Reagent combination or kit for preparing gamma delta T cells and application of reagent combination or kit

Abstract

The invention discloses a reagent combination or a kit for preparing gamma delta T cells and application of the reagent combination or the kit. In the gamma delta T cell population obtained by the invention, most cells show CD4-CD8a-double negative phenotypes, part of the cells express CD56, and TCR alpha beta and CD41a are not expressed at all, which are consistent with the phenotypes of natural human gamma delta T cells. The gamma delta T cell obtained by the invention has the capability of secreting TNF-alpha and IL-2 cell factors. The gamma delta T cell obtained by the invention has a relatively strong cytotoxic effect on liver cancer cell lines, such as HepG2 cells and Huh-7 cells. The gamma delta T cells can effectively inhibit the proliferation of cancer cells, but have little influence on normal cells, and show high targeting and selectivity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a reagent combination or kit for preparing γδT cells and its application. Background Technology

[0002] Cancer is one of the leading causes of death worldwide due to its high incidence and mortality rates. In 2022, there were 20 million new cancer cases and 9.7 million cancer deaths globally. Therefore, there is an urgent need to develop new anti-tumor strategies to reduce mortality and improve patients' quality of life. Among various emerging treatment strategies, adoptive cell transfer therapy (ACT) has attracted widespread attention. ACT involves collecting the patient's own immune cells, culturing and modifying them in vitro to enhance their targeted killing function, and then reinfusing them into the patient to eliminate tumor cells. Currently, the adoption of αβT cells carrying engineered TCRs has achieved some success, but it also has limitations. Clinical results show that αβT cell ACT therapy has demonstrated excellent results in hematological malignancies; however, solid tumors are highly heterogeneous, rarely expressing only one tumor-specific antigen, and due to the signaling limitations of immunosuppressive checkpoints within tumors, the use of αβT cell ACT therapy in the treatment of solid tumors remains significantly limited.

[0003] Recently, there has been considerable interest in the application of γδT cells in cancer immunotherapy. γδT cells recognize target cells independently of human leukocyte antigen (HLA), and release relatively few cytokines, resulting in a lower probability of cytokine release syndrome. Furthermore, γδT cells can directly kill target cells without the involvement of dendritic cells. Therefore, γδT cells have significant clinical value and advantages in tumor immunotherapy. To date, γδT cells have undergone clinical trials in many cancers, such as liver cancer, malignant leukemia, advanced lung cancer, lymphoma, and cholangiocarcinoma, with most trials showing good tolerability and high safety. In recent years, in vitro expansion of human peripheral blood-derived γδT cells has become a candidate for adoptive immunotherapy (ACT). However, due to limitations in the availability and source of human peripheral blood, it is often difficult to expand γδT cells to the clinically required quantities for cancer immunotherapy. Therefore, it is urgent to find new ways to obtain γδT cells.

[0004] Based on this, this study aims to establish a new system for in vitro induction of human embryonic stem cells (hESCs) into γδT cells, providing a new technical means to solve the problem of γδT cell source in ACT. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a reagent combination or kit for preparing γδT cells and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a reagent combination comprising a fifth culture medium, the fifth culture medium comprising a basal culture medium supplemented with magnesium ascorbate phosphate (MAP), hematopoietic growth factor and Notch signaling pathway activator.

[0007] In some embodiments, the hematopoietic growth factor in the fifth culture medium is selected from at least one of stem cell factor, thrombopoietin, interleukin-3, interleukin-6, interleukin-7, and FMS-associated tyrosine kinase 3 ligand.

[0008] In some embodiments, the hematopoietic growth factors in the fifth culture medium include stem cell factors, interleukin-7, and FMS-associated tyrosine kinase 3 ligand.

[0009] In some embodiments, the Notch signaling pathway activator is selected from at least one of DLL1, DLL3, DLL4, JAG1, or JAG2.

[0010] The fifth culture medium includes a basal medium supplemented with MAP, stem cell factor, interleukin-7, FMS-associated tyrosine kinase 3 ligand, and DLL4.

[0011] In some embodiments, the concentration of MAP in the fifth culture medium is 10-50 μM. For example, the working concentration of MAP in the fifth culture medium is 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM.

[0012] In some embodiments, the concentration of stem cell factors in the fifth culture medium is 5-20 ng / mL. For example, the working concentration of stem cell factors in the fifth culture medium is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL.

[0013] In some embodiments, the concentration of interleukin-7 in the fifth culture medium is 1-10 ng / mL. For example, the working concentration of interleukin-7 in the fifth culture medium is 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL.

[0014] In some embodiments, the concentration of FMS-associated tyrosine kinase 3 ligand in the fifth culture medium is 1-10 ng / mL. For example, the working concentration of FMS-associated tyrosine kinase 3 ligand in the fifth culture medium is 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL.

[0015] In some embodiments, the concentration of the Notch signaling pathway activator in the fifth culture medium is 5-20 ng / mL. For example, the working concentration of the Notch signaling pathway activator in the fifth culture medium is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL.

[0016] In some embodiments, the fifth culture medium also contains FBS and a glutamine additive. The fifth culture medium comprises a basal medium supplemented with FBS, a glutamine additive, MAP, stem cell factors, interleukin-7, FMS-associated tyrosine kinase 3 ligand, and DLL4.

[0017] In some embodiments, the concentration of FBS in the fifth culture medium is 10-30%. For example, the working concentration of FBS in the fifth culture medium is 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

[0018] In some embodiments, the concentration of the glutamine additive in the fifth culture medium is 1-3 mM. For example, the working concentration of the glutamine additive in the fifth culture medium is 1 mM, 1.5 mM, 2 mM, 2.5 mM, or 3 mM. The concentration of the glutamine additive in the fifth culture medium is 2 mM.

[0019] In some embodiments, antibiotics are also added to the fifth culture medium.

[0020] The fifth culture medium includes a basal medium supplemented with FBS, glutamine additive, MAP, stem cell factor, interleukin-7, FMS-associated tyrosine kinase 3 ligand, DLL4, and antibiotics.

[0021] In some embodiments, the antibiotics in the fifth culture medium include penicillin, streptomycin, and amphotericin B. In some embodiments, the concentration of penicillin in the fifth culture medium is 50-150 U / mL, the concentration of streptomycin is 0.05-0.15 mg / mL, and the concentration of amphotericin B is 0.125-0.375 µg / mL. For example, the working concentrations of penicillin in the fifth culture medium are 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, or 150 U / mL; the working concentrations of streptomycin are 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, or 0.15 mg / mL; and the working concentrations of amphotericin B are 0.125 µg / mL, 0.15 µg / mL, 0.175 µg / mL, 0.2 µg / mL, 0.225 µg / mL, 0.25 µg / mL, or 0.275 µg / mL. µg / mL, 0.3 µg / mL, 0.325 µg / mL, 0.35 µg / mL, or 0.375 µg / mL. In some embodiments, the antibiotic is a penicillin-streptomycin-amphotericidal B mixed solution (100× triple antibody), wherein the penicillin content is 10 kU / mL, the streptomycin content is 10 mg / mL, and the amphotericin B content is 25 µg / mL. In some embodiments, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the fifth basal culture medium is 0.5 vol%-1.5 vol%. For example, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the fifth basal culture medium is 0.5 vol%, 0.6 vol%, 0.7 vol%, 0.8 vol%, 0.9 vol%, 1 vol%, 1.1 vol%, 1.2 vol%, 1.3 vol%, 1.4 vol%, or 1.5 vol%.

[0022] In some embodiments, the basal medium in the fifth culture medium is selected from at least one of RPMI-1640 and MEM-α basal medium.

[0023] The fifth culture medium includes MEM-α basal medium supplemented with FBS, glutamine additive, MAP, stem cell factor, interleukin-7, FMS-associated tyrosine kinase 3 ligand, DLL4 and antibiotics.

[0024] In some embodiments, the reagent combination further comprises a first culture medium, which includes a basal culture medium supplemented with bone morphogenetic protein, GSK-3 inhibitor, Activin A and Rock inhibitor.

[0025] In some embodiments, the reagent combination further comprises a second culture medium, which includes a basal culture medium supplemented with vascular endothelial growth factor.

[0026] In some embodiments, the reagent combination further comprises a third culture medium, which includes a basal culture medium supplemented with vascular endothelial growth factor and fibroblast growth factor.

[0027] In some embodiments, the reagent combination further comprises a fourth culture medium, which includes a basal culture medium supplemented with vascular endothelial growth factor, hematopoietic growth factor, fibroblast growth factor and TGF-β signaling pathway inhibitors.

[0028] In some embodiments, the bone morphogenetic protein in the first culture medium is selected from at least one of BMP4 and BMP2.

[0029] In some embodiments, the GSK-3 inhibitor in the first culture medium is selected from SB216763 (CAS No.: 280744-09-4), TWS119 (CAS No.: 601514-19-6), NP031112 (CAS No.: 865854-05-3), CHIR-98014 (CAS No.: 252935-94-7), AZD2858 (CAS No.: 486424-20-8), AZD1080 (CAS No.: 612487-72-6), SB415286 (CAS No.: 264218-23-7), LY2090314 (CAS No.: 603288-22-8), and CHIR-99021 (CAS No.: 280744-09-4). At least one of (252917-06-9).

[0030] In some embodiments, the Rock inhibitor in the first culture medium is selected from at least one of Y-27632 (CAS No.: 146986-50-7), Thiazovivin (CAS No.: 1226056-71-8), Fasudil HCl (CAS No.: 105628-07-7), GSK429286A (CAS No.: 864082-47-3), RKI-1447 (CAS No.: 1342278-01-6), and Azaindole 1 (CAS No.: 867017-68-3).

[0031] In some embodiments, the first culture medium includes a basal medium supplemented with BMP4, CHIR-99021, Activin A, and Y-27632.

[0032] In some embodiments, the concentration of bone morphogenetic protein in the first culture medium is 5-20 ng / mL. For example, the working concentration of bone morphogenetic protein in the first culture medium is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, or 20 ng / mL. The concentration of bone morphogenetic protein in the first culture medium is 10 ng / mL.

