Construction method and application of human iAGM hematopoietic organoid

Abstract

The invention discloses a culture medium composition for inducing human pluripotent stem cells to differentiate into human iAGM hematopoietic organoid, human embryonic stem cells are differentiated into the human iAGM hematopoietic organoid through combination and matching of different cell factors in stages, and the generated hematopoietic stem cells have high primitiveness and authenticity, so that the functionality of the hematopoietic stem cells is finally guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for constructing a human iAGM hematopoietic organoid and its application. Background Art

[0002] Allogeneic or syngeneic hematopoietic stem cell transplantation (alloSCT) is a clinical treatment method for repairing hematopoietic function defects. However, due to the severe shortage of donor bone marrow cells, the urgent clinical transplantation needs are hindered. Fully functional hematopoietic multipotent progenitor cells (MPPs) derived from human embryonic stem cells (hESCs) have emerged as a great therapeutic potential, having a strong impact on the study of hematopoiesis and potentially becoming an unlimited cell source for alternative therapeutic applications. Although recent studies have provided new insights into the mechanisms underlying hematopoietic development signals and specification, the comprehensive understanding of the cellular and molecular mechanisms of hematopoiesis remains insufficient. Generating abundant, functional hESC-derived MPP cells that can be long-term transplanted and differentiated into multi-lineage hematopoietic cells is still challenging, which hinders the clinical effective utilization of hESC-derived MPP cells as potential stem cell therapeutic agents.

[0003] Hematopoiesis is a highly regulated hierarchical process, which is now decomposed into two sequential stages: primitive hematopoiesis and definitive hematopoiesis. Some evidence suggests that the first defined hematopoietic stem cells (HSCs) are generated from a special subset of blood-derived endothelial cells (ECs) located in the aorta-gonad-mesonephros (AGM) region. In the late stage of development, hematopoietic stem cell precursors (pre-HSCs) mature when they migrate to the fetal liver (FL) before colonizing the bone marrow (BM) during embryogenesis. The ventral domain of the dorsal aorta (AoV) has been identified as a functional HSC niche in humans and mice, in which there are a large number of intra-aortic hematopoietic cell clusters (IAHCs). These cell populations consist of pre-HSCs expressing hematopoietic functional characteristics such as CD45 and CD41, and also express endothelial cell markers such as CD31 (PECAM1), VE-Cadherin (CD144), and CD34, etc., indicating that there may be a post-hematopoietic endothelial cell intermediate in these structures that transitions from endothelial cells to hematopoietic stem and progenitor cells (HSPCs). However, the exact cell composition and function of IAHCs in vivo, as well as the molecular steps involved in the generation of hematopoietic stem cells from endothelial cells, are not fully understood.

[0004] Currently, researchers are conducting a large number of studies to obtain a stable source of HSCs through the differentiation of human pluripotent stem cells, but strictly functional HSCs have not yet been generated. This may be because previous hematopoiesis research has mainly focused on the "flat" analysis of specific cell populations, lacking a comprehensive "three-dimensional" analysis. The generation and development of HSCs in the AGM region require the regulation of surrounding microenvironmental cell signals, including at least mesenchymal cells, epithelial cells, and endothelial cells, etc., that is, the fate regulation of hematopoiesis development by specific microenvironments and niche signals. Summary of the Invention

[0005] Therefore, the objective of the present invention is to establish a technical system for constructing and deriving AGM hematopoietic organoids (iAGM hematopoietic organoids) through the three-dimensional differentiation of human embryonic stem cells in vitro. This differentiation system can simulate the physiological environment of in vivo embryonic hematopoiesis and the microstructural and functional characteristics similar to natural organs in vitro, thereby initiating the generation of hematopoiesis.

[0006] To achieve the above objective, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, a culture medium combination is provided. The culture medium combination includes a first culture medium, a second culture medium, and a third culture medium; the first culture medium includes a first basal medium supplemented with Activin A, vascular endothelial growth factor, bone morphogenetic protein, fibroblast growth factor, TGF-β signaling pathway inhibitor, GSK-3 inhibitor, and Rock inhibitor; the second culture medium includes a second basal medium supplemented with vascular endothelial growth factor, bone morphogenetic protein, and fibroblast growth factor; the third culture medium includes a third basal medium supplemented with hematopoietic growth factor.

[0008] In some embodiments, the vascular endothelial growth factor 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.

[0009] In some embodiments, the vascular endothelial growth factor is VEGF-A.

[0010] In some embodiments, the bone morphogenetic protein is selected from at least one of BMP4 and BMP2.

[0011] In some embodiments, the bone morphogenetic protein is BMP4.

[0012] In some embodiments, the fibroblast growth factor 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, FGF23.

[0013] In some embodiments, the fibroblast growth factor is FGF2.

[0014] In some embodiments, the TGF-β signaling pathway inhibitor is selected from at least one of LY2157299, EW-7197, LY3200882, SB-431542, LY2109761, TP-0427736, IN-1130, R-268712, A-83-01, SB-525334, GW788388, RepSox, A-77-01, SB-505124, SD-208, LY364947.

[0015] In some embodiments, the TGF-β signaling pathway inhibitor is SB-431542.

[0016] In some embodiments, the GSK-3 inhibitor is selected from at least one of TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR-99021.

[0017] In some embodiments, the GSK-3 inhibitor is CHIR-99021.

[0018] In some embodiments, the Rock inhibitor is selected from at least one of Y-27632, Thiazovivin, Fasudil HCl, GSK429286A, RKI-1447, and Azaindole 1.

[0019] In some embodiments, the Rock inhibitor is Y-27632.

[0020] In some embodiments, the hematopoietic growth factor is selected from at least one of stem cell factor, thrombopoietin, interleukin-3, interleukin-6, FLT3-ligand.

[0021] In some embodiments, the hematopoietic growth factors are stem cell factor, interleukin-3, and FLT3-ligand.

[0022] In some embodiments, the first culture medium comprises a first basal medium supplemented with Activin A, VEGF-A, BMP4, FGF2, SB-431542, CHIR-99021, and Y-27632.

[0023] In some embodiments, the addition concentration of Activin A in the first basal medium is 5-15 ng / mL.

[0024] In some embodiments, the addition concentration of VEGF-A in the first basal medium is 5-15 ng / mL.

[0025] In some embodiments, the addition concentration of BMP4 in the first basal medium is 5-15 ng / mL.

[0026] In some embodiments, the addition concentration of FGF2 in the first basal medium is 5-15 ng / mL.

[0027] In some embodiments, the addition concentration of SB-431542 in the first basal medium is 2-6 μM.

[0028] In some embodiments, the addition concentration of CHIR-99021 in the first basal medium is 2-4 μM.

[0029] In some embodiments, the addition concentration of Y-27632 in the first basal medium is 5-15 μM.

[0030] In some embodiments, the first culture medium comprises a first basal medium supplemented with 5-15 ng / mL Activin A, 5-15 ng / mL VEGF-A, 5-15 ng / mL BMP4, 5-15 ng / mL FGF2, 2-6 μM SB-431542, 2-4 μM CHIR-99021, and 5-15 μM Y-27632.

[0031] In some embodiments, the first culture medium comprises a first basal medium supplemented with 10 ng / mL Activin A, 10 ng / mL VEGF-A, 10 ng / mL BMP4, 10 ng / mL FGF2, 4 μM SB-431542, 3 μM CHIR-99021, and 10 μM Y-27632.

[0032] In some embodiments, the second culture medium comprises a second basal medium supplemented with VEGF-A, BMP4, and FGF2.

[0033] In some embodiments, the addition concentration of VEGF-A in the second basal medium is 5 - 15 ng / mL.

[0034] In some embodiments, the addition concentration of BMP4 in the second basal medium is 5 - 15 ng / mL.

[0035] In some embodiments, the addition concentration of FGF2 in the second basal medium is 5 - 15 ng / mL.

[0036] In some embodiments, the second medium comprises a second basal medium supplemented with 5 - 15 ng / mL VEGF-A, 5 - 15 ng / mL BMP4, and 5 - 15 ng / mL FGF2.

[0037] In some embodiments, the second medium comprises a second basal medium supplemented with 10 ng / mL VEGF-A, 10 ng / mL BMP4, and 10 ng / mL FGF2.

[0038] In some embodiments, the third medium comprises a third basal medium supplemented with SCF, IL3, and Flt3L.

[0039] In some embodiments, the addition concentration of SCF in the third basal medium is 50 - 150 ng / mL.

[0040] In some embodiments, the addition concentration of IL3 in the third basal medium is 50 - 150 ng / mL.

[0041] In some embodiments, the addition concentration of Flt3L in the third basal medium is 50 - 150 ng / mL.

[0042] In some embodiments, fetal bovine serum, fungal antibiotics, glutamine, and β-mercaptoethanol are further added to the third basal medium.

[0043] In some embodiments, the fungal antibiotics include penicillin, streptomycin, and amphotericin B.

[0044] In some embodiments, the addition concentration of fetal bovine serum in the third basal medium is 10 vol% - 30 vol%.

[0045] In some embodiments, the addition concentration of penicillin in the third basal medium is 50 - 150 U / mL, the addition concentration of streptomycin is 0.05 - 0.15 mg / mL, and the addition concentration of amphotericin B is 0.125 - 0.375 μg / mL.

[0046] In some embodiments, the fungal antibiotic is a penicillin-streptomycin-amphotericin B mixed solution (100× triple antibiotic), wherein the penicillin content is 10 kU / ml, the streptomycin content is 10 mg / ml, and the amphotericin B content is 25 μg / ml.

