and / or CD11b - , and / or CD11c-, and / or CD56 - , and / or CD20 - , and / or CD16 - , and / or CD2 - , and / or CD235a - iHSC.

34. An iHSC produced according to the method of any one of claims 30-33.

35. One or more cells, said one or more cells being obtained from a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising: a device for seeding human γδ T cell-derived iPSCs in a tissue culture dish, wherein the iPSCs are maintained for up to about 6 days; a device for initiating redifferentiation of the iPSCs into iHSCs by culturing in the tissue culture dish for about 2 days to obtain iPSC-derived cell intermediates; a device for culturing the iPSC-derived cell intermediate for about 8 days; a device for collecting non-adherent iPSC-derived cell intermediates and adding them back into culture; as well as Device for harvesting redifferentiated iHSCs into two distinct cell fractions - a non-adherent cell fraction and an adherent cell fraction - at approximately day 10 of culture.

Images