[0033] In some embodiments, the concentration of the GSK-3 inhibitor in the first culture medium is 1-5 μM. For example, the working concentration of the GSK-3 inhibitor in the first culture medium is 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM. The concentration of the GSK-3 inhibitor in the first culture medium is 3 μM.

[0034] In some embodiments, the concentration of Activin A in the first culture medium is 1-5 ng / mL. For example, the working concentration of Activin A in the first culture medium is 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, or 5 ng / mL. The concentration of Activin A in the first culture medium is 2 ng / mL.

[0035] In some embodiments, the concentration of the rock inhibitor in the first culture medium is 5-20 μM. For example, the working concentration of the rock inhibitor in the first culture medium is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM. The concentration of the rock inhibitor in the first culture medium is 10 μM.

[0036] In some embodiments, antibiotics are also added to the first culture medium.

[0037] In some embodiments, the first culture medium includes a basal culture medium supplemented with BMP4, CHIR-99021, Activin A, Y-27632 and antibiotics.

[0038] In some embodiments, the antibiotics in the first culture medium include penicillin, streptomycin, and amphotericin B. The concentration of penicillin in the first culture medium is 50-150 U / mL, the concentration of streptomycin is 0.05-0.15 mg / mL, and the concentration of amphotericin B is 0.125-0.375 µg / mL. For example, the working concentrations of penicillin in the first culture medium are 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, or 150 U / mL; the working concentrations of streptomycin are 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, or 0.15 mg / mL; and the working concentrations of amphotericin B are 0.125 µg / mL, 0.15 µg / mL, 0.175 µg / mL, 0.2 µg / mL, 0.225 µg / mL, 0.25 µg / mL, or 0.275 µg / mL. µg / mL, 0.3 µg / mL, 0.325 µg / mL, 0.35 µg / mL, or 0.375 µg / mL. In some embodiments, the antibiotic is a penicillin-streptomycin-amphotericidal B mixed solution (100× triple antibody), wherein the penicillin content is 10 kU / mL, the streptomycin content is 10 mg / mL, and the amphotericin B content is 25 µg / mL. In some embodiments, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the first basal culture medium is 0.5 vol%-1.5 vol%. For example, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the first basal culture medium is 0.5 vol%, 0.6 vol%, 0.7 vol%, 0.8 vol%, 0.9 vol%, 1 vol%, 1.1 vol%, 1.2 vol%, 1.3 vol%, 1.4 vol%, or 1.5 vol%.

[0039] In some embodiments, the vascular endothelial growth factor in the second culture medium is selected from at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor 1, and placental growth factor 2.

[0040] In some embodiments, the concentration of vascular endothelial growth factor (VEGF) in the second culture medium is 20-60 ng / mL. For example, the working concentration of VEGF in the second culture medium is 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, or 60 ng / mL. The concentration of VEGF in the second culture medium is 40 ng / mL.

[0041] In some embodiments, antibiotics are also added to the second culture medium.

[0042] In some embodiments, the second culture medium comprises a basal culture medium supplemented with VEGF-A and antibiotics.

[0043] In some embodiments, the antibiotics in the second culture medium include penicillin, streptomycin, and amphotericin B. In some embodiments, the concentration of penicillin in the second culture medium is 50-150 U / mL, the concentration of streptomycin is 0.05-0.15 mg / mL, and the concentration of amphotericin B is 0.125-0.375 µg / mL. For example, the working concentrations of penicillin in the second culture medium are 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, or 150 U / mL; the working concentrations of streptomycin are 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, or 0.15 mg / mL; and the working concentrations of amphotericin B are 0.125 µg / mL, 0.15 µg / mL, 0.175 µg / mL, 0.2 µg / mL, 0.225 µg / mL, 0.25 µg / mL, or 0.275 µg / mL. µg / mL, 0.3 µg / mL, 0.325 µg / mL, 0.35 µg / mL, or 0.375 µg / mL. In some embodiments, the antibiotic is a penicillin-streptomycin-amphotericidal B mixture (100× triple antibody), wherein the penicillin content is 10 kU / mL, the streptomycin content is 10 mg / mL, and the amphotericin B content is 25 µg / mL. In some embodiments, the working concentration of the penicillin-streptomycin-amphotericidal B mixture in the second basal culture medium is 0.5 vol%-1.5 vol%. For example, the working concentration of the penicillin-streptomycin-amphotericidal B mixture in the second basal culture medium is 0.5 vol%, 0.6 vol%, 0.7 vol%, 0.8 vol%, 0.9 vol%, 1 vol%, 1.1 vol%, 1.2 vol%, 1.3 vol%, 1.4 vol%, or 1.5 vol%.

[0044] In some embodiments, the vascular endothelial growth factor in the third culture medium is selected from at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor 1, and placental growth factor 2.

[0045] In some embodiments, the fibroblast growth factor in the third culture medium is selected from at least one of FGF1, FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0046] In some embodiments, the third culture medium includes a basal culture medium supplemented with VEGF-A and FGF2.

[0047] In some embodiments, the concentration of vascular endothelial growth factor (VEGF) in the third culture medium is 20-60 ng / mL. For example, the working concentration of VEGF in the third culture medium is 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, or 60 ng / mL. The concentration of VEGF in the third culture medium is 40 ng / mL.

[0048] In some embodiments, the concentration of fibroblast growth factor in the third culture medium is 10-30 ng / mL. For example, the working concentration of fibroblast growth factor in the third culture medium is 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, or 30 ng / mL. The concentration of fibroblast growth factor in the third culture medium is 20 ng / mL.

[0049] In some embodiments, antibiotics are also added to the third culture medium.

[0050] In some embodiments, the third culture medium includes a basal culture medium supplemented with VEGF-A, FGF2 and antibiotics.

[0051] In some embodiments, the antibiotics in the third culture medium include penicillin, streptomycin, and amphotericin B. In some embodiments, the concentration of penicillin in the third culture medium is 50-150 U / mL, the concentration of streptomycin is 0.05-0.15 mg / mL, and the concentration of amphotericin B is 0.125-0.375 µg / mL. For example, the working concentrations of penicillin in the third culture medium are 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, or 150 U / mL; the working concentrations of streptomycin are 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, or 0.15 mg / mL; and the working concentrations of amphotericin B are 0.125 µg / mL, 0.15 µg / mL, 0.175 µg / mL, 0.2 µg / mL, 0.225 µg / mL, 0.25 µg / mL, or 0.275 µg / mL. µg / mL, 0.3 µg / mL, 0.325 µg / mL, 0.35 µg / mL, or 0.375 µg / mL. In some embodiments, the antibiotic is a penicillin-streptomycin-amphotericidal B mixed solution (100× triple antibody), wherein the penicillin content is 10 kU / mL, the streptomycin content is 10 mg / mL, and the amphotericin B content is 25 µg / mL. In some embodiments, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the third basal culture medium is 0.5 vol%-1.5 vol%. For example, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the third basal culture medium is 0.5 vol%, 0.6 vol%, 0.7 vol%, 0.8 vol%, 0.9 vol%, 1 vol%, 1.1 vol%, 1.2 vol%, 1.3 vol%, 1.4 vol%, or 1.5 vol%.

[0052] In some embodiments, the vascular endothelial growth factor in the fourth culture medium is selected from at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor 1, and placental growth factor 2.

[0053] In some embodiments, the hematopoietic growth factor in the fourth culture medium is selected from at least one of stem cell factor, thrombopoietin, interleukin-3, interleukin-6, interleukin-7, and FMS-associated tyrosine kinase 3 ligand.

[0054] In some embodiments, the hematopoietic growth factors in the fourth culture medium include stem cell factors, interleukin-7, and FMS-associated tyrosine kinase 3 ligand.

[0055] In some embodiments, the fibroblast growth factor in the fourth culture medium is selected from at least one of FGF1, FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0056] In some embodiments, the TGF-β signaling pathway inhibitor in the fourth culture medium is selected from LY2157299 (CAS No.: 700874-72-2), EW-7197 (CAS No.: 1352608-82-2), LY3200882 (CAS No.: 1898283-02-7), SB-431542 (CAS No.: 301836-41-9), LY2109761 (CAS No.: 700874-71-1), TP-0427736 (CAS No.: 864374-00-5), IN-1130 (CAS No.: 868612-83-3), R-268712 (CAS No.: 879487-87-3), and A-83-01 (CAS No.: 700874-72-2). At least one of the following: 909910-43-6), SB-525334 (CAS No.: 356559-20-1), GW788388 (CAS No.: 452342-67-5), RepSox (CAS No.: 446859-33-2), A-77-01 (CAS No.: 607737-87-1), SB-505124 (CAS No.: 694433-59-5), SD-208 (CAS No.: 627536-09-8), and LY364947 (CAS No.: 396129-53-6).

[0057] In some embodiments, the fourth culture medium comprises a basal medium supplemented with VEGF-A, stem cell factor, interleukin-7, FMS-associated tyrosine kinase 3 ligand, FGF2, and SB-431542.

[0058] In some embodiments, the concentration of vascular endothelial growth factor (VEGF) in the fourth culture medium is 40-60 ng / mL. For example, the working concentration of VEGF in the fourth culture medium is 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 51 ng / mL, 52 ng / mL, 53 ng / mL, 54 ng / mL, 55 ng / mL, 56 ng / mL, 57 ng / mL, 58 ng / mL, 59 ng / mL, or 60 µg / mL. The concentration of VEGF in the fourth culture medium is 50 ng / mL.

[0059] In some embodiments, the concentration of stem cell factors in the fourth culture medium is 10-30 ng / mL. For example, the working concentration of stem cell factors in the fourth culture medium is 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, or 30 ng / mL. The concentration of stem cell factors in the fourth culture medium is 20 ng / mL.

[0060] In some embodiments, the concentration of interleukin-7 in the fourth culture medium is 5-15 ng / mL. For example, the working concentration of interleukin-7 in the fourth culture medium is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, or 15 ng / mL. The concentration of interleukin-7 in the fourth culture medium is 10 ng / mL.