[0047] In some embodiments, the added concentration of the penicillin-streptomycin-amphotericin B mixed solution in the third basal medium is 0.5 vol% - 1.5 vol%.

[0048] In some embodiments, the added concentration of L-glutamine in the third basal medium is 1 - 3 mM.

[0049] In some embodiments, the added concentration of β-mercaptoethanol in the third basal medium is 50 - 150 mM.

[0050] In some embodiments, the third medium comprises a third basal medium supplemented with 10 vol% - 30 vol% fetal bovine serum, 50 - 150 U / mL penicillin, 0.05 - 0.15 mg / mL streptomycin, 0.125 - 0.375 μg / mL amphotericin B, 1 - 3 mM L-glutamine, 50 - 150 mM β-mercaptoethanol, 50 - 150 ng / mL SCF, 50 - 150 ng / mL IL3, and 50 - 150 ng / mL Flt3L.

[0051] In some embodiments, the third medium comprises a third basal medium supplemented with 20 vol% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 2 mM L-glutamine, 100 mM β-mercaptoethanol, 100 ng / mL SCF, 100 ng / mL IL3, and 100 ng / mL Flt3L.

[0052] In some embodiments, the first basal medium is selected from at least one of X-VIVO15 basal medium and X-VIVO20.

[0053] In some embodiments, the first basal medium is X-VIVO15 basal medium.

[0054] In some embodiments, the first medium is X-VIVO15 basal medium supplemented with 10 ng / mL Activin A, 10 ng / mL VEGF-A, 10 ng / mL BMP4, 10 ng / mL FGF2, 4 μM SB-431542, 3 μM CHIR-99021, and 10 μM Y-27632.

[0055] In some embodiments, the second basal medium is selected from at least one of X-VIVO15 basal medium and X-VIVO20.

[0056] In some embodiments, the second basal medium is X-VIVO15 basal medium.

[0057] In some embodiments, the second medium is X-VIVO15 basal medium supplemented with 10 ng / mL VEGF-A, 10 ng / mL BMP4, and 10 ng / mL FGF2.

[0058] In some embodiments, the third basal medium is selected from at least one of IMDM basal medium and RPMI-1640.

[0059] In some embodiments, the third basal medium is IMDM basal medium.

[0060] In some embodiments, the third medium is IMDM basal medium supplemented with 20 vol% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 2 mM L-glutamine, 100 mM β-mercaptoethanol, 100 ng / mL SCF, 100 ng / mL IL3, and 100 ng / mL Flt3L.

[0061] The second aspect of the present invention provides a kit, which contains the medium combination of the first aspect.

[0062] In some embodiments, it further contains trophoblast cells.

[0063] The third aspect of the present invention provides the use of the medium combination of the first aspect or the kit of the second aspect:

[0064] (1) To prepare iAGM hematopoietic organoids;

[0065] (2) To prepare products for inducing the differentiation of human pluripotent stem cells into iAGM hematopoietic organoids.

[0066] The fourth aspect of the present invention provides a method for preparing iAGM hematopoietic organoids, which obtains iAGM hematopoietic organoids by culturing human pluripotent stem cells with the kit of the second aspect; the culture is suspension culture.

[0067] The fifth aspect of the present invention provides the use of the iAGM hematopoietic organoids prepared by the method of the fourth aspect in any one of the following:

[0068] 1) For constructing humanized blood and / or immune system animals;

[0069] 2) Products for preparing animals with humanized blood and / or immune systems;

[0070] 3) Simulating human blood and / or immune system diseases;

[0071] 4) Products for preparing products that simulate human blood and / or immune system diseases;

[0072] 5) Preparing human blood cells including red blood cells and immune cells;

[0073] 6) Products for preparing human blood cells;

[0074] 7) Products for preparing products that induce transplantation tolerance;

[0075] 8) Products for preparing products that restore hematopoietic, blood, and immune functions;

[0076] 9) Generating humanized antibodies;

[0077] 10) Products for preparing products that generate humanized antibodies;

[0078] 11) Drug screening, research and development, and / or toxicity analysis related to hematopoiesis, blood, and immunity;

[0079] 12) Studying the pathogenic mechanisms of hematopoietic, blood, and immune diseases;

[0080] 13) Preparing products for treating hematopoietic, blood, and immune diseases;

[0081] 14) Constructing models of hematopoietic, blood, and immune diseases.

[0082] The beneficial effects of the present invention are as follows:

[0083] The present invention provides a medium combination for inducing the differentiation of human pluripotent stem cells into iAGM hematopoietic organoids. The differentiation of human embryonic stem cells into iAGM hematopoietic organoids is achieved through the combination of different cytokines in different stages. The generated hematopoietic stem cells have high primitiveness and authenticity, thus ultimately ensuring their functionality.

[0084] The present invention provides a method for preparing iAGM hematopoietic organoids. This method simulates the 3D microenvironment of the generation and development of hematopoietic stem cells in the human body under 3D suspension culture conditions through the combination of different cytokines in different stages, and realizes the 3D differentiation growth of human pluripotent stem cell-derived hematopoietic stem cells. In the iAGM hematopoietic organoids of the present invention, the hematopoietic niche microenvironment supports the generation of hematopoietic stem cells, ensuring the primitiveness and authenticity of hematopoietic stem cells. Description of the Drawings

[0085] Figure 1 It is a roadmap for the directional induction and differentiation of human embryonic stem cells into hematopoietic organoids.

[0086] Figure 2 It is a picture of cell spheres during the directed induction and differentiation of human embryonic stem cells into hematopoietic organoids.

[0087] Figure 3 It is the result diagram of the directed induction and differentiation of human embryonic stem cells into mesoderm cell spheres: Among them, A is the flow cytometry detection diagram of Epcam - CD56 + mesoderm progenitor cells; B is the result of KDR immunofluorescence staining of mesoderm cell spheres (magnification, 20×, scale bar 40μm, upper; 40×, scale bar 50μm, lower); D is the result diagram of the expression levels of BMP4, Wnt5a, KDR, APLNR, CXCR4 and CDX4 in mesoderm cell spheres.

[0088] Figure 4 It is the result diagram of the directed induction and differentiation of mesoderm cell spheres into hematopoietic endothelial cells: Among them, A is the fluorescence picture of the CD31-positive reticular structure in hematopoietic endothelial cell spheres on the 7th day of differentiation (overlay of bright field and CD31-EGFP fluorescence, scale bar 40μm); B is the result of CD31 and CD34 immunofluorescence staining of hematopoietic endothelial cell spheres on the 7th day of differentiation (magnification, 20×, scale bar 40μm, upper; 40×, scale bar 50μm, lower); C is the result diagram of the expression levels of DLL4, SOX7, NRP2, Notch1, EPHB4 and NR2F2 in hematopoietic endothelial cell spheres on the 7th day of differentiation; D is the CD31 + CD34 + and CD144 + CD34 + flow cytometry detection diagram of hematopoietic endothelial cells; E is the statistical analysis result of the above D diagram, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0089] Figure 5Results of the differentiation of hematopoietic endothelial cell spheres into AGM hematopoietic organoids: Among them, A shows the results of immunofluorescence staining of CD45, RUNX1, and VE-Cadherin in iAGM hematopoietic organoids on the 11th day of differentiation (magnification, 20×, scale bar 40 μm, top; 40×, scale bar 50 μm, bottom); B shows the results of immunofluorescence staining of CD45, RUNX1, and VE-Cadherin in natural CS16 (aborted embryo at 37 days of gestation) AGM samples (magnification, 20×, scale bar 40 μm, top; 40×, scale bar 50 μm, bottom); C shows the results of the expression levels of ANGPT1, ICAM1, RUNX2, and GATA1 in iAGM hematopoietic organoids on the 11th day of differentiation; D shows the results of + CD45 + and CD43 + CD45 + Flow cytometry detection of hematopoietic cells; E shows the statistical analysis results of the above Figure D, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0090] Figure 6 Results of the hematopoietic potential of iAGM hematopoietic organoids shown in vitro: Among them, A shows the determination of the Methocult colony formation assay of single cells collected from iAGM hematopoietic organoids; B shows the results of the counting and statistical analysis of the colonies (including CFU-GEMM, CFU-E, and CFU-G / M) in the above A; C shows the detection of the potential of iAGM hematopoietic organoids and cord blood CD34 + cells to be induced to produce CD45 + cells in vitro; D shows the detection of the potential of iAGM hematopoietic organoids and cord blood CD34 + cells to be induced to produce T cells in vitro. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0091] Figure 7 Results of verifying the in vivo multi-lineage potential of iAGM hematopoietic organoids: Among them, A shows the detection of human CD45 + cells in the bone marrow of mice transplanted with CB-CD34 + cells and iAGM hematopoietic organoids 4 weeks after transplantation; B shows the statistical analysis diagram of the above A results; C shows the myeloid and lymphoid reconstitution of CB-CD34 + cells and iAGM hematopoietic organoid cells in the bone marrow of transplanted mice 4 weeks after transplantation. Further analysis of myeloid cells (CD11b + ) and granulocytic cells (CD16 + ) in CD45+ ) B cells (CD19 + ) NK cells (CD56 + ) and T cells (CD3 + ) ratios. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0092] Figure 8 This is a result graph showing the similarity of hematopoietic lineages differentiated in the bone marrow of mice transplanted with iAGM hematopoietic organoids and natural AGM samples 4 weeks after transplantation.