[0061] In some embodiments, the concentration of FMS-associated tyrosine kinase 3 ligand in the fourth culture medium is 5-15 ng / mL. For example, the working concentration of FMS-associated tyrosine kinase 3 ligand in the fourth culture medium is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, or 15 ng / mL. The concentration of FMS-associated tyrosine kinase 3 ligand in the fourth culture medium is 10 ng / mL.

[0062] In some embodiments, the concentration of fibroblast growth factor in the fourth culture medium is 10-30 ng / mL. For example, the working concentration of fibroblast growth factor in the fourth culture medium is 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, or 30 ng / mL. The concentration of fibroblast growth factor in the fourth culture medium is 20 ng / mL.

[0063] In some embodiments, the concentration of the TGF-β signaling pathway inhibitor in the fourth culture medium is 2-6 μM. For example, the working concentration of the TGF-β signaling pathway inhibitor in the fourth culture medium is 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, or 6 μM. The concentration of the TGF-β signaling pathway inhibitor in the fourth culture medium is 4 μM.

[0064] In some embodiments, antibiotics are also added to the fourth culture medium.

[0065] In some embodiments, the fourth culture medium comprises a basal medium supplemented with VEGF-A, stem cell factor, interleukin-7, FMS-associated tyrosine kinase 3 ligand, FGF2, SB-431542 and antibiotics.

[0066] In some embodiments, the antibiotics in the fourth culture medium include penicillin, streptomycin, and amphotericin B. In some embodiments, the concentration of penicillin in the fourth culture medium is 50-150 U / mL, the concentration of streptomycin is 0.05-0.15 mg / mL, and the concentration of amphotericin B is 0.125-0.375 µg / mL. For example, the working concentrations of penicillin in the fourth culture medium are 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, or 150 U / mL; the working concentrations of streptomycin are 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, or 0.15 mg / mL; and the working concentrations of amphotericin B are 0.125 µg / mL, 0.15 µg / mL, 0.175 µg / mL, 0.2 µg / mL, 0.225 µg / mL, 0.25 µg / mL, or 0.275 µg / mL. µg / mL, 0.3 µg / mL, 0.325 µg / mL, 0.35 µg / mL, or 0.375 µg / mL. In some embodiments, the antibiotic is a penicillin-streptomycin-amphotericidal B mixed solution (100× triple antibody), wherein the penicillin content is 10 kU / mL, the streptomycin content is 10 mg / mL, and the amphotericin B content is 25 µg / mL. In some embodiments, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the fourth basal culture medium is 0.5 vol%-1.5 vol%. For example, the working concentration of the penicillin-streptomycin-amphotericidal B mixed solution in the fourth basal culture medium is 0.5 vol%, 0.6 vol%, 0.7 vol%, 0.8 vol%, 0.9 vol%, 1 vol%, 1.1 vol%, 1.2 vol%, 1.3 vol%, 1.4 vol%, or 1.5 vol%.

[0067] In some embodiments, the basal medium in the first culture medium is APEL™2 basal medium.

[0068] In some embodiments, the first culture medium comprises APEL™2 basal medium supplemented with BMP4, CHIR-99021, Activin A, Y-27632 and antibiotics.

[0069] In some embodiments, the basal medium in the second culture medium is APEL™2 basal medium.

[0070] In some embodiments, the second culture medium comprises APEL™2 basal medium supplemented with VEGF-A and antibiotics.

[0071] In some embodiments, the basal medium in the third culture medium is APEL™2 basal medium.

[0072] In some embodiments, the third culture medium comprises APEL™2 basal medium supplemented with VEGF-A, FGF2 and antibiotics.

[0073] In some embodiments, the basal medium in the fourth culture medium is APEL™2 basal medium.

[0074] In some embodiments, the fourth culture medium comprises APEL™2 basal medium supplemented with VEGF-A, stem cell factor, interleukin-7, FMS-associated tyrosine kinase 3 ligand, FGF2, SB-431542 and antibiotics.

[0075] A second aspect of the present invention provides a reagent kit comprising the reagent combination of the first aspect.

[0076] The third aspect of the present invention provides the application of the reagent combination of the first aspect or the kit of the second aspect in any of the following aspects: (1) preparing γδT cells; (2) preparing products that induce pluripotent stem cells to differentiate into γδT cells.

[0077] The fourth aspect of the present invention provides a method for preparing γδT cells, wherein γδT cells are obtained by culturing pluripotent stem cells using the reagent combination of the first aspect or the kit of the second aspect; wherein the culture is a suspension culture.

[0078] In some embodiments, the method includes: culturing hematopoietic progenitor cells using a fifth culture medium to obtain γδT cells.

[0079] In some embodiments, hematopoietic progenitor cells are cultured using the fifth culture medium to obtain γδT cells for 5-6 days.

[0080] In some embodiments, the method further includes: First culture stage: Pluripotent stem cells are cultured using the first culture medium, then the culture medium is changed to the second culture medium for further culture, and then the culture medium is changed to the third culture medium for continued culture to obtain hematopoietic endothelial cells; Second culture stage: Hematopoietic endothelial cells obtained by culturing them in the fourth culture medium are used to obtain hematopoietic progenitor cells.

[0081] In some embodiments, culturing hematopoietic progenitor cells to obtain γδT cells using the fifth culture medium includes: resuspending the hematopoietic progenitor cells and mixing them with hematopoietic endothelial cells obtained from the first stage of culture, followed by further culture.

[0082] In some embodiments, pluripotent stem cells are cultured in the first culture medium for 2 days, then the culture medium is changed to the second culture medium for 1 day, and then the culture medium is changed to the third culture medium for 3 days to obtain hematopoietic endothelial cells.

[0083] In some embodiments, hematopoietic endothelial cells obtained by culturing the fourth culture medium for 4 days are used to obtain hematopoietic progenitor cells.

[0084] In some embodiments, the suspension culture is carried out under hypoxic conditions, which are defined as an O2 concentration of 5% and a CO2 concentration of 5%.

[0085] The fifth aspect of the present invention provides the use of the γδT cells prepared by the method of the fourth aspect in any of the following: preparation of a drug for treating cancer; preparation of a drug for treating diseases caused by pathogenic microorganism infection; preparation of an immunomodulatory drug.

[0086] In some implementations, the cancer includes hematologic malignancies (such as leukemia, lymphoma, multiple myeloma) and solid tumors (such as breast cancer, prostate cancer, ovarian cancer, etc.).

[0087] In some embodiments, the pathogenic microorganism includes at least one of bacteria (such as Mycobacterium tuberculosis, Escherichia coli, Staphylococcus aureus, Listeria, etc.), fungi (such as Candida albicans), viruses (such as cytomegalovirus, influenza virus, human immunodeficiency virus, EB virus), and parasites (such as Plasmodium malariae, Leishmaniasis).

[0088] The beneficial effects of this invention are: it provides a reagent combination for preparing γδT cells and a novel method for differentiating γδT cells from hematopoietic stem / progenitor cells derived from hESCs. In the γδT cell population obtained by this invention, most cells exhibit CD4+. - CD8a - The cells exhibit a double-negative phenotype, partially expressing CD56 and completely lacking expression of TCRαβ and CD41a, consistent with the natural human γδT cell phenotype. The γδT cells obtained in this invention possess the ability to secrete TNF-α and IL-2 cytokines. These γδT cells demonstrate strong cytotoxicity against liver cancer cell lines, such as HepG2 and Huh-7 cells. These γδT cells effectively inhibit cancer cell proliferation while having negligible effects on normal cells, exhibiting high targeting and selectivity. Attached Figure Description

[0089] Figure 1 A novel system for in vitro differentiation of human embryonic stem cells (hESCs) into γδT cells was demonstrated.

[0090] Figure 2 This image shows the differentiation of human embryonic stem cells (hESCs) into hematopoietic endothelial cells (HECs). (A) Cell morphology of hESCs from day 0 to day 6 of differentiation; scale bar = 100 μm. (B) Immunostaining images of CD34 and CD31 in HECs; scale bar = 100 μm, magnified view scale bar = 10 μm. (C) Flow cytometry analysis of KDR in hESCs during the first 8 days of differentiation. + Cells and CD31 + CD34 + Representative image of cells. (D) Quantitative PCR analysis of the expression levels of key genes in hESCs from day 0 to day 8 of differentiation.

[0091] Figure 3 This image shows the differentiation of hematopoietic endothelial cells (hESCs) into hematopoietic progenitor cells (HESCs). Among them, (A) morphology of cell spheroids on day 10 of hESC differentiation; scale bar = 100 μm. (B) Flow cytometry analysis of the proportion of each cell type within the cell spheroids on day 10 of hESC differentiation. (C) Morphology of a single floating cell on day 10 of hESC differentiation; scale bar = 100 μm. (D) Flow cytometry analysis of the proportion of each cell type within a single floating cell on day 10 of hESC differentiation. (E) Immunostaining images of CD45 and CD34 in a single floating cell on day 10 of hESC differentiation. Scale bar = 100 μm.

[0092] Figure 4 This study demonstrated the differentiation of hematopoietic progenitor cells into γδ T cells. Specifically, (A) flow cytometry was used to detect CD45 under different culture conditions. + Cell ratio, 7AAD - Live cells enter. (B) GraphPad Prism software for CD45 + Cell proportions were statistically analyzed (n=3), and data are presented as mean ± SEM. (C) Flow cytometry detection of CD3 under different culture conditions. + TCRγδ + Cell percentage, CD45 + Cells enter the portal. (D) GraphPad Prism software for CD3 + TCRγδ + Cell proportions were statistically analyzed (n=3), and data are presented as mean ± SEM. (E) Cell morphology of hESCs from day 10 to day 25 of differentiation; scale bar = 100 μm. (F) Flow cytometry analysis of CD45 at different culture time points. +Cell ratio, 7AAD - Live cells enter. (G) GraphPad Prism software for CD45 + Cell proportions were statistically analyzed (n=3), and data are presented as mean ± SEM. (H) Flow cytometry was used to detect CD3 at different culture time points. + TCRγδ + Cell percentage, CD45 + Cells enter the portal. (I) GraphPad Prism software for CD3 + TCRγδ + Cell proportions were statistically analyzed (n=3), and data are presented as mean ± SEM. (J) Flow cytometry detection of TCRγδ in hESCs on day 25 of differentiation. + TCRαβ + CD8a + CD4 + CD56 + and CD41a + Representative cell images, all 7AAD - Live cells were introduced (three independent replicate experiments).