[0093] Figure 9 This is a result graph showing the similarity of hematopoietic lineages differentiated in the spleen of mice transplanted with iAGM hematopoietic organoids and natural AGM samples 8 weeks after transplantation.

[0094] Figure 10 This is a result graph of the similarity analysis between iAGM hematopoietic organoids and natural AGM samples: Among them, A intuitively shows the similarity between hematopoietic endothelial cell spheres on the 7th day of differentiation, iAGM hematopoietic organoids on the 11th day of differentiation, and cell clusters in the natural AGM sample area by PCA principal component analysis; B is a Umap graph integrating cells of iAGM hematopoietic organoids and cells in the CS16 natural AGM area. C is a Umap graph that respectively shows the clustering of all cells of iAGM hematopoietic organoid cells (right) and CS16 natural AGM area cells (left), and the name of each cell cluster is determined according to the expression of cell-specific markers; D is the expression of specific genes of iAGM hematopoietic organoid cells and CS16 natural AGM area cells in all cell clusters; E is the GO functional enrichment analysis of similar expressed genes of iAGM hematopoietic organoid cells and CS16 natural AGM area cells in each cell cluster. Detailed implementation mode

[0095] In the first aspect of the present invention, a culture medium combination is provided. The culture medium combination includes a first culture medium, a second culture medium, and a third culture medium; the first culture medium includes a first basal medium supplemented with Activin A, vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), TGF-β signaling pathway inhibitor, GSK-3 inhibitor, and Rock inhibitor; the second culture medium includes a second basal medium supplemented with vascular endothelial growth factor, bone morphogenetic protein, and fibroblast growth factor; the third culture medium includes a third basal medium supplemented with hematopoietic growth factor.

[0096] In the present invention, the first basal medium and the second basal medium refer to commercial media that can be used for stem cell culture. Among them, the "first basal medium supplemented with Activin A, vascular endothelial growth factor, bone morphogenetic protein, fibroblast growth factor, TGF-β signaling pathway inhibitor, GSK-3 inhibitor, and Rock inhibitor" is obtained by additionally adding Activin A, vascular endothelial growth factor, bone morphogenetic protein, fibroblast growth factor, TGF-β signaling pathway inhibitor, GSK-3 inhibitor, and Rock inhibitor to a commercial medium that can be used for stem cell culture. The "second basal medium supplemented with vascular endothelial growth factor, bone morphogenetic protein, and fibroblast growth factor" is obtained by additionally adding vascular endothelial growth factor, bone morphogenetic protein, and fibroblast growth factor to a commercial medium that can be used for stem cell culture. In the present invention, the third basal medium refers to a commercial medium that can be used for endothelial cell culture. The "third basal medium supplemented with fetal bovine serum, fungal antibiotic, glutamine, β-mercaptoethanol, and hematopoietic growth factor" is obtained by additionally adding hematopoietic growth factor to a commercial medium that can be used for endothelial cell culture.

[0097] The medium combination provided in the present invention can be used to induce the differentiation of human pluripotent stem cells into induced AGM (iAGM) hematopoietic organoids.

[0098] "Pluripotent stem cells" refer to stem cells that have the pluripotency to differentiate into all cells existing in an organism, namely the three germ layers of endoderm, mesoderm, and ectoderm, and also have the ability to proliferate. There is no particular limitation on the above pluripotent stem cells, and examples thereof include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, and multipotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. The pluripotent stem cells are preferably at least one selected from the group consisting of ES cells, ntES cells, and human iPS cells. In one aspect of the embodiment of the present invention, the pluripotent stem cells are preferably iPS cells or ES cells.

[0099] The human embryonic stem cells in the present invention are pluripotent stem cells derived from early embryos. Mouse embryonic stem cells were initially established in 1981, and human embryonic stem cells were established in 1998 and are now widely used in regenerative medicine.

[0100] In some embodiments, the vascular endothelial growth factor 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.

[0101] In some embodiments, the vascular endothelial growth factor is VEGF-A.

[0102] In some embodiments, the bone morphogenetic protein is selected from at least one of BMP4 and BMP2.

[0103] In some embodiments, the bone morphogenetic protein is BMP4.

[0104] In some embodiments, the fibroblast growth factor 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.

[0105] In some embodiments, the fibroblast growth factor is FGF2.

[0106] In some embodiments, the TGF-β signaling pathway inhibitor 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), A-83-01 (CAS No.: 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).

[0107] In some embodiments, the TGF-β signaling pathway inhibitor is SB-431542.

[0108] In some embodiments, the GSK-3 inhibitor is selected from at least one of 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), CHIR-99021 (CAS No.: 252917-06-9).

[0109] In some embodiments, the GSK-3 inhibitor is CHIR-99021.

[0110] In some embodiments, the Rock inhibitor 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).

[0111] In some embodiments, the Rock inhibitor is Y-27632.

[0112] In some embodiments, the hematopoietic growth factor is selected from at least one of stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and FLT3-ligand (Flt3L).

[0113] In some embodiments, the hematopoietic growth factor is stem cell factor, interleukin-3, and FLT3-ligand.

[0114] In some embodiments, the first culture medium comprises a first basal medium supplemented with Activin A, VEGF-A, BMP4, FGF2, SB-431542, CHIR-99021, and Y-27632.

[0115] In some embodiments, the addition concentration of Activin A in the first basal medium is 5 - 15 ng / mL. Here, the addition concentration = the mass of added Activin A / the volume of the first basal medium.

[0116] In some embodiments, the addition concentration of VEGF-A in the first basal medium is 5 - 15 ng / mL. Here, the addition concentration = the mass of added VEGF / the volume of the first basal medium.

[0117] In some embodiments, the addition concentration of BMP4 in the first basal medium is 5 - 15 ng / mL. Here, the addition concentration = the mass of added BMP4 / the volume of the first basal medium.

[0118] In some embodiments, the addition concentration of FGF2 in the first basal medium is 5 - 15 ng / mL. Here, the addition concentration = the mass of added FGF / the volume of the first basal medium.

[0119] In some embodiments, the addition concentration of SB-431542 in the first basal medium is 2 - 6 μM. Here, the addition concentration = the amount of substance of added SB-431542 / the volume of the first basal medium.

[0120] In some embodiments, the addition concentration of CHIR-99021 in the first basal medium is 2 - 4 μM. Here, the addition concentration = the amount of substance of added CHIR-99021 / the volume of the first basal medium.

[0121] In some embodiments, the addition concentration of Y-27632 in the first basal medium is 5 - 15 μM. Here, the addition concentration = the amount of substance of added VEGF / the volume of the first basal medium.

[0122] In some embodiments, the first medium comprises the first basal medium added with 5 - 15 ng / mL Activin A, 5 - 15 ng / mL VEGF-A, 5 - 15 ng / mL BMP4, 5 - 15 ng / mL FGF2, 2 - 6 μM SB-431542, 2 - 4 μM CHIR-99021, and 5 - 15 μM Y-27632.

[0123] In some embodiments, the first medium comprises the first basal medium added with 10 ng / mL Activin A, 10 ng / mL VEGF-A, 10 ng / mL BMP4, 10 ng / mL FGF2, 4 μM SB-431542, 3 μM CHIR-99021, and 10 μM Y-27632.

[0124] In some embodiments, the second culture medium comprises a second basal medium supplemented with VEGF-A, BMP4, and FGF2.

[0125] In some embodiments, the addition concentration of VEGF in the second basal medium is 5-15 ng / mL. Here, the addition concentration = the mass of added VEGF / the volume of the second basal medium.

[0126] In some embodiments, the addition concentration of BMP4 in the second basal medium is 5-15 ng / mL. Here, the addition concentration = the mass of added BMP4 / the volume of the second basal medium.

[0127] In some embodiments, the addition concentration of FGF2 in the second basal medium is 5-15 ng / mL. Here, the addition concentration = the mass of added FGF / the volume of the second basal medium.

[0128] In some embodiments, the second culture medium comprises a second basal medium supplemented with 5-15 ng / mL VEGF-A, 5-15 ng / mL BMP4, and 5-15 ng / mL FGF2.

[0129] In some embodiments, the second culture medium comprises a second basal medium supplemented with 10 ng / mL VEGF-A, 10 ng / mL BMP4, and 10 ng / mL FGF2.

[0130] In some embodiments, the third culture medium comprises a third basal medium supplemented with SCF, IL3, and Flt3L.

[0131] In some embodiments, the addition concentration of SCF in the third basal medium is 50-150 ng / mL. Here, the addition concentration = the mass of added SCF / the volume of the third basal medium.

[0132] In some embodiments, the addition concentration of IL3 in the third basal medium is 50-150 ng / mL. Here, the addition concentration = the mass of added IL-3 / the volume of the third basal medium.

[0133] In some embodiments, the addition concentration of Flt3L in the third basal medium is 50-150 ng / mL. Here, the addition concentration = the mass of added Flt3L / the volume of the third basal medium.

[0134] In some embodiments, fetal bovine serum, fungal antibiotics, glutamine, and β-mercaptoethanol are further added to the third basal medium.

[0135] In some embodiments, the fungal antibiotics include penicillin, streptomycin, and amphotericin B.

[0136] In some embodiments, the added concentration of fetal bovine serum in the third basal medium is 10 vol% - 30 vol%. Here, the added concentration = the volume of added fetal bovine serum / the volume of the third basal medium.

[0137] In some embodiments, the added concentration of penicillin in the third basal medium is 50 - 150 U / mL, the added concentration of streptomycin is 0.05 - 0.15 mg / mL, and the added concentration of amphotericin B is 0.125 - 0.375 μg / mL.

[0138] In some embodiments, the fungal antibiotic is a penicillin - streptomycin - amphotericin B mixed solution (100× triple antibiotic), wherein the penicillin content is 10 kU / ml, the streptomycin content is 10 mg / ml, and the amphotericin B content is 25 μg / ml.

[0139] In some embodiments, the added concentration of the penicillin - streptomycin - amphotericin B mixed solution in the third basal medium is 0.5 vol% - 1.5 vol%. Here, the added concentration = the volume of added penicillin - streptomycin - amphotericin B mixed solution / the volume of the third basal medium.

[0140] In some embodiments, the added concentration of L - glutamine in the third basal medium is 1 - 3 mM. Here, the added concentration = the amount of added L - glutamine / the volume of the third basal medium.

[0141] In some embodiments, the added concentration of β - mercaptoethanol in the third basal medium is 50 - 150 mM. Here, the added concentration = the amount of added β - mercaptoethanol / the volume of the third basal medium.

[0142] In some embodiments, the third medium includes a third basal medium supplemented with 10 vol% - 30 vol% fetal bovine serum, 50 - 150 U / mL penicillin, 0.05 - 0.15 mg / mL streptomycin, 0.125 - 0.375 μg / mL amphotericin B, 1 - 3 mM L - glutamine, 50 - 150 mM β - mercaptoethanol, 50 - 150 ng / mL SCF, 50 - 150 ng / mL IL3, and 50 - 150 ng / mL Flt3L.

[0143] In some embodiments, the third culture medium comprises a third basal medium supplemented with 20 vol% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 2 mM L-glutamine, 100 mM β-mercaptoethanol, 100 ng / mL SCF, 100 ng / mL IL3, and 100 ng / mL Flt3L.

[0144] In some embodiments, the first basal medium is selected from at least one of X-VIVO15 basal medium and X-VIVO20.

[0145] In some embodiments, the first basal medium is X-VIVO15 basal medium.

[0146] In some embodiments, the first culture medium is X-VIVO15 basal medium supplemented with 10 ng / mL Activin A, 10 ng / mL VEGF-A, 10 ng / mL BMP4, 10 ng / mL FGF2, 4 μM SB-431542, 3 μM CHIR-99021, and 10 μM Y-27632.

[0147] In some embodiments, the second basal medium is selected from at least one of X-VIVO15 basal medium and X-VIVO20.

[0148] In some embodiments, the second basal medium is X-VIVO15 basal medium.

[0149] In some embodiments, the second culture medium is X-VIVO15 basal medium supplemented with 10 ng / mL VEGF-A, 10 ng / mL BMP4, and 10 ng / mL FGF2.

[0150] In some embodiments, the third basal medium is selected from at least one of IMDM basal medium and RPMI-1640.

[0151] In some embodiments, the third basal medium is IMDM basal medium.

[0152] In some embodiments, the third culture medium is IMDM basal medium supplemented with 20 vol% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B, 2 mM L-glutamine, 100 mM β-mercaptoethanol, 100 ng / mL SCF, 100 ng / mL IL3, and 100 ng / mL Flt3L.

[0153] The second aspect of the present invention provides a kit, which contains the culture medium combination of the first aspect.

[0154] In some embodiments of the present invention, the trophoblast cells include at least one of OP9 cells, MS5 cells, HS-5 or other fibroblasts.

[0155] In some embodiments, it further contains trophoblast cells.

[0156] The third aspect of the present invention provides the use of the culture medium combination of the first aspect or the kit of the second aspect:

[0157] (1) To prepare iAGM hematopoietic organoids;

[0158] (2) To prepare a product for inducing the differentiation of human pluripotent stem cells into iAGM hematopoietic organoids.

[0159] The fourth aspect of the present invention provides a method for preparing iAGM hematopoietic organoids, which uses the kit of the second aspect to culture human pluripotent stem cells to obtain iAGM hematopoietic organoids; the culture is suspension culture.

[0160] The method provided by the present invention is a three-dimensional (3D) suspension culture-directed differentiation method. This method simulates the 3D microenvironment of hematopoietic stem cell generation and development in the human body through different combinations of cytokines in different stages, and realizes the 3D differentiation and growth of human pluripotent stem cell-derived hematopoietic stem cells. In the iAGM hematopoietic organoids of the present invention, the hematopoietic niche microenvironment supports the generation of hematopoietic stem cells, ensuring the primitiveness and authenticity of hematopoietic stem cells.

[0161] In some embodiments, the method includes:

[0162] Culturing human pluripotent stem cells in the first culture medium in the first stage;

[0163] Culturing the human pluripotent stem cells after the first-stage culture in the second culture medium in the second stage;

[0164] Culturing the human pluripotent stem cells after the second-stage culture with inactivated trophoblast cells in the third culture medium in the third stage;

[0165] Culturing the human pluripotent stem cells after the third-stage culture in the third culture medium in the fourth stage.

[0166] In some embodiments, the time of the first-stage culture is 1 day.

[0167] In some embodiments, the time of the second-stage culture is 3 days.

[0168] In some embodiments, the time of the third-stage culture is 4 days.

[0169] In some embodiments, the culture time of the fourth stage is 4 - 5 days.

[0170] The fifth aspect of the present invention provides the application of the iAGM hematopoietic organoids prepared by the method of the fourth aspect in any one of the following:

[0171] 1) For constructing humanized blood and / or immune system animals;

[0172] 2) For preparing products for constructing humanized blood and / or immune system animals;

[0173] 3) Simulating human blood and / or immune system diseases;

[0174] 4) For preparing products for simulating human blood and / or immune system diseases;

[0175] 5) Preparing human blood cells including red blood cells and immune cells;

[0176] 6) For preparing products for human blood cells;

[0177] 7) For preparing products for inducing transplantation tolerance;

[0178] 8) For preparing products for restoring hematopoietic, blood and immune functions;

[0179] 9) Generating humanized antibodies;

[0180] 10) For preparing products for generating humanized antibodies;

[0181] 11) Screening, research and / or toxicity analysis of hematopoietic, blood and immune related drugs;

[0182] 12) Studying the pathogenic mechanisms of hematopoietic, blood and immune diseases;

[0183] 13) Preparing products for treating hematopoietic, blood and immune diseases;

[0184] 14) Constructing models of hematopoietic, blood and immune diseases.

[0185] The content of the present invention will be further described in detail through specific examples below. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or testing methods are conventional methods in the art.

[0186] In the examples of the present invention, "hESC" is human embryonic stem cells.

[0187] Example 1: Inducing the differentiation of human embryonic stem cells into iAGM hematopoietic organoids

[0188] The roadmap for the directed induction and differentiation of hESCs into iAGM hematopoietic organoids is as follows Figure 1 shown, including the following steps:

[0189] S1. Single-cell passage of hESCs: Select a well-conditioned hESC well (well-conditioned: when the cell coverage reaches 70% - 80% 4 - 5 days after hESC passage, and the hPSC with regular clone edges and no differentiated cells). After aspirating the culture medium (hESC medium), add 1 mL of calcium- and magnesium-free PBS to wash the cells. After aspirating, add 1 mL of gentle cell dissociation reagent (GCDR, purchased from STEMCELL), place it back in the carbon dioxide incubator and incubate for 5 minutes. Then aspirate the GCDR in the well, add 1 mL of mTeSR1 medium (purchased from STEM CELL) containing 10 μM of Y-27632 (Rock inhibitor, CAS No.: 146986-50-7), and pipette the cells until single cells drop off to finally obtain a single-cell suspension;

[0190] S2. Embryoid body differentiation stage: Aspirate 20 μL of the single-cell suspension, stain it with trypan blue, and count it using a hemocytometer; centrifuge the above single-cell suspension, aspirate the supernatant, add calcium- and magnesium-free PBS to wash the cell pellet, and centrifuge again to discard the supernatant; use a Corning low-adhesion six-well culture plate, add 3 mL of embryoid body differentiation stage medium to each well; aspirate about 1×10 6 single-cell suspension and inoculate it into the well, cross-shake the well plate to evenly suspend the cells in the culture medium, record it as day 0, and cell spheres with a diameter of 50 - 70 microns will form the next day;

[0191] S3. Hematopoietic mesoderm differentiation stage: After 24 hours (day 1), wash the cell spheres in each well with 1 mL of calcium- and magnesium-free PBS, aspirate it, and add 3 mL of hematopoietic mesoderm differentiation medium; after 48 hours (day 2), wash the cell spheres in each well with 1 mL of calcium- and magnesium-free PBS, aspirate it, and add 3 mL of hematopoietic mesoderm differentiation medium; after 72 hours (day 3), replace with 3 mL of hematopoietic mesoderm differentiation medium, and continue to culture for another 24 hours to obtain hematopoietic mesoderm cell spheres;

[0192] S4. Hematopoietic endothelium differentiation stage: When the confluence of mouse OP9 stromal cells reaches 80%, culture them with OP9 medium containing 10 μg / mL of mitomycin C for 2 hours, wash away the residual mitomycin C with PBS, digest with trypsin for 5 minutes and then terminate the digestion, collect the cell suspension, centrifuge at 1500 rpm for 5 minutes and then discard the supernatant, and resuspend and count with hematopoietic endothelium medium. Collect the mesoderm organoids on day 4 and the mouse OP9 stromal cells treated with mitomycin C (3*10 5Co-culture was carried out with (the hole). From day 4 to day 7, the hematopoietic mesoderm cell spheres (day 4) / hematopoietic endothelial cell spheres (days 4 to 7) were washed with 1 mL of calcium- and magnesium-free PBS per well per day. After aspiration, 3 mL of hematopoietic endothelial medium was added to finally obtain hematopoietic endothelial cell spheres;

[0193] S5. iAGM hematopoietic organoid differentiation stage: From day 7 to day 11, the hematopoietic endothelial cell spheres were washed with 1 mL of calcium- and magnesium-free PBS per well per day. After aspiration, 3 mL of iAGM hematopoietic organoid medium was added for suspension culture to obtain iAGM hematopoietic organoids.