[0093] Figure 5 The expression levels of genes related to different developmental stages of hESCs from day 10 to day 25 of differentiation were shown by quantitative PCR.

[0094] Figure 6 This study demonstrates the phenotypic identification and functional analysis of γδT cells. Specifically, (A) quantitative PCR was used to detect the expression levels of γδT cell function-related genes from day 10 to day 25 of hESC differentiation. (B) flow cytometry was used to detect TNF-α on day 25 of hESC differentiation. + IL-2 + granzyme B + perforin + and IFN-γ + Representative image of cells; Stimulated group from CD45 + Cell entry (three independent replicate experiments).

[0095] Figure 7Representative graphs (three independent replicates) show the effects of flow cytometry on the cytotoxicity of hESC-derived γδT cells against cancer cells. (A) Results of 293T cell culture alone; (B) Results of co-culture with γδT cells; (C) Results of HepG2 cell culture alone; (D) Results of co-culture with γδT cells; (E) Results of Hun7 cell culture alone; (F) Results of co-culture with γδT cells. Target cells were labeled with CFSE, and dead cells were labeled with DAPI. Scale bar = 100 μm.

[0096] Figure 8 Transcriptome sequencing analysis during γδT cell differentiation is presented. (A) Principal component analysis of γδT cell differentiation. The scatter plot shows the projections of the first two principal components (PC1 and PC2) at different time points. These points represent individual samples and are colored according to time points. (B) A bubble plot shows the significantly enriched KEGG pathway in DEGs upregulated on days 2, 6, 10, and 25 after hESC differentiation. The y-axis represents the KEGG pathway, and the x-axis represents the gene proportion. p Pathways with values ​​<0.05 were considered significantly enriched. The (CF)STRING database was analyzed to reveal protein-protein interaction networks of differentially upregulated genes among hESCs on days 2 (C), 6 (D), 10 (E), and 25 (F). Detailed Implementation

[0097] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0098] The experimental reagents and materials used in the embodiments of this invention are as follows: The human embryonic stem cell (hESC) H9 cell line was obtained from the WiCell Research Center in the United States according to the material transfer agreements (numbers 19-W0512, 24-W0162, 24-W0163).

[0099] The MEF cell line was prepared and cryopreserved in our laboratory. The specific experimental steps are as follows: (1) First, primary mouse embryonic fibroblasts (MEF) were prepared: CF-1 pregnant mice at 13.5 days of gestation were taken, and after disinfection and dissection, the fetal mice were removed, the head, limbs and internal organs were removed, and the back tissue was collected; after washing with PBS, digesting with trypsin and cutting, the cells were collected by centrifugation, and the single-cell suspension and tissue blocks were obtained by filtration. They were cultured in DMEM high glucose medium containing serum, and after reaching confluence, the primary cells were cryopreserved. (2) Cell resuscitation and expansion were then carried out: cryopreserved primary MEF were taken, and after resuscitation, they were cultured and expanded to F3 generation; after the cells reached confluence, they were treated with mitomycin C, washed thoroughly with PBS, digested and counted, resuspended in cryopreservation solution containing DMSO, frozen at -80℃ and then transferred to liquid nitrogen for long-term storage.

[0100] Human hepatoblastoma (HepG2), human liver cancer cell line (Huh-7), human cervical cancer cell line (HeLa), human T-lymphocytic leukemia cells (Jurkat), and human embryonic kidney cells (293T) were all purchased from the Cell Bank of the Chinese Academy of Sciences.

[0101] STEMdiff™ APEL™2 medium (STEMCELL TECHNOLOGIES, 05275), DMEM medium (Gibco, 10569010), MEM-α medium (Gibco, 12561049), DMEM-F12 (Gibco, C11330500BT), type IV collagenase (Gibco, 17104019), Gentle Cell Dissociation Reagent (GCDR) (STEMCELLTECHNOLOGIES, 07174), BMP4 (Peprotech, 120-05), CHIR99021 (MCE, HY-10182), Activin A (Peprotech, 120-14), Y-27632 (Selleckchem, S1049), Penicillin-Streptomycin-Gentamicin Solution (Triple Antibody) (Solarbio, P1410), VEGF-A (Peprotech, 100-20), bFGF (Peprotech, 100-18B), Flt3L (Peprotech, 300-07), IL-7 (Peprotech, 200-07), SCF (Peprotech, 300-07), SB431542 (Selleckchem, S1067), FBS (Excell Bio, FND500), GlutaMAX (Thermo) Fisher, 35050061), Magnesium Ascorbate Phosphate (MAP) (Sigma, A8960-5G), DLL4 (Peprotech, 140-07), PMA (Biolegend, 423303), CFSE (Biolegend, 423801).

[0102] The nucleotide sequence primers used in the embodiments of this invention are shown in Table 1.

[0103] Table 1 Primers designed based on nucleotide sequences

[0104] The flow cytometry antibodies used in the embodiments of this invention include CD34 (BioLegend, 343606), CD45 (BioLegend, 04014), CD43 (BioLegend, 315204), 7-AAD (BD, 59925), TCRα / β (BioLegend, 306721), TCRγ / β (BioLegend, 331222), CD5 (BioLegend, 364022), CD7 (BioLegend, 343106), CD56 (BioLegend, 362516), and CD3 (B ioLegend, 317332), CD8a (BioLegend, 300920), CD4 (BioLegend, 300556), CD31 (BioLegend, 303134), KDR (BD, 560494), CD41a (BD, 56 1852), CD235a (BioLegend, 349112), CD56 (BioLegend, 362516), CD45 (BioLegend, 304014), Perforin (BioLegend, 353323), Granzyme B Recombinant (BioLegend, 372205), TNF-α (Biolegend, 502912), IFN-γ (Biolegend, 502509), IL-2 (Biolegend, 500347).

[0105] The embodiments of this invention mainly use detection techniques such as RT-qPCR, immunofluorescence staining, and flow cytometry. The specific steps are as follows: 1. RT-qPCR detection: RNA was extracted using the Trizol method throughout the process. Cell spheres at different differentiation time points were collected, 1 mL of Trizol was added, and the mixture was incubated at room temperature for 5 minutes, followed by vortexing to ensure complete cell lysis. The cells were then centrifuged at 12000 g at 4°C for 15 minutes, and the supernatant was transferred to a new 1.5 mL enzyme-free centrifuge tube. 200 μL of chloroform was added to the centrifuge tube, and the mixture was vortexed until it turned a milky pink color. The mixture was incubated at room temperature for 15 minutes, followed by centrifugation at 12000 g at 4°C for 15 minutes. At this point, the homogenate separated into three layers: a clear RNA supernatant on top, a white protein layer in the middle, and a red organic layer at the bottom. Transfer the clear supernatant to a new 1.5 mL enzyme-free centrifuge tube (approximately 400-500 μL, being careful not to aspirate the protein layer). Add 500 μL of isopropanol and mix well. Incubate at room temperature for 10 minutes, then centrifuge at 12000 g, 4°C for 10 minutes. Discard the supernatant and invert the centrifuge tube, aspirating as much liquid as possible. A white RNA precipitate will form at the bottom of the tube. Add 1 mL of 75% ethanol solution, vortex to mix, allowing the RNA precipitate to float in the liquid. Centrifuge at 8000 g, 4°C for 5 minutes. After centrifugation, aspirate as much liquid as possible, retaining only the RNA precipitate. Allow to dry at room temperature for 10 minutes until the alcohol has completely evaporated. Then, add 10-30 μL of RNase-free water to dissolve the RNA precipitate. After testing the RNA concentration and integrity, store at -80°C for a short period or use directly for reverse transcription. Once the RNA test is satisfactory, use High-Capacity Prime Script. TM The RT cDNA kit reverse transcribed RNA into cDNA (total reaction volume: 20 μL). Using the synthesized cDNA as a template, amplification was performed on a real-time quantitative PCR instrument using SYBR Green qPCR premix. The relative expression level of the target gene was analyzed by comparing the Ct (ΔΔCt) method and normalized to the expression level of the internal reference gene.

[0106] 2. Immunofluorescence staining: At different differentiation time points, 20 μL of cell suspension was aspirated, and the suspended cells were placed on a glass slide, while cell spheres were placed in 1.5 mL EP tubes. Cells were immersed in 4% paraformaldehyde and fixed at room temperature for 30 minutes, then washed three times with 1×PBS buffer. Cells were then immersed in 0.5% Triton X-100 and incubated at room temperature for 20 minutes, followed by three washes with 1×PBS buffer. Goat serum was added, and the cells were incubated at room temperature for 30 minutes, then aspirated without washing. Primary antibody dilution buffer was added, and the cells were placed in a humidified chamber and incubated overnight at 4°C. After 12 hours, the humidified chamber was placed in a 37°C oven for 1 hour to rewarm, and the primary antibody was washed away with 1×PBS buffer, repeating the washing process three times. Then, diluted secondary antibody was added, and the cells were returned to the humidified chamber and incubated at room temperature in the dark for 1 hour (subsequent steps should also be performed in the dark). After incubation, 1 mL of 1×PBS buffer was added to wash away the secondary antibody, repeating the washing process three times. Add an appropriate amount of DAPI staining solution, let stand at room temperature for 5 minutes, wash 3 times with 1×PBS buffer, and then cover the cells with anti-fluorescence quencher. The cells can then be directly photographed using a confocal microscope or placed in a humidified chamber for short-term storage at 4°C.

[0107] 3. Flow Cytometry Detection: During hESC differentiation, cells were collected at different time points for flow cytometry detection. The steps were as follows: Cell spheres were collected, washed with 1 mL of 1×PBS, and incubated at room temperature for 3 minutes. The PBS was then discarded. 500 μL of TrypLE digestion solution was added to digest the cell spheres into single cells. After incubation at room temperature for 5 minutes, the cells were gently pipetted with 1 mL of PBS until no cell pellet was visible. The cells were diluted with 1 mL of 1×PBS and centrifuged at 450 g, 4°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of 1×PBS and centrifuged at 450 g, 4°C for 5 minutes. The suspended cells were collected, washed with 1×PBS, and centrifuged again. Approximately 100 μL of liquid remained. The bottom of the centrifuge tube was gently tapped to resuspend the cells. The tubes were then divided into two equal portions: a blank control group and an experimental group. The flow cytometry antibody targeting the protein was added to the experimental group, vortexed, and incubated on ice in the dark for 25-35 minutes. Add 1 mL of 1×PBS buffer to each tube to wash away unbound antibodies, and centrifuge at 450 g, 4°C for 5 minutes. Discard the supernatant, resuspend the cell pellet in 200 μL of 1×PBS buffer, filter through a 200-mesh filter, and transfer to flow cytometry tubes (cells should be kept away from light and incubated at 4°C). Perform flow cytometry analysis; before flow cytometry, add 1 μL of 7-AAD to the experimental group to remove dead cells.

[0108] Example 1: Method for in vitro induction of human embryonic stem cells (hESCs) into γδT cells This embodiment provides a novel system for in vitro differentiation of human embryonic stem cells (hESCs) into γδT cells, and the technical roadmap is as follows: Figure 1 As shown in the figure. By simulating the in vivo development process of γδT cells, a 3D suspension three-stage differentiation system combined with hypoxia was established, and hESCs were differentiated into γδT cells after 25 days.

[0109] Phase 1 (Days 0-6: Hematopoietic Endothelial Cell Differentiation): hESCs were first induced to differentiate using APEL-1 medium in a 3D suspension culture. After two days, the medium was changed to APEL-2 medium for further differentiation. After one day, the medium was changed to APEL-3 medium for another three days of differentiation. After six days of induction, hematopoietic endothelial cells (HECs) were formed.

[0110] Phase 2 (Days 6-10: Hematopoietic progenitor cell differentiation): HECs differentiated to day 6 were resuspended in freshly prepared APEL-4 medium, and single suspended hematopoietic progenitor cells (HPCs) were obtained 4 days later.

[0111] Phase 3 (Days 10-25: γδT cell differentiation): HPCs differentiated to day 10 were resuspended as single suspension cells in MEM-α medium and mixed with HECs. The mixture was then placed in a low-adhesion six-well plate to continue differentiation, and γδT cells were obtained after 15 days.

[0112] APEL-1 medium: APEL TM 2. The basal culture medium was supplemented with BMP4 at a final concentration of 10 ng / mL, CHIR99021 at 3 μM, Activin A at 2 ng / mL, Y-27632 at 10 μM, and triple antibodies at 1%.

[0113] APEL-2 medium: APEL TM 2. The basal culture medium was supplemented with a final concentration of 40 ng / mL VEGF and 1% triple antibiotics.

[0114] APEL-3 medium: APEL TM 2. The basal culture medium was supplemented with a final concentration of 40 ng / mL VEGF, 20 ng / mL bFGF and 1% triple antibiotics.

[0115] APEL-4 medium: APEL TM 2. The basal culture medium was supplemented with a final concentration of 50 ng / mL VEGF, 10 ng / mL LFlt3L, 10 ng / mL IL-7, 20 ng / mL SCF, 20 ng / mL bFGF, 4 μM SB431542 and 1% triple antibody.

[0116] MEM-α medium: MEM-α basal medium was supplemented with a final concentration of 20% FBS, 1% GlutaMAX, 30 μM MAP, 10 ng / mL SCF, 5 ng / mL IL-7, 5 ng / mL Flt3L, 10 ng / mL DLL4 and 1% triple antibodies.

[0117] RPMI 1640 medium (Thermo, C11875500BT): RPMI 1640 basal medium supplemented with a final concentration of 4% B-27 TM Additives, 1% GlutaMAX, 30 μM MAP, 10 ng / mL SCF, 5 ng / mL IL-7, 5 ng / mL Flt3L, 10 ng / mL DLL4 and 1% triple antibody.

[0118] I. The induction and differentiation of human embryonic stem cells into hematopoietic endothelial cells specifically includes the following steps: 1. Resuscitation, culture, and passage of human embryonic stem cells The hESCs used in this experiment were feeder cell-dependent cultures.

[0119] Feeder cell resuscitation: Prepare a 0.1% gelatin solution using 1×PBS buffer and add it to a six-well plate for coating. Allow the plate to stand at room temperature for 1-2 hours before seeding. Thaw frozen MEF cells rapidly in a 37°C water bath. Transfer the cell suspension to a centrifuge tube, add 3 mL of room-temperature MEF complete medium, and centrifuge at 1500 rpm for 5 minutes at 22°C. Discard the supernatant. Resuspend the cells in an appropriate amount of MEF complete medium and seed them into gelatin-coated culture plates. Mix well and return to the incubator. Incubate at 37°C and 5% CO2 for 1 day. The seeding density in the six-well plate is 2-3 × 10⁶ cells / well. 5 Cells / well

[0120] Thawing and passage of hESCs: Thaw frozen hESCs rapidly in a 37°C water bath, transfer to centrifuge tubes, add 3 mL of hESC complete culture medium at room temperature, centrifuge at 1500 rpm for 5 minutes at 22°C without stopping the flow; discard the supernatant, and gently resuspend the cells in 6 mL of hESC complete culture medium (containing 10 μM Rocki). Discard the original culture medium from pre-layered MEF cells, wash the MEF cells with an appropriate amount of hESC complete culture medium, discard the medium, add the hESC cell suspension to the MEF cells, add culture medium to 2 mL / well, mix well, and incubate at 37°C and 5% CO2, changing the medium daily. When the hESC confluence reaches approximately 80%, passage at a 1:6 ratio. Discard the original culture medium from the six-well plates, wash the cells with an appropriate amount of 1×PBS buffer, add 1 mL / well of type IV collagenase, and incubate at 37°C for 40 minutes. After observing cell edge lifting or detachment under a microscope, the collagenase was aspirated, and the cells were washed once with hESCs complete medium. Then, all cells were carefully blown off using the same medium, avoiding repeated blowing that could damage the cells. The cell clusters were seeded into pre-coated MEF cells in six-well plates, shaken well, and returned to the incubator. The plates were then cultured at 37°C and 5% CO2, with the medium changed daily.

[0121] 2. Induction of human embryonic stem cells into hematopoietic endothelial cells After 5-6 days of passage, when the cell colony reaches approximately 80% confluence, the original culture medium is aspirated, and the cells are washed with 1×PBS buffer. 1 mL of GCDR is added to each well, and the cells are incubated at 37°C for approximately 5 minutes for digestion. When cell colony edges are observed to be bright or detached, the GCDR is discarded. 2 mL of APEL-1 medium is added to each well, and the cell colonies are pipetted off, mixed, and seeded into low-adhesion six-well plates for static suspension culture (the number of pipetting cycles should be kept to less than 10). Two days later, the culture medium is changed. The six-well plate is tilted and allowed to stand for 5 minutes, then the original culture medium is slowly aspirated. At this point, the cell clusters are small; a small amount of liquid can be retained to avoid cell loss. 2 mL / well of APEL-2 medium is added to continue differentiation. One day later, the medium is changed to APEL-3 medium and differentiation continues for 3 days, with 3 mL / well of APEL-3 medium added, following the same medium change method. The entire differentiation process is carried out under a hypoxic environment of 37°C, 5% O2, and 5% CO2.

[0122] 3. Cell detection (1) Changes in cell morphology: Take pictures of the cell spheres every day for 6 days before differentiation to record changes in cell morphology.

[0123] (2) Cell RT-qPCR detection: Six days before differentiation, cell spheres were collected every two days to detect the expression levels of mesoderm-specific gene KDR, endothelial-specific gene VE-Cadherin, endothelial-related genes CD31, DLL4 and SOX17; at the same time, the expression levels of transcription factors ERG and TIE1, which are related to early hematopoiesis, were also detected.

[0124] (3) Cell flow cytometry: Cell spheroids were collected every two days for 8 days before differentiation to detect KDR. + Mesodermal-specific markers and CD31 + CD34 + Expression levels of hematopoietic endothelial-specific markers.

[0125] (4) Immunofluorescence staining of cells: On day 6 of differentiation, cell spheroids were collected to detect CD31. + CD34 + Expression of hematopoietic endothelial-specific markers.

[0126] like Figure 2 As shown in Figure A, it can be clearly seen that on day 1 of culture, hESCs begin to form small cell spheroids. As differentiation continues, these cell spheroids gradually increase in size, and by day 4, vacuolar structures begin to appear inside the cell spheroids. This may be due to the accumulation of intracellular fluid or the formation of intercellular spaces. By day 6, the vacuolar structures become more pronounced, and the diameter of the cell spheroids reaches approximately 500 μm. At this point, the cells have undergone significant morphological changes.

[0127] After 6 days of directed differentiation induction under hypoxic conditions, immunofluorescence staining analysis was performed on the 6th day of differentiation. Figure 2 As shown in Figure B, a high proportion of CD34 is present in the cell spheroids. + and CD31 + Cellular expression. This finding indicates that hESCs can be converted into HECs with endothelial cell characteristics through a hypoxia-induced strategy.