[0194] The entire above differentiation process was cultured at 37 °C, 5% CO2, and 100% humidity.

[0195] Embryoid body differentiation stage medium (the first medium): X-VIVO15 basal medium containing 10 ng / mL Activin A (purchased from Peprotech), 10 ng / mL VEGF-A (purchased from Peprotech), 10 ng / mL BMP4 (purchased from Peprotech), 10 ng / mL bFGF2 (purchased from Peprotech), 4 μM SB-431542 (purchased from MedChemExpress), 3 μM CHIR-99021 (purchased from MedChemExpress), and 10 μM Y-27632 (purchased from MedChemExpress). The X-VIVO15 basal medium was purchased from Lonza.

[0196] Hematopoietic mesoderm differentiation medium (the second medium): X-VIVO15 basal medium containing 10 ng / mL VEGF-A (purchased from Peprotech), 10 ng / mL BMP4 (purchased from Peprotech), and 10 ng / mL bFGF2 (purchased from Peprotech).

[0197] Hematopoietic endothelial medium (the third medium): IMDM basal medium containing 20 vol% fetal bovine serum (purchased from ExCell Bio), 1 vol% fungal antibiotic (100× triple antibiotic) (purchased from Solarbio), 1 vol% L-glutamine (purchased from Gibco, the concentration of glutamine is 200 mM), 100 mM β-mercaptoethanol (purchased from Sigma), 100 ng / mL SCF (purchased from Peprotech), 100 ng / mL IL3 (purchased from Peprotech), and 100 ng / mL Flt3L (purchased from Peprotech). The IMDM basal medium was purchased from Sigma-Aldrich.

[0198] iAGM Hematopoietic Organoid Medium (Third Medium): IMDM basal medium containing 20 vol% fetal bovine serum (purchased from ExCell Bio), 1 vol% fungal antibiotic (purchased from Solarbio), 1 vol% L-glutamine (purchased from Gibco, with a concentration of 200 mM for glutamine), 100 mM β-mercaptoethanol (purchased from Sigma), 100 ng / mL SCF (purchased from Peprotech), 100 ng / mL IL3 (purchased from Peprotech), and 100 ng / mL Flt3L (purchased from Peprotech). The IMDM basal medium was purchased from Gibco.

[0199] hESC Medium: DMEM / F12 basal medium (purchased from Gibco) containing 20 vol% serum replacement KSR (purchased from Thermo), 1 vol% non-essential amino acids (100×, purchased from Solarbio), 1 vol% L-glutamine (purchased from Gibco, with a concentration of 200 mM for glutamine), 0.1 mM β-mercaptoethanol (purchased from Sigma), and 8 ng / mL bFGF (purchased from Peprotech).

[0200] Example 2: Characterization of Cell Sphere Structure during Culture

[0201] On the 4th, 7th, and 11th days, the cell spheres were taken, embedded in agarose, sectioned, and stained with H&E to observe the structure of the cell spheres. The specific steps were as follows: The cell spheres were slowly shaken and suspended in 10% neutral formalin for fixation for 1 hour. After standing and precipitating for 5 minutes, the supernatant was aspirated, and a little eosin was added to label the cell spheres. After heating the centrifuge tube in a 45°C water bath for 10 minutes, a little melted agar was added, and the centrifuge tube was shaken to mix the agar and the cell spheres thoroughly. After cooling, the agar solidified. The bottom of the centrifuge tube was placed in a water bath for 5 - 10 seconds, and the centrifuge tube was inverted and gently tapped to make the agar block fall out. The agar block was cut into an appropriate size, embedded in embedding paper, dehydrated with gradient ethanol, cleared with xylene, infiltrated with wax, and then placed in an embedding machine to embed into blocks. Sections with a thickness of 5 μm were cut using a microtome, spread, fished, and baked; Subsequently, H&E staining was performed. The steps were as follows: dewaxing with xylene, hydrating with gradient decreasing concentration ethanol, staining with Harris hematoxylin solution for 15 minutes, differentiating with 0.5% hydrochloric acid alcohol for 30 seconds, bluing with water washing for 20 minutes, pre-treating with 80% ethanol for 2 minutes before eosin staining, staining the cytoplasm with eosin solution for 5 seconds, bleaching with 80% ethanol for 2 minutes after eosin staining, dehydrating with gradient ethanol, clearing, dropping neutral gum for mounting, air-drying naturally in a cool and ventilated place, and scanning the sections with a digital pathology scanning system.

[0202] And by obtaining aborted embryos (CS16, 37 days of gestation) donated by volunteers from the First People's Hospital of Guangzhou, carefully dissecting the AGM structure, embedding it in paraffin, sectioning, and performing HE staining in the same manner as above. Finally, morphologically compare the iAGM hematopoietic organoids with the natural AGM structure.

[0203] The results are as Figure 2 shown: As differentiation progresses, the cell spheres gradually present a hollow structure, and in the later stage of differentiation, the vesicles are larger, and this structure is similar to the natural AGM, and both contain clustered cell mass structures inside.

[0204] Example 3: Characterization of mesoderm cell spheres

[0205] On the 1st, 2nd, and 4th days, the cell spheres were embedded in paraffin, sectioned, and HE stained. The specific steps were the same as in Example 1. Randomly sample and take n photos (n > 10). The representative images are as Figure 3 shown in A of Figure 3 A shows that the size of the cell spheres continuously increases during differentiation.

[0206] After that, collect the cell spheres on the 1st, 2nd, and 4th days respectively, let them settle naturally for 1 minute, discard the supernatant, add 1 mL of PBS to wash the mesoderm cell spheres, let them settle naturally for 1 minute, discard the supernatant, and then add 1 mL of TrypLE TM Express (Gibco). After digesting at room temperature for 10 minutes, add 1 mL of IMDM medium containing 10 vol% fetal bovine serum to terminate the digestion. After trypan blue staining, count using a hemocytometer. Take 1×10 6 cells for flow cytometry detection of PE-CD326 and PE / Cy5-CD56. The specific steps are as follows: First, treat with TrypLE TM Express (Gibco) to dissociate the cell spheres into single cells, incubate with antibodies (PE-CD326, Cat#324206, Biolegend; PE / Cy5-CD56, Cat#362516, Biolegend), and finally use a BD FACS Celesta twelve-color high-end flow cytometer to detect fluorescent positive cells, and at the same time detect hESC as a control. The results are as Figure 3 shown in A of

[0207] Collect the cell spheres on the 4th day, let them sediment naturally for 1 minute, discard the supernatant, add 1 mL of PBS to wash the cell spheres, let them sediment naturally for 1 minute, discard the supernatant, add 1 mL of 4% paraformaldehyde to fix at room temperature for 30 minutes. After discarding the supernatant respectively, add 1 mL of PBS to wash the cell spheres 3 times. After discarding the supernatant, add 1 mL of 0.5% Triton X-100 to permeabilize for 20 minutes. After discarding the supernatant respectively, add 1 mL of PBS to wash the cell spheres 3 times. After discarding the supernatant, add 200 μL of goat serum to block and incubate for 30 minutes. After discarding the supernatant, add 500 μL of diluted antibodies (KDR, Cat#MAB3571-SP, RD; Brachyury, Cat#81694S, CST), put them in a dark box and incubate overnight at 4°C; then warm up for 45 minutes, add fluorescent secondary antibody and incubate for 1 hour at 37°C in the dark. After discarding the supernatant respectively, add 1 mL of PBS to wash the cell spheres 3 times. Subsequently, stain the cell nuclei with DAPI (Beyotime) for 5 minutes. After discarding the supernatant respectively, add 1 mL of PBS to wash the cell spheres 3 times. Then dropwise add anti-fluorescence quenching agent. Finally, observe and take pictures using a Zeiss LSM900 laser confocal microscope. The results are as Figure 3 shown in B of Figure 3 : On the 4th day, most cells in the cell spheres expressed KDR and Brachyury. It shows that most cells in the spheres are mesodermal cells.

[0208] Separately, take the cell spheres on the 4th, 7th, and 11th days for RNA extraction, and then use qPCR to detect the expression levels of mesodermal cell-specific genes BMP4, Wnt5a, KDR, APLNR, CXCR4, and CDX4. The specific steps are as follows: Use a universal RNA extraction kit (TaKaRa) to extract the total RNA of the cell precipitate; after quantification on a NanoDrop microspectrophotometer (Thermo Fisher Scientific), use the High-Capacity PrimeScript TM RT cDNA Reverse Transcriptase Kit (TaKaRa) to reverse 1 μg of RNA into cDNA; use PowerUp TM SYBR TM qPCR Green Master Mix (Thermo Fisher Scientific) and primers (as shown in Table 1) to perform real-time fluorescence quantitative PCR in a Quant StudioTM 1 real-time PCR system (Thermo Fisher Scientific). At the same time, use qPCR to detect the mesodermal cell-specific genes in hESC and CS16 AGM as controls.

[0209] Table 1 Primer sequences

[0210]

[0211] The results are as Figure 3 shown in C: On the 4th day, the expression levels of mesoderm marker genes BMP4, Wnt5a, KDR, APLNR, CXCR4, and CDX4 were significantly increased. It can be seen that human pluripotent stem cells were successfully and highly differentiated into mesoderm cell spheres.

[0212] Example 4: Characterization of hematopoietic endothelial cell spheres

[0213] According to Figure 2 the representative images of the cell spheres on the 7th day in [reference], it can be seen that the cell spheres gradually changed from solid cell spheres (mesoderm cell spheres) to vesicular structures (hematopoietic endothelial cell spheres) as the number of days increased.

[0214] On the 7th day, the cell spheres were taken to detect the expression of CD34 and CD31 by immunofluorescence staining and single-photon confocal microscopy. The specific steps are as follows: The cell spheres were immersed in 4% paraformaldehyde (PFA) and incubated for 15 minutes to fix the cell spheres, and then permeabilized with 0.5% TritonX-100 (Sigma-Aldrich) for 20 minutes; between each step, the samples were washed 3 times with PBS; after washing, the samples were placed in 10% goat serum and incubated at room temperature for 30 minutes; then the treated samples were incubated overnight at 4°C in PBS containing primary antibodies (rabbit anti-CD31, 1:1000, Cat#102-PA07S, ReliaTech GmbH; mouse anti-CD34, 1:1000, Cat#ab54208, Abcam), then placed in a 37°C oven for rewarming, and incubated in PBS containing secondary antibodies (AlexaFluor488-conjugated goat anti-rabbit IgG, 1:800, Cat#4412S, CST; AlexaFluor594-conjugated goat anti-mouse IgG, 1:800, Cat#8890S, CST) at 37°C in the dark for 1 - 2 hours. Finally, DAPI (Cell Signaling Technology) was used for staining for 5 minutes, and the hematopoietic endothelial cell spheres were transferred to a confocal dish through an anti-quencher (S2100, Solarbio), and the prepared samples were photographed with a single-photon confocal microscope (Ti-EA1, Nikon).

[0215] The results are as Figure 4 shown in A and B: The hematopoietic endothelial cell spheres in the differentiation system could be stained, presenting as a reticular structure highly expressing CD31, and co-expressing the hematopoietic endothelial cell-specific markers CD31 and CD34 in local regions.

[0216] Collect cell spheres on the 4th, 7th, and 11th days respectively. Let them settle naturally for 1 minute. After discarding the supernatant, add 1 mL of PBS to wash the cell spheres, let them settle naturally for 1 minute, discard the supernatant. Take a part of the cell spheres for RNA extraction, and then detect the expression levels of DLL4, SOX7, NRP2, Notch1, EPHB4, and NR2F2 genes by qPCR. The specific steps are as follows: Use a universal RNA extraction kit (TaKaRa) to extract the total RNA of the cell precipitate; after quantification on a NanoDrop microspectrophotometer (Thermo Fisher Scientific), use the High-Capacity PrimeScript TM RT cDNA Reverse Transcriptase Kit (TaKaRa) to reverse 1 μg of RNA into cDNA; Use PowerUp TM SYBR TM qPCR Green Master Mix (Thermo Fisher Scientific) and primers (as shown in Table 2) to perform real-time fluorescence quantitative PCR in a Quant StudioTM 1 real-time PCR system (Thermo Fisher Scientific); Normalize the cycle threshold (Ct) value of each condition to the corresponding expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to generate ΔCt; The RNA level is calculated as 2-ΔΔCt. At the same time, use qPCR to detect the expression of hematopoietic endothelial-specific genes in hESC and CS16 AGM as a control.

[0217] Table 2 Primer sequences

[0218]

[0219] The results are as shown in Figure 4 C: The expression levels of hematopoietic endothelial genes such as DLL4, SOX7, NRP2, Notch1, EPHB4, and NR2F2 are relatively the highest on the 7th day. Therefore, the 7th day may be the best time point for hematopoietic endothelial cell differentiation. Therefore, it is determined to enter the hematopoietic endothelial stage on the 7th day.

[0220] Collect cell spheres on the 1st, 4th, 7th, 9th, and 11th days respectively. Let them settle naturally for 1 minute. After discarding the supernatant, add 1 mL of PBS to wash the cell spheres, let them settle naturally for 1 minute. After discarding the supernatant, add 1 mL of TrypLE TM Express (Gibco), digest at room temperature for 10 minutes, then add 1 mL of IMDM medium containing 10 vol% fetal bovine serum to terminate the digestion. After trypan blue staining, count using a hemocytometer, and take 1×10 6Flow cytometry detection of cells with PE - CD34, Alexa Fluor 700 - CD31, and APC - CD144 was performed. The specific steps are as follows: First, the cell spheres were dissociated into single cells using TrypLE TM Express (Gibco). The cells were then incubated with antibodies (PE - CD34, Cat#343606, Biolegend; Alexa Fluor 700 - CD31, Cat#303134, Biolegend; and APC - CD144, Cat#348508, Biolegend). Finally, fluorescence - positive cells were detected using a BD FACS Celesta twelve - color high - end flow cytometer, and hESCs were detected as a control.

[0221] The results are shown in Figure 4 D and E below: On day 7, the proportions of CD34 + CD31 + / CD34 + CD144 + positive cells in the cell spheres were 22.4% and 16.5% respectively. This indicates that the cell spheres on day 7 contain a certain proportion of hematopoietic endothelial cells.

[0222] Example 5: Characterization of Hematopoietic Organoids

[0223] iAGM hematopoietic organoids (11-day cell spheres) and native AGM (CS16 AGM) samples were taken to detect the expression of CD45, RUNX1, and VE-Cadherin by immunofluorescence staining and single-photon confocal microscopy. The specific steps are as follows: The iAGM hematopoietic organoids were immersed in 4% paraformaldehyde (PFA) and incubated for 15 minutes to fix the iAGM hematopoietic organoids. Then, they were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 20 minutes. Between each step, the samples were washed 3 times with PBS. After washing, the samples were placed in 10% goat serum and incubated at room temperature for 30 minutes. Then, the treated samples were incubated overnight at 4°C in PBS containing primary antibodies (rabbit anti-CD45, 1:1000, Cat#13917T, CST; mouse anti-RUNX1, 1:1000, Cat#MAB23991, RD; rabbit anti-VE-Cadherin, 1:1000, Cat#2500S, CST), then placed in a 37°C oven for rewarming, and incubated in PBS containing secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit IgG, 1:800, Cat#4412S, CST; Alexa Fluor 488-conjugated goat anti-mouse IgG, 1:800, Cat#4408S, CST; Alexa Fluor 594-conjugated goat anti-rabbit IgG, 1:800, Cat#8889S, CST; Alexa Fluor 594-conjugated goat anti-mouse IgG, 1:800, Cat#8890S, CST) in the dark at 37°C for 1-2 hours. Finally, DAPI (Cell Signaling Technology) was used for staining for 5 minutes. The iAGM hematopoietic organoids were transferred to a confocal dish through an anti-quencher (S2100, Solarbio), and the prepared samples were photographed with a single-photon confocal microscope (Ti-EA1, Nikon).

[0224] The results are as Figure 5 shown in A and B in the figure: The iAGM hematopoietic organoids in the differentiation system could be stained and were similar in structure to native AGM, highly expressing CD45, RUNX1, and VE-Cadherin.

[0225] Harvest cell spheres on days 4, 7, and 11 respectively. Let them settle naturally for 1 - 2 minutes, discard the supernatant, add 1 mL of PBS to wash the cell spheres, let them settle naturally for 1 minute, discard the supernatant, extract RNA, and then detect the expression levels of ANGPT1, ICAM1, RUNX2, and GATA1 by qPCR. The specific steps are as follows: Use a universal RNA extraction kit (TaKaRa) to extract the total RNA from the cell pellet; after quantification on a NanoDrop microspectrophotometer (Thermo Fisher Scientific), use the High-Capacity PrimeScript TM RT cDNA Reverse Transcriptase Kit (TaKaRa) to reverse 1 μg of RNA into cDNA; use PowerUp TM SYBR TM qPCR Green Master Mix (Thermo Fisher Scientific) and primers (shown in Table 3). Perform real-time fluorescence quantitative PCR in a Quant StudioTM 1 real-time PCR system (Thermo Fisher Scientific); normalize the cycle threshold (Ct) value of each condition to the corresponding expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to generate ΔCt; the RNA level is calculated as 2-ΔΔCt. At the same time, use qPCR to detect the hematopoietic gene expression in hESC and CS16 AGM as a control.

[0226] Table 3 Primer Sequences

[0227]

[0228] The results are as shown in Figure 5 C: The expression levels of hematopoietic genes such as ANGPT1, ICAM1, RUNX2, and GATA1 are relatively the highest on day 11. It can be seen that day 11 may have entered the hematopoietic generation stage.

[0229] Collect cell spheres on days 1, 4, 7, 9, and 11 respectively. Let them settle naturally for 1 minute, discard the supernatant, add 1 mL of PBS to wash the cell spheres, let them settle naturally for 1 minute, discard the supernatant, and then add 1 mL of TrypLE TM Express (Gibco). After digestion at room temperature for 10 minutes, add 1 mL of IMDM medium containing 10 vol% fetal bovine serum to terminate digestion. After trypan blue staining, count using a hemocytometer, and take 1×10 6 cells for flow cytometry detection of PE-CD34, APC / Cyanine7-CD45, and FITC-CD43. The specific steps are as follows: First, use TrypLE TMThe cell spheres were dissociated into single cells using Express (Gibco), incubated with antibodies (PE-CD34, Cat#343606, Biolegend; APC / Cyanine7-CD45, Cat#304014, Biolegend and FITC-CD43, Cat#315204, Biolegend), and finally, fluorescence-positive cells were detected using a BD FACS Celesta twelve-color high-end flow cytometer. hESC and CS16 AGM were detected simultaneously as controls.