[0128] Subsequently, KDR was detected by flow cytometry 8 days before hESC differentiation. + The ratio of mesodermal cells to CD31+CD34+ HECs. The results indicate that KDR... + The proportion of cells reached its peak on day 4, at approximately 22.3%, and gradually decreased as differentiation progressed. Meanwhile, on day 6 of differentiation, 34.8% of CD31+CD34+ HECs were detected, and their proportion rose to approximately 39.2% on day 8. Figure 2 (C)

[0129] To systematically analyze the induced differentiation process of hESCs into HECs, cell spheres from hESCs during the differentiation process were collected and subjected to quantitative PCR. On day 2 of hESC differentiation, the expression level of the early mesoderm-related gene Brachyury significantly increased. As differentiation progressed, on day 4, the expression levels of the mesoderm-specific gene KDR and the endothelial-specific gene VE-Cadherin reached their highest levels, indicating that cells at this stage differentiated along the mesoderm direction and gradually tended towards endothelial cell differentiation. By day 6 of differentiation, the expression levels of endothelial-related genes CD31, DLL4, and SOX17 reached their highest levels; simultaneously, the expression levels of transcription factors ERG and TIE1, which are related to early hematopoiesis, also increased significantly, reaching their peak on day 6 as well. This indicates that the differentiated cells at this time possess HEC characteristics. Therefore, this method, to a certain extent, simulates the developmental process of HECs in the in vivo hematopoietic system. Figure 2 (D).

[0130] II. The stage of induction of hematopoietic endothelial cells into hematopoietic progenitor cells 1. Induction of hematopoietic endothelial cell differentiation into hematopoietic progenitor cells Cell spheres differentiated to day 6 were collected into 15 mL centrifuge tubes and allowed to settle for 5 minutes. The original culture medium was then discarded. The cells were resuspended in 12 mL of freshly prepared APEL-4 medium and transferred to low-adhesion six-well plates, 2 mL per well. After mixing, the plates were returned to the incubator and cultured under a hypoxic environment of 37°C, 5% O2, and 5% CO2. HPCs were obtained after 4 days.

[0131] 2. Cell detection (1) Changes in cell morphology: Cell spheres and single floating cells were photographed on day 10 of differentiation to record cell morphology.

[0132] (2) Cell flow cytometry: Collect cell spheroids on day 10 of differentiation and detect CD31. + CD34 + CD45 + CD43 + and CD7 + Cell proportions; single floating cells were collected on day 10 of differentiation, and CD34 levels were measured. + CD43 + CD7 + CD5 + and CD45 + Cell ratio.

[0133] (3) Immunofluorescence staining of cells: single floating cells on day 10 of differentiation, CD45 was detected. + CD34 + The expression of a marker.

[0134] like Figure 3 As shown, these differentiated HECs were placed under the same hypoxic environment for 4 days for hematopoietic induction, successfully differentiating HECs into HPCs. After 4 days, the hollow structure of HECs became increasingly apparent, and a large number of single suspended HPCs and some cell aggregates were observed to have been produced. Figure 3 (A and C in the middle).

[0135] To further investigate the expression of specific surface markers in cell spheroids on day 10 of differentiation, cell spheroids from each group were collected, and the proportion of cells positive for each marker was determined by flow cytometry. The results showed that CD34 was detected in up to 39.1% of the cell spheroids. + CD31 + HECs were detected, and the results on day 8 were similar, indicating that HECs maintained a relatively stable proportion during differentiation. Furthermore, 28.8% CD45 was also detected. + Cells, a typical marker of white blood cells. Also, a small amount of CD43. + and CD7 + Cells were also detected, further demonstrating the presence of hematopoietic cells at various stages of differentiation within the cell spheres. Figure 3 (Medium B). Because a large number of round, suspended cells were observed in the culture medium on day 10 of differentiation, flow cytometry was used to detect the proportion of cells positive for each marker to further investigate whether these cells possess hematopoietic characteristics. Among them, CD43... + The proportion of HPCs was as high as 99.4%, indicating the homogeneity and high hematopoietic potential of this cell population. Furthermore, the cell population at this stage contained 22.67% CD34. + HSPCs. Meanwhile, 90.1% of CD7 were observed. + Cells and 10.2% CD45 + CD7 is a hallmark molecule of pro-T cells, while CD45 is expressed in most lymphocytes, suggesting that this cell population has the potential to differentiate into T cell lineages. Figure 3 (D).

[0136] Immunofluorescence staining analysis was performed on HPCs on day 10 to detect the expression of cell surface proteins CD34 and CD45 at this stage. The results are as follows: Figure 3 As shown in Figure E, HPCs at this stage significantly overexpressed the markers CD34 and CD45. Notably, most cells co-expressed CD34 and CD45, indicating that HPCs maintained strong pluripotency and had the potential to differentiate into multiple hematopoietic lineages on day 10 of hematopoietic differentiation.

[0137] III. Establishment of methods for differentiating hematopoietic progenitor cells into γδT cells (a) Differentiation efficiency of different culture methods 1. Establishment of a method for differentiating hematopoietic progenitor cells into γδT cells: hESCs differentiated to day 10 were collected into 15 mL centrifuge tubes. After standing for 1 minute, the cell spheres settled naturally, and the single suspended cells remained in the supernatant. The supernatant was transferred to a new 15 mL centrifuge tube and centrifuged at 900 rpm and 4°C for 5 minutes. The original culture medium was discarded after centrifugation. Four different culture methods were set up: (1) R+ / - HECs group: Umbilical cord blood-derived γδT cell differentiation medium (RPMI 1640 basal medium with a final concentration of 4% B-27) was used. TM (1) Additives, 1% GlutaMAX, 30 μMMAP, 10 ng / mL SCF, 5 ng / mL IL-7, 5 ng / mL Flt3L, 10 ng / mL DLL4 and 1% triple antibody) were used to resuspend single suspension cells and then placed in six-well plates for further differentiation; 2 mL of culture medium per well. (2) R+ / +HECs group: Single suspension cells were resuspended in umbilical cord blood-derived γδT cell differentiation medium and then mixed with HECs and placed in low adhesion six-well plates for further differentiation; 2 mL of culture medium per well. (3) M+ / - HECs group: Single suspension cells were resuspended in hESCs-derived γδT cell differentiation medium (MEM-α basal medium with a final concentration of 20% FBS, 1% GlutaMAX, 30 μM MAP, 10 ng / mL SCF, 5 ng / mL IL-7, 5 ng / mL Flt3L, 10 ng / mL DLL4 and 1% triple antibody) and placed in six-well plates for further differentiation; 2 mL of medium per well. (4) M+ / + HECs group: Single suspension cells were resuspended in hESCs-derived γδT cell differentiation medium and then mixed with HECs and placed in low-adhesion six-well plates for further differentiation; 2 mL of medium per well.

[0138] Then, 1 mL of culture medium was added every 2 days, and the culture medium was replaced every 5 days using a half-medium medium replacement method. After shaking well, the cells were returned to the incubator and cultured in a normoxic environment of 37℃, 20% O2 and 5% CO2. γδT cells were obtained after 15 days.

[0139] 2. Cell detection: Flow cytometry is used to perform precise phenotypic analysis on these cells.

[0140] Suspension cells were collected on day 15 after induced differentiation, and precise phenotypic analysis of these cells was performed using flow cytometry. Figure 4 (A) Through Figure 4Results from studies B and D clearly show that, 15 days after induction of differentiation, the MEM-α medium (M+ / -HECs) produced a higher proportion of CD45 cells compared to the RPMI 1640 medium group (R+ / -HECs). + The presence of these cells, to some extent, indicates the advantage of MEM-α medium in promoting hematopoietic cell differentiation. Figure 4 Further studies C and D showed that when HPCs and HECs were co-cultured (M+HECs), 42.4% of CD3+ was induced. + TCRγδ + The presence of these cells indicates efficient production of mature γδT cells at this stage. Notably, this result is significantly superior to the other three groups. Based on the above experimental results, it has been determined that co-culturing HPCs with HECs using MEM-α medium is the optimal strategy for inducing the differentiation of hESC-derived HPCs into mature γδT cells. Therefore, this differentiation method will be used in subsequent experiments to ensure efficient production of γδT cells.

[0141] (ii) Differentiation of hematopoietic progenitor cells into γδT cells 1. Differentiation of hematopoietic progenitor cells into γδ T cells Cells differentiated from hESCs to day 10 were collected into 15 mL centrifuge tubes. After standing for 1 minute, the cell spheroids settled naturally, leaving individual suspended cells in the supernatant. The supernatant was transferred to a new 15 mL centrifuge tube and centrifuged at 900 rpm for 5 minutes at 4°C. The original culture medium was discarded after centrifugation. The individual suspended cells were resuspended in hESC-derived γδT cell differentiation medium and mixed with HECs. The mixture was then placed in low-adhesion six-well plates for further differentiation, with 2 mL of medium per well. 1 mL of medium was added every 2 days, and the medium was replaced every 5 days using a half-medium medium change method. After mixing well, the plates were returned to the incubator and cultured under normoxic conditions of 37°C, 20% O2, and 5% CO2. γδT cells were obtained after 15 days.

[0142] 2. Cell detection (1) Changes in cell morphology: Suspension cells on days 15, 20 and 25 of differentiation were photographed to record cell morphology.

[0143] (2) Cell flow cytometry: Suspension cells were collected on days 15, 20, and 25 of differentiation, and CD45 was detected. + CD3 + TCRγδ + The proportion of cells.

[0144] On day 10 of induction of hESC differentiation, a large number of single suspension HPCs were obtained. Subsequently, these cells and hematopoietic endothelial cell spheres were continuously induced to differentiate in MEM-α medium supplemented with multiple cytokines including DLL4, SCF, Flt3L, IL-7, and MAP until day 25. During this period, the cell size and morphology remained relatively stable without significant changes. Figure 4 (E). Of particular note is the aggregation of cell clusters in some areas during days 10 to 15 of differentiation, indicating that the cells are undergoing active proliferation. As differentiation progresses further, around day 20, a decrease in cell number is observed, likely due to biological processes such as cell differentiation, apoptosis, or migration. Furthermore, some cells begin to adhere to the culture surface.