[0230] The results are shown in Figure 5 D and E of: On day 11, the proportions of CD34 + CD45 + / CD43 + CD45 + cells in the cell spheres were 11% and 15.3% respectively. This indicates that the cell spheres on day 11 contained a certain proportion of hematopoietic stem and progenitor cells.

[0231] Example 6: In vitro functional verification of iAGM hematopoietic organoids

[0232] iAGM hematopoietic organoids (cell spheres on day 11) were collected and subjected to an in vitro colony formation assay with cord blood-derived CD34+ cells to verify their in vitro hematopoietic potential. The specific steps were as follows: The colony formation assay was performed according to the kit instructions to evaluate hematopoietic potential. iAGM hematopoietic organoids were collected, allowed to sediment naturally for 1 minute, the supernatant was discarded, and then 1 mL of PBS was added to wash the iAGM hematopoietic organoids. After allowing them to sediment naturally for 1 minute again, the supernatant was discarded, and then 1 mL of TrypLE TM Express (Gibco) was added, and after digestion at room temperature for 10 minutes, 1 mL of IMDM medium containing 10 vol% fetal bovine serum was added to terminate the digestion. After trypan blue staining, cell counting was performed using a hemocytometer. 5×10 3 single cells of iAGM hematopoietic organoids and cord blood-derived CD34+ cells were seeded into 1 mL of MethoCult H4435 (StemCell Technologies), and the additional cytokines added were 10 ng / mL Flt3L, 10 ng / mL IL6, and 50 ng / mL TPO (PeproTech). After 14 days, the hematopoietic colonies were counted and scored, the number of colony-forming cells (CFC) in the plated cell population was calculated, and the CFC cells were further collected for Wright-Giemsa staining.

[0233] The results are shown in Figure 6As shown in A and B: iAGM hematopoietic organoid single cells showed clonogenic potential in methylcellulose assays and produced different types of colonies, with a slightly lower number than cord blood-derived CD34+ cells. And in the colony formation assay, the iAGM hematopoietic organoid single cell group produced almost all types of hematopoietic colonies, including erythroid CFU, granulocyte CFU (CFU-G), macrophage CFU (CFU-M), granulocyte / macrophage CFU (CFU-GM), and granulocyte / erythroid / macrophage / megakaryocyte CFU.

[0234] To evaluate the production potential of hematopoietic progenitor cells, iAGM hematopoietic organoid single cells and cord blood-derived CD34+ cells were collected separately and induced in vitro in CD45 induction medium, because strong CD45 expression indicates the maturation of bone marrow progenitor cells. The specific steps are as follows: Flow-sort CD34 + CD45 - cells from iAGM hematopoietic organoids and induce culture in the presence of 10 vol% fetal bovine serum (FBS; Gibco), 50 ng / mL SCF (PeproTech), 50 ng / mL Flt-3L (PeproTech), 10 ng / mL IL-3 (PeproTech), and 10 ng / mL IL-6 (PeproTech), and replace half of the medium every 2 days.

[0235] The results are as Figure 6 shown in C: As differentiation proceeded, CD34 + CD45 - cells gradually differentiated into CD45+ hematopoietic cells, and by day 7, the percentage of CD45-positive cells was close to 60%, demonstrating that iAGM hematopoietic organoids have hematopoietic potential to a certain extent.

[0236] T lineage potential has been identified as a marker of definitive HSCs. Therefore, the T lineage potential of iAGM hematopoietic organoids (day 11 cell spheres) was further verified in vitro. The iAGM hematopoietic organoids will be collected, allowed to sediment naturally for 1 minute, the supernatant discarded, and then 1 mL of PBS added to wash the iAGM hematopoietic organoids, allowed to sediment naturally for 1 minute, and the supernatant discarded to collect 30 iAGM hematopoietic organoids (containing 1×10 5 CD45 + cells) and 3×10 5OP9 cells were centrifuged and aggregated to form 3D cell aggregates, which were then seeded in 0.4-mm Millicell transwell EMD inserts and placed in a six-well plate. 1 mL of T cell differentiation medium (RPMI1640 (Gibco), 4% B27 supplement (Thermo Fisher Scientific), 30 mM l-ascorbic acid 2-phosphate magnesium salt hydrate (Sigma-Aldrich), 1% penicillin / streptomycin (Thermo Fisher Scientific), stem cell factor (20 ng / mL; PeproTech), FLT3L (5 ng / mL; PeproTech), IL-7 (5 ng / mL; PeproTech), and DLL4 (10 ng / mL; PeproTech)) was added. The medium was changed every 2 - 3 days and cultured for 8 weeks. CD45 was detected by flow cytometry on specific days. + CD3 + TCRαβ + The proportion of cells.

[0237] The results were as Figure 6 shown in D: As differentiation progressed, the iAGM hematopoietic organoid group produced approximately 17.2% CD45 + CD3 + TCRαβ + T cells at week 4, slightly lower than that of the cord blood-derived CD34+ cell aggregate group (24% CD45 + CD3 + TCRαβ + T cells).

[0238] Example 7: In vivo functional verification of iAGM hematopoietic organoids

[0239] iAGM hematopoietic organoids (cell spheres on day 11) and cord blood-derived CD34 + cells were collected for transplantation experiments to verify their multi-lineage hematopoietic reconstitution ability in vivo. The specific steps were as follows: First, iAGM hematopoietic organoids were collected, allowed to settle naturally for 1 minute, the supernatant was discarded, and then 1 mL of PBS was added to wash the iAGM hematopoietic organoids. After allowing to settle naturally for 1 minute, the supernatant was discarded, and then 1 mL of TrypLE TM Express (Gibco) was added. After digestion at room temperature for 10 minutes, 1 mL of IMDM medium containing 10 vol% fetal bovine serum was added to terminate the digestion. After trypan blue staining, counting was performed using a hemocytometer. 3×10 5 single cells of iAGM hematopoietic organoids and cord blood-derived CD34 +Cells were washed and resuspended in PBS containing 2 vol% fetal bovine serum, and then transplanted into immunodeficient mice via the tail vein for multi-lineage hematopoietic reconstitution. Four weeks later, cells from bone marrow tissue (BM) were flow sorted to evaluate hematopoietic reconstitution, that is, BM cells were collected and stained with APC / Cyanine7-CD45 (Cat#304014, BioLegend), PerCP / Cyanine5.5-CD16 (Cat#302028, BioLegend), PE / Cy7-CD11b (Cat#301322, BioLegend), PE / Cy5-CD56 (Cat#362516, BioLegend), Brilliant Violet 785-CD19 (Cat#302240, BioLegend) and Brilliant Violet 510-CD3 (Cat#317332, BioLegend) antibodies. Dead cells were excluded using DAPI (Sigma) stained with the above markers. After incubating in the dark at 4 °C for 30 minutes, the cells were washed twice with PBS. Cell positive expression was analyzed by BD FACSCanto II (BD Biosciences), and the data was analyzed using FlowJo software.

[0240] The results are as Figure 7 shown in A, B, and C: In addition, 3.56% of human CD45 + cells were detected in the bone marrow of mice in the iAGM hematopoietic organoid transplantation group 4 weeks after transplantation, and all hematopoietic cell lines including the T lymphatic system were reconstituted in vivo.

[0241] Collect the bone marrow of mice in the iAGM hematopoietic organoid and cord blood-derived CD34 + cell transplantation group, and isolate CD235a + cells and CD45 +Cells were used for single-cell sequencing to compare the similarity of the differentiation lineages of implanted cells. The specific steps were as follows: The sorted cells were loaded into a Chromium Single Cell B chip (10X Genomics, PN-120262), and single-cell gel beads were generated by processing in a Chromium Single Cell controller (10X Genomics). Library construction was performed using Chromium Single Cell 30 Reagent Kits v3 (10X Genomics, PN-1000092). The Cell Ranger v3.0.2 (10x Genomics) software was used to process the iAGM hematopoietic organoid dataset to generate a feature barcode matrix. The R package Seurat v4.0 was used to read and analyze the feature barcode matrix. The steps were as follows: First, cells with a unique feature count of more than 5000 were screened according to the quality control matrix plot; then the UMI counts were normalized using the NormalizeData function with default settings; the RunUMAP function of Seurat was used to perform non-linear dimensionality reduction and clustering, and the resolution was set to 0.2.

[0242] The results showed that Figure 8 the comprehensive analysis of SEURAT indicated that there was a large overlap between the cell differentiation profiles of iAGM hematopoietic organoids and natural AGM samples, suggesting a high similarity in the differentiation lineages of the implanted cells in vivo between the two samples.

[0243] After 8 weeks, the spleens of mice in the iAGM hematopoietic organoid and cord blood-derived CD34 + cell transplantation groups were collected, and CD235a + cells and CD45 +Cells were subjected to single-cell sequencing to further compare the similarity of the differentiation lineages of implanted cells. The specific steps are as follows: The sorted cells were loaded into a Chromium Single Cell B chip (10X Genomics, PN-120262) and processed in a Chromium Single Cell controller (10X Genomics) to generate single-cell gel beads. A library was constructed using Chromium Single Cell 30 Reagent Kits v3 (10X Genomics, PN-1000092). The CellRanger v3.0.2 (10x Genomics) software was used to process the iAGM hematopoietic organoid dataset to generate a feature barcode matrix. The R package Seurat v4.0 was used to read and analyze the feature barcode matrix. The steps are as follows: First, cells with unique feature counts above 5000 were selected according to the quality control matrix plot; then the UMI counts were normalized using the NormalizeData function with default settings; the RunUMAP function of Seurat was used to perform non-linear dimensionality reduction and clustering, and the resolution was set to 0.2.

[0244] The results are as Figure 9 shown in the SEURAT comprehensive analysis. The cell differentiation profiles of iAGM hematopoietic organoids and natural AGM samples were almost identical after 8 weeks, indicating a high degree of consistency in the differentiation lineages of the implanted cells in the two samples in vivo.

[0245] Example 8: iAGM hematopoietic organoids and natural AGM have a high degree of similarity in composition and development

[0246] 1. Transcriptome sequencing and analysis

[0247] To analyze the similarity between iAGM hematopoietic organoids and natural AGM samples, transcriptome sequencing and analysis were first performed. The specific steps are as follows: First, mRNA was extracted from hESCs, hematopoietic mesoderm cell spheres (day 4 cell spheres), hematopoietic endothelial cell spheres (day 7 cell spheres), and iAGM hematopoietic organoids (day 11 cell spheres), and then whole transcriptome sequencing analysis (RNA-seq) was performed. Using Ultra TMThe RNA library preparation kit (NEB, USA) was used to generate RNA sequencing libraries, and the sequencing was performed by Novogene (China). Sequencing was carried out on an Illumina HiSeq X-Ten sequencer, and the paired-end sequencing reaction was 150 bp. Subsequent analysis was performed using R / Bioconductor software through the graphical user interface Chipster (v3.8, chipstercsc.fi). The differential expression threshold was set at log2 fold change ≥ 1 (upregulation) or ≤ 1 (downregulation), and the adjusted p-value ≤ 0.05. The R package ("gplots") was used to generate heatmaps. The heatmaps were clustered using the average-linkage hierarchical clustering method based on the Euclidean distance metric. The TBtools software and DAVID database were used to detect the statistical enrichment of differentially expressed genes in KEGG pathways and gene ontology.

[0248] The results are as Figure 10 shown in A, where hematopoietic endothelial cell spheres have higher similarity with VC + CD45 - cells in the native AGM, and iAGM hematopoietic organoids have higher similarity with VC + CD45 + and VC - CD45 + cells.

[0249] 2. Single-cell RNA-seq

[0250] To further analyze the similarities and differences between iAGM hematopoietic organoids and native AGM samples in each lineage, single-cell RNA-seq of iAGM hematopoietic organoids was performed as follows: iAGM hematopoietic organoids were digested into single-cell suspensions using TrypLE (Thermo Fischer Scientific), and then the cells were resuspended in PBS containing 0.1 vol% fetal bovine serum. DAPI staining was used to exclude dead cells. The specific procedures for library construction and sequencing were the same as those in transcriptome sequencing and analysis.

[0251] The results are as Figure 10 shown in B - D. SEURAT comprehensive analysis showed that there was a large overlap between iAGM hematopoietic organoids and native AGM samples. According to the expression patterns of marker genes, the above cells were divided into 4 clusters: "Mes", "Endo", "Epi", and "Hem". In addition, unsupervised clustering analysis confirmed the clustering of iAGM organoids and similar lineages in the CS16 AGM region, as well as the significant enrichment of related specific genes in specific cell populations.

[0252] In summary, these data confirm that iAGM hematopoietic organoids contain four different cell lines, which is similar to the microenvironment of natural AGM samples. And by performing GO functional enrichment analysis on the similarly expressed genes of iAGM hematopoietic organoid cells and CS16 natural AGM region cells in each cell cluster, the results are as Figure 10 shown in E below, and the results also confirm that the biological functions are consistent with the biological characteristics of each cluster.

[0253] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A culture medium combination, which includes a first culture medium, a second culture medium and a third culture medium; the first culture medium includes a first basal medium added with Activin A, vascular endothelial growth factor, bone morphogenetic protein, fibroblast growth factor, TGF-β signaling pathway inhibitor, GSK-3 inhibitor and Rock inhibitor; the second culture medium includes a second basal medium added with vascular endothelial growth factor, bone morphogenetic protein, fibroblast growth factor; the third culture medium includes a third basal medium added with hematopoietic growth factor.

2. The culture medium combination according to claim 1, wherein, The vascular endothelial growth factor 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; Preferably, the bone morphogenetic protein is selected from at least one of BMP4 and BMP2; Preferably, the fibroblast growth factor 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, FGF23; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of LY2157299, EW-7197, LY3200882, SB-431542, LY2109761, TP-0427736, IN-1130, R-268712, A-83-01, SB-525334, GW788388, RepSox, A-77-01, SB-505124, SD-208, LY364947; Preferably, the GSK-3 inhibitor is selected from at least one of TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR-99021; Preferably, the Rock inhibitor is selected from at least one of Y-27632, Thiazovivin, Fasudil HCl, GSK429286A, RKI-1447 and Azaindole 1; Preferably, the hematopoietic growth factor is selected from at least one of stem cell factor, thrombopoietin, interleukin-3, interleukin-6, FLT3-ligand; 3. The culture medium composition according to claim 1, wherein The first culture medium includes a first basal medium added with Activin A, VEGF-A, BMP4, FGF2, SB-431542, CHIR-99021 and Y-27632; Preferably, the addition concentration of Activin A in the first basal medium is 5-15 ng / mL; Preferably, the addition concentration of VEGF-A in the first basal medium is 5 - 15 ng / mL; Preferably, the addition concentration of BMP4 in the first basal medium is 5 - 15 ng / mL; Preferably, the addition concentration of FGF2 in the first basal medium is 5 - 15 ng / mL; Preferably, the addition concentration of SB-431542 in the first basal medium is 2 - 6 μM; Preferably, the addition concentration of CHIR-99021 in the first basal medium is 2 - 4 μM; Preferably, the addition concentration of Y-27632 in the first basal medium is 5 - 15 μM.

4. The culture medium composition according to claim 1, wherein The second medium comprises a second basal medium supplemented with VEGF-A, BMP4 and FGF2; Preferably, the addition concentration of VEGF-A in the second basal medium is 5 - 15 ng / mL; Preferably, the addition concentration of BMP4 in the second basal medium is 5 - 15 ng / mL; Preferably, the addition concentration of FGF2 in the second basal medium is 5 - 15 ng / mL.

5. The culture medium composition according to claim 1, characterized in that, The third medium comprises a third basal medium supplemented with SCF, IL3 and Flt3L; Preferably, the addition concentration of SCF in the third basal medium is 50 - 150 ng / mL; Preferably, the addition concentration of IL3 in the third basal medium is 50 - 150 ng / mL; Preferably, the addition concentration of Flt3L in the third basal medium is 50 - 150 ng / mL; Preferably, fetal bovine serum, fungal antibiotics, glutamine and β-mercaptoethanol are further added to the third basal medium; Preferably, the fungal antibiotics include penicillin, streptomycin and amphotericin B; Preferably, the addition concentration of fetal bovine serum in the third basal medium is 10 vol% - 30 vol%; Preferably, the addition concentration of penicillin in the third basal medium is 50 - 150 U / mL, the addition concentration of streptomycin is 0.05 - 0.15 mg / mL, and the addition concentration of amphotericin B is 0.125 - 0.375 μg / mL; Preferably, the addition concentration of L-glutamine in the third basal medium is 1 - 3 mM; Preferably, the addition concentration of β-mercaptoethanol in the third basal medium is 50 - 150 mM.

6. The culture medium composition according to claim 1, wherein The first basal medium is selected from at least one of X-VIVO15 basal medium and X-VIVO20; Preferably, the second basal medium is selected from at least one of X-VIVO15 basal medium and X-VIVO20; Preferably, the third basal medium is selected from at least one of IMDM basal medium and RPMI-1640.

7. A kit, characterized in that, The kit contains the medium combination according to any one of claims 1 - 6; Preferably, it further contains trophoblast cells.

8. Use of the medium combination according to any one of claims 1 - 6 or the kit according to claim 7: (1) To prepare iAGM hematopoietic organoids; (2) To prepare a product for inducing the differentiation of human pluripotent stem cells into iAGM hematopoietic organoids.

9. A method for preparing iAGM hematopoietic organoids, which obtains iAGM hematopoietic organoids by culturing human pluripotent stem cells with the kit described in claim 7; the culturing is suspension culturing.

10. Use of the iAGM hematopoietic organoids prepared by the method described in claim 9 in any one of the following: 1) For constructing humanized blood and / or immune system animals; 2) For preparing products for constructing humanized blood and / or immune system animals; 3) Simulating human blood and / or immune system diseases; 4) For preparing products for simulating human blood and / or immune system diseases; 5) Preparing human blood cells including red blood cells and immune cells; 6) For preparing products for human blood cells; 7) For preparing products for inducing transplantation tolerance; 8) For preparing products for restoring hematopoietic, blood, and immune functions; 9) Generating humanized antibodies; 10) For preparing products for generating humanized antibodies; 11) Hematopoietic, blood, and immune-related drug screening, research and development, and / or toxicity analysis; 12) Studying the pathogenic mechanisms of hematopoietic, blood, and immune diseases; 13) Preparing products for treating hematopoietic, blood, and immune diseases; 14) Constructing hematopoietic, blood, and immune disease models.

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