[0145] During the induction of HPCs into γδ T cells, single suspension cells were collected, and the lymphocyte marker CD45, as well as the mature γδ T cell markers CD3 and TCRγδ, were detected by flow cytometry at days 15, 20, and 25 of differentiation. Results are as follows: Figure 4 As shown in F and G, CD45... + The proportion of cells (i.e. lymphocytes) increased significantly, reaching approximately 74.2% by day 25. This indicates that this differentiation strategy effectively promotes the differentiation of HPCs into lymphocytes.

[0146] Further analysis revealed that CD3 + TCRγδ + Mature γδT cells (i.e., mature γδT cells) began to appear in the cell population on day 20 of differentiation, and reached 37.4% on day 25. Figure 4 (H, I). This finding confirms that HPCs can be successfully induced to differentiate into mature γδ T cells. Notably, almost all detected CD3... + TCRγδ + All cells are derived from CD45 + The cell population further supports our observation that HPCs initially differentiate into lymphocytes, which then further differentiate into mature γδT cells. To further identify the expression of specific markers in the differentiated γδT cells, flow cytometry was used to analyze hESCs on day 25 of differentiation. The results are as follows: Figure 4 As shown in Figure J, TCRαβ was not detected. + This result clearly indicates that this differentiation system does not induce the production of αβT cells. Up to 88.6% of CD8α cells were detected. - CD4 -The cells were analyzed, and 20.2% of them expressed CD56, consistent with the phenotype of native γδT cells. Notably, CD41a, a specific marker for megakaryocytes, was almost not expressed in our final differentiated cells, further supporting the conclusion that the differentiated cells were γδT cells.

[0147] Example 2: Phenotypic identification and functional analysis of γδT cells I. Cellular RT-qPCR Detection During the differentiation of HPCs into γδT cells, the expression levels of relevant specific genes and changes in the expression of γδT cell function-related genes at different stages were detected by quantitative PCR. The specific steps are as follows: During the differentiation of hESCs, single cells were collected on days 10, 15, 20, and 25, and RNA was extracted for RT-qPCR detection. First, the expression levels of megakaryocytic and erythroid transcription factors FLI1 and KLF1; hematopoietic marker genes CD43 and RUNX1; pro-T marker gene CD7; genes NOTCH1, IL7R, and RUNX1 that play a key or important role in the regulation of TCR γ and δ chain rearrangement; T cell development-related transcription factors TCF7 and BCL11B; lymphocyte-related genes CD45 and CD3E; and NK cell marker gene CD56 were detected. Second, the expression levels of key transcription factors for γδT cell differentiation EGR1, EGR2, EGR3, and TBET; TNFSF11 (tumor necrosis factor ligand superfamily member 11); and ZAP70, a tyrosine kinase of the SYK family, were detected.

[0148] During the differentiation of HPCs into γδT cells, the expression levels of relevant specific genes at different stages were detected by quantitative PCR. Figure 5 Megakaryocytic and erythroid transcription factors FLI1 and KLF1 were gradually downregulated during HPC differentiation into γδT cells. Hematopoietic markers CD43 and RUNX1 were also gradually downregulated during differentiation, as was the pro-T marker CD7. In mouse developmental studies, NOTCH1, IL7R, and RUNX1 genes play key or important roles in the regulation of TCR γ and δ chain rearrangements, and these genes maintained high expression levels during development. T cell development-related transcription factors TCF7 and BCL11B were significantly upregulated from day 15 to 20 of differentiation, and significantly highly expressed on day 25. Lymphocyte-related gene CD45 was gradually upregulated during γδT cell differentiation. High expression of the NK cell marker CD56 was also detected on day 25. Therefore, these results indicate that this differentiation method can induce HPCs to develop into γδT cells, and exhibits a similar gene expression profile to the in vivo γδT cell development process.

[0149] Secondly, the expression changes of γδT cell function-related genes were further examined. Figure 6 (A) During the differentiation of hESCs into γδT cells, the expression of key transcription factors EGR1, EGR2, EGR3, and TBET was significantly upregulated. These transcription factors play a crucial role in the development of γδT cells, and their upregulated expression indicates that the differentiation protocol effectively promotes γδT cell maturation. Furthermore, TNFSF11 (tumor necrosis factor ligand superfamily member 11), as a member of the TNF superfamily, is expressed in activated lymphocytes and is closely associated with high cytotoxic potential. During differentiation, TNFSF11 expression was also gradually upregulated, reaching a significantly high level on day 25, suggesting that the differentiated γδT cells may possess high cytotoxic potential. Finally, the ZAP70 gene, a tyrosine kinase of the SYK family, is mainly expressed on T cells and NK cells and plays a key role in T cell development and activation. During our differentiation, ZAP70 expression was also gradually upregulated, further supporting the effectiveness of this differentiation protocol in promoting γδT cell development and activation. In summary, the hESCs-derived γδT cell induction differentiation protocol successfully promoted the upregulation of key transcription factors, thereby facilitating the development of mature and functional γδT cells.

[0150] II. In vitro stimulation experiment of γδT cells To comprehensively evaluate the functionality of hESCs-derived γδ T cells, a classic T cell function activation assay was employed. Figure 6(Medium B). Specifically, all cells differentiated to day 25 were exposed to phorbol ester (PMA) stimulation for 6 hours to simulate signal transduction during T cell activation. Subsequently, these stimulated cells were collected, and flow cytometry was used to detect the cytokines TNF-α and IL-2. The specific steps are as follows: γδT cells differentiated from hESCs to day 25 were collected in 15 mL centrifuge tubes, centrifuged at 900 rpm and 4°C for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in culture medium and seeded into 24-well plates. 2 μL of PMA was added to each mL of cell suspension to stimulate γδT cells, and after mixing, the cells were returned to the incubator and cultured at 37°C and 5% CO2 for 6 hours. Activated cells were then harvested for detection. The cell suspension was centrifuged at 900 rpm and 4°C for 5 minutes, the supernatant was discarded, and 1 mL of 1×PBS buffer was added to the pellet to resuspend the cells. The cells were then centrifuged at 900 rpm and 4°C for 5 minutes, and the supernatant was discarded. To label cells with antibodies on the cell membrane surface, resuspend cells in 50 μL of 1×PBS buffer, add 1 μL of CD45 antibody, mix well, and incubate on ice in the dark for 45 minutes. Wash once with 1×PBS buffer before labeling cells with antibodies inside the cell membrane. Add 200 μL of fixation / permeabilization working solution (prepared fresh before use) to fix and perforate the cells, mix well, and incubate at room temperature in the dark for 1 hour. Do not wash; add 600 μL of 1×perm buffer (diluted with double-distilled water) directly to each tube, centrifuge at 400 g, 4°C for 5 minutes, then at 8500 g, 4°C for 2 minutes, discard the supernatant, and repeat the washing process twice. Resuspend the cells in a mixture of IFN-γ, TNF-α, Perforin, Granzyme B, and IL-2 prepared with 1×perm buffer, and incubate at room temperature in the dark for 45 minutes. Wash the cells with 1 mL of 1×PBS buffer, centrifuge at 400 g, 4°C for 5 minutes, then at 8500 g, 4°C for 2 minutes, and discard the supernatant. Resuspend the cells in 200 μL of 1×PBS buffer, filter the cells through a 200-mesh filter into flow cytometry tubes, and analyze using a flow cytometer.

[0151] like Figure 6 As shown in B, in these CD45 + In the cells, 30.4% expressed TNF-α, and 13.4% expressed IL-2. These experimental data indicate that the differentiated hESC-derived γδT cells are functionally highly similar to natural γδT cells. They can not only directly kill or inhibit tumors by secreting TNF-α, but also activate and enhance the activity of immune cells by releasing IL-2, thus playing an important role in the immune system.

[0152] III. Cytotoxicity assay of γδT cells γδT cells act in a manner unrestricted by human leukocyte antigen (HLA) in various cancer types. Therefore, the cytotoxicity of γδT cells against four cancer cell lines was evaluated, including one non-solid tumor Jurkat cell line and three solid tumor lines: HepG2 cells, Huh-7 cells, and HeLa cells. Figure 7 These cell lines have different HLA types from the hESCs used in this study. Cancer cell lines were used as target cells (Target, T), and hESC-derived γδT cells were used as effector cells (E). Target cells were labeled with CFSE and seeded into 12-well plates for culture. After target cells adhered, γδT cells were added at an E:T ratio of 2:1 for co-culture. After 48 hours, all cells were collected for flow cytometry analysis, and dead cells were labeled with DAPI. First, 293T cells were used as a control to detect the cytotoxic effect of γδT cells on normal cells. The specific steps are as follows: Several human cancer cell lines were used for killing experiments, including HepG2 cells, Huh-7 cells, HeLa cells, and Jurkat cells. 292T cells were used as a control to detect the cytotoxic effect of γδT cells on normal human cells. First, target cells (Target, T) were collected, washed with 1×PBS buffer, and centrifuged at 1500 rpm and 22°C for 5 minutes. Cells were resuspended in 1 mL of 1×PBS buffer with 1 μL CCFSE (5 μM) added, stained at 37°C in the dark for 15 minutes, then 3 mL of completely pre-chilled culture medium was added and the cells were incubated at 4°C for 10 minutes. After centrifugation at 1500 rpm and 22°C for 5 minutes, the supernatant was discarded, the cells were washed twice with 1×PBS buffer, resuspended in culture medium, and counted. Target cells were seeded into 12-well plates at a specific quantity, shaken well, and returned to the incubator for culture at 37°C and 5% CO2. After 24 hours, when the cells were fully adherent, γδT cells were added to the target cells at an E:T ratio of 2:1, and co-cultured for another 48 hours. All cells were collected for flow cytometry analysis, and dead cells were labeled with DAPI to analyze the cell death status of the target cells.

[0153] from Figure 7 According to the results from the Chinese study, 1.7% of 293T cells died when cultured alone. Furthermore, as... Figure 7 As shown in Figure B, 3.39% of 293T cells died after the addition of hESCs-derived γδT cells, indicating that γδT cells had no significant killing effect on 293T cells. When γδT cells were co-cultured at an E:T ratio of 2:1, 66% CFSE was detected after 48 hours. - γδT cells and 54% CFSE+ The proportion of 293T cells increased significantly at this point, indicating substantial proliferation of 293T cells. Further investigation was conducted to examine the cytotoxic effect of γδT cells on cancer cells. Results Figure 7 As shown in the CF study, when cancer cells were cultured alone, only 4.26% of HepG2 cells and 0.91% of Huh-7 cells died. However, after adding hESC-derived γδT cells, 26.1% of HepG2 cells and 23.4% of Huh-7 cells died. Furthermore, it was found that compared to 293T cells, co-culturing with γδT cells resulted in only 23.7% CFSE after 48 hours. + HepG2 cells and 16.2% CFSE + Huh-7 cells, indicating that γδT cells inhibited the proliferation of this tumor cell.

[0154] In summary, differentiated γδT cells have significant cytotoxic effects on liver cancer cells, inhibiting their proliferation without affecting the growth of normal cells.

[0155] Example 3: Transcriptome sequencing analysis during γδT cell differentiation 1. Transcriptome sequencing sample delivery: During the differentiation of hESCs into γδT, RNA was extracted from undifferentiated hESCs and from differentiation stages 2, 6, 10, 15, 20, and 25, and then sent to Novogene Biotech for transcriptome sequencing (bulk RNAseq).

[0156] 2. Methods for analyzing transcriptome sequencing data: Gene expression level quantification: Clean data after quality control was aligned to a reference genome, and the alignment results were statistically analyzed. Hisat2 software was used to align reads to a human reference genome, and feature counts software was used to calculate the expression level of each gene. The results were then combined to obtain an expression matrix.

[0157] Differential expression analysis: The analysis was performed in RStudio. The DESeq2 package in R was used to perform differential expression analysis between the two groups, and the statistical method used was hypothesis testing. Differentially expressed genes were screened using corrected p-values ​​and log² foldchange. Volcano plots of differential expression were generated using ggplot, and heatmaps of differentially expressed genes were generated using pheatmap.

[0158] Differential gene enrichment analysis: Differentially upregulated genes compared to the previous stage were analyzed for KEGG pathway enrichment using the KOBAS online database (http: / / bioinfo.org / kobas / ). The adjusted values ​​were then used for further analysis. PA value <0.01 was considered a significant enrichment threshold. Data visualization was performed using the R package ggplot in RStudio. To construct the protein-protein interaction network for upregulated differentially expressed genes at each time point, the STRING database (Search Tool for the Retrieval of Interacting Genes / Proteins) was used. Upregulated differentially expressed genes were input into the STRING database, which provides comprehensive information on known and predicted protein-protein interactions.

[0159] The results are as follows Figure 8 As shown, PCA results indicated that samples at different differentiation time points exhibited significant clustering, with samples from day 0 and day 2 clustering closely together, suggesting similar gene expression patterns in the initial differentiation stage. As differentiation progressed, the gene expression profiles of samples from days 6, 10, 15, 20, and 25 showed progressive changes, reflecting the dynamic changes in γδT cell differentiation. This indicates that gene expression undergoes significant changes during differentiation as cells mature into γδT cells. Figure 8 (A)

[0160] To investigate whether the differentiation mechanism of human embryonic stem cell-derived γδT cells is similar to that of human γδT cells, KEGG pathway analysis was performed on differentially regulated genes at the following four differentiation stages: significantly upregulated genes on day 2 compared to day 0, significantly upregulated genes on day 6 compared to day 2, significantly upregulated genes on day 10 compared to day 6, and significantly upregulated genes on day 25 compared to day 10. Figure 8 (B) KEGG pathway analysis was performed on differentially expressed genes. On day 6, differentially expressed genes were significantly enriched in several key signaling pathways closely related to stem cell proliferation and differentiation, such as the PI3K-AKT, MAPK, and Hippo signaling pathways. These pathways were also enriched in core pathways of mesoderm development, such as WNT and TGF-β, which play crucial roles in embryogenesis and organ development. On day 10, differentially expressed genes were significantly enriched in calcium signaling pathways, which regulate stem cell proliferation, differentiation, and maturation during hematopoietic development and play an important role in maintaining the normal function of the hematopoietic system. The TRP channel is a calcium ion channel involved in immune cell migration and cytokine release, promoting the functional development of immune cells. On day 25, differentially expressed genes were significantly enriched in signaling pathways related to cell proliferation and differentiation, such as PI3K-AKT, MAPK, Jak-STAT, Hippo, and Ras. Jak-STAT is involved in T cell development and immune regulatory responses. Nod-like receptors, leukocyte transendothelial migration (TEM), FC-γr-mediated phagocytosis, TNF, NF-κB, Toll-like receptors, and T-cell receptors are all involved in immune responses, lymphocyte differentiation, and functional development.

[0161] Furthermore, the protein-protein interaction network (PPI) of differentially upregulated genes in cells at different induction stages was analyzed using the STRING database, further verifying the association between genes and biological functions. Figure 8 (CF). PPI analysis showed that WNT3A, MSGN1, TBXT, and other genes were tightly linked on day 2 of induction, acting as hub genes (highly linked genes). Studies indicate that WNT3A is essential for the formation of the mesoderm in normal embryos, while MSGN1 may regulate the expression of T-box transcription factors required for mesoderm formation and differentiation, and TBXT is involved in the transcriptional regulation of genes required for mesoderm formation and differentiation. On day 6 of induction, PPI analysis showed that ESAM, CDH5, and CLDN5 were tightly linked as hub genes at this stage. ESAM is an endothelial cell selective adhesion molecule that mediates cell aggregation. CDH5 binds to α-catenin to form a link with the cytoskeleton and, together with KRIT1 and MPP5, maintains appropriate endothelial cell polarity and vascular lumen integrity. Tight junction protein 5 (CLDN5) plays a crucial role in the tight junction-specific closure of intercellular spaces. On day 10 of induction, PPI analysis showed that CCR2, CSF2, and PTPRC were tightly linked, acting as hub genes at this stage. CCR2 regulates the expression of T cell inflammatory factors and influences T cell differentiation, particularly promoting T cell differentiation into helper T cell 17 (Th17) cells during inflammation. CSF2, also known as granulocyte-macrophage colony-stimulating factor, is a cytokine that promotes the growth and differentiation of hematopoietic progenitor cells from multiple lineages, including granulocytes, macrophages, eosinophils, and erythrocytes. PPI analysis on day 25 of induction showed tight linkage between genes such as FCGR3A, PDCD1LG2, and TLR8, indicating they are hub genes for this stage. FCGR3A is a low-affinity immunoglobulin γ Fc region receptor iii-a that binds to complexed or aggregated IgG as well as monomeric IgG. It is crucial for promoting antibody-dependent cytotoxicity (ADCC) and other antibody-dependent responses such as phagocytosis. PDCD1LG2, also known as programmed cell death 1 ligand 2, plays a role in the co-stimulatory signaling pathway essential for T cell proliferation and IFNG production, functioning independently of PDCD1.

[0162] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A reagent combination comprising a fifth culture medium, the fifth culture medium comprising a basal culture medium supplemented with magnesium ascorbate phosphate, hematopoietic growth factor and Notch signaling pathway activator.

2. The reagent combination according to claim 1, characterized in that, The hematopoietic growth factor is selected from at least one of stem cell factor, thrombopoietin, interleukin-3, interleukin-6, interleukin-7, and FMS-associated tyrosine kinase 3 ligand. Preferably, the hematopoietic growth factors include stem cell factors, interleukin-7, and FMS-associated tyrosine kinase 3 ligand.

3. The reagent combination according to claim 1, characterized in that, The Notch signaling pathway activator is selected from at least one of DLL1, DLL3, DLL4, JAG1, or JAG2.

4. The reagent combination according to claim 1, characterized in that, The concentration of magnesium ascorbate phosphate in the fifth culture medium is 10-50 μM; Preferably, the concentration of the stem cell factor in the fifth culture medium is 5-20 ng / mL; Preferably, the concentration of interleukin-7 in the fifth culture medium is 1-10 ng / mL; Preferably, the concentration of the FMS-associated tyrosine kinase 3 ligand in the fifth culture medium is 1-10 ng / mL; Preferably, the concentration of the Notch signaling pathway activator in the fifth culture medium is 5-20 ng / mL.

5. The reagent combination according to any one of claims 1-4, characterized in that, The basal medium in the fifth culture medium is selected from at least one of RPMI-1640 and MEM-α basal medium.

6. The reagent combination according to claim 5, characterized in that, The reagent combination also includes a first culture medium, which comprises a basal culture medium supplemented with bone morphogenetic protein, GSK-3 inhibitor, Activin A and Rock inhibitor; Preferably, the reagent combination further comprises a second culture medium, which includes a basal culture medium supplemented with vascular endothelial growth factor; Preferably, the reagent combination further comprises a third culture medium, which includes a basal culture medium supplemented with vascular endothelial growth factor and fibroblast growth factor. Preferably, the reagent combination further comprises a fourth culture medium, which includes a basal culture medium supplemented with vascular endothelial growth factor, hematopoietic growth factor, fibroblast growth factor and TGF-β signaling pathway inhibitors.

7. A reagent kit, characterized in that, The kit comprises the reagent combination as described in any one of claims 1-6.

8. The use of the reagent combination according to any one of claims 1-6 or the kit according to claim 7 in any of the following aspects: (1) Preparation of γδT cells; (2) Prepare products that induce pluripotent stem cells to differentiate into γδT cells.

9. A method for preparing γδT cells, wherein γδT cells are obtained by culturing pluripotent stem cells using a combination of reagents according to any one of claims 1-6 or a kit according to claim 7; wherein the culture is a suspension culture.

10. The use of the method of claim 9 or the γδT cells prepared by the method of claim 9 in any of the following: To prepare drugs for treating cancer; Preparation of drugs for diseases caused by pathogenic microorganisms; To prepare immunomodulatory drugs.

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