A medium composition and method for inducing differentiation of pluripotent stem cells into hematopoietic stem cells

Abstract

This invention relates to the field of hematopoietic stem cell technology, specifically to a culture medium combination and method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells. Addressing the problems of low efficiency and low yield associated with existing methods that require digestion and sorting for pluripotent stem cell differentiation into hematopoietic stem cells, resulting in only one harvest after induction, this invention provides a culture medium combination and method that enables "one-time induction, multiple harvests." This invention adds CHIR99021 to culture medium I and SB431542 to culture medium III, combined with other culture medium components and dynamic culture, to achieve highly efficient mesoderm induction. Combined with periodic replacement of culture medium V, digestion and sorting are unnecessary, allowing for more than three periodic harvests of high-purity (CD34) stem cells in the same container from a single induction. + CD45 + >95% hematopoietic stem cells. This invention simplifies the operation steps, eliminating the need for re-seeding, digestion, or sorting and enrichment, significantly increasing the yield per batch and making it suitable for industrial production; the entire process is free of foreign substances, meets GMP standards, and the resulting cells meet clinical application requirements.

Description

Technical Field

[0001] This invention relates to the field of hematopoietic stem cell technology, specifically to a culture medium combination and method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells. Background Technology

[0002] Hematopoietic stem cells (HSCs) are a rare type of adult stem cell with the dual potential for self-renewal and multi-lineage differentiation. After transplantation into the body, they can support lifelong hematopoiesis and reconstitution of the blood system. They are considered a fundamental treatment for hematological malignancies and other hematological diseases.

[0003] Hematopoietic stem cell transplantation, also known as bone marrow transplantation, involves collecting hematopoietic stem cells (HSCs) and progenitor cells from a donor, purifying them, and then infusing them back into the patient. The cells home to the bone marrow stem cell niche and proliferate and differentiate into all blood cell lineages. Currently, there are three sources of HSCs available for transplantation: bone marrow, mobilized peripheral blood, and umbilical cord blood, but these are limited. Adult HSC transplantation therapy typically involves >1.5 × 10⁻⁶ HSCs. 5 CD34 + The infusion rate is typically 1 cells / kg, but a single donor sample often fails to reach this dosage threshold, usually requiring dual umbilical cord blood transplantation or multi-donor mixed infusion. Furthermore, the low success rate of HLA-matched donor typing further exacerbates the donor shortage. Although researchers have developed various in vitro expansion methods, such as co-culturing HSCs with bone marrow stromal cells, adding cytokines and small molecule compounds to the culture medium, and conducting three-dimensional culture in hydrogels, the expansion effects and clinical translation of these methods remain limited. Therefore, inducing pluripotent stem cells to differentiate into true HSCs that "reconstruct the entire blood system in the transplant recipient" has become a research hotspot in bone marrow transplantation therapy.

[0004] In 1991, researchers used embryonic stem cells to form embryoid bodies (EBs), and for the first time induced blood cells in vitro in a semi-solid culture medium containing 10% fetal bovine serum. Over the past 35 years, researchers have developed various protocols for inducing the differentiation of pluripotent stem cells into hematopoietic stem cells, primarily using two-dimensional systems, three-dimensional systems, and in vivo teratoma models. With technological advancements, current protocols have eliminated stromal cells, fetal bovine serum, and viral vectors, better meeting clinical translational needs and improving safety and controllability. The two-dimensional induction system uses a monolayer culture mode, while the three-dimensional induction system uses EBs. By sequentially adding specific cytokines and small molecule compounds, the hematopoietic lineage is directionally differentiated to obtain induced HSCs, referred to as iHSCs. However, existing two-dimensional induction systems cannot simulate the three-dimensional topological characteristics of HSCs' niches in vivo, resulting in iHSCs that are immature and underfunctional. The experimental conditions of three-dimensional culture systems are harsh (requiring a hypoxic environment), with significant batch-to-batch differences, and the operation process is relatively complex (requiring digestion and sorting of EBs). This requires researchers to have extensive experimental experience. More importantly, most current methods can only collect iHSCs once, making them extremely inefficient.

[0005] Against this backdrop, the key to overcoming the bottleneck in iHSC acquisition lies in developing a targeted differentiation scheme that is simple, less demanding, and highly stable, thereby achieving a direct and sustainable harvest of purified iHSCs. Summary of the Invention

[0006] The technical problem this invention aims to solve is as follows: Regarding existing technologies for differentiating pluripotent stem cells into hematopoietic stem cells (iHSCs), mainstream methods (such as cytokine-driven two-dimensional and three-dimensional cultures) are generally limited by stringent experimental conditions and complex operational procedures. Because digestion and sorting steps must be introduced during the process, not only are cell yield and survival rates reduced, but more importantly, the "one-time induction, one-time harvest" model results in a lack of necessary stability and reproducibility in induction efficiency.

[0007] The technical solution of this invention to solve the above-mentioned technical problems is as follows: a culture medium combination and method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells. The culture medium combination includes: culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V, wherein culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V are added in the first, second, third, fourth, and fifth stages of differentiation, respectively.

[0008] (1) The culture medium I includes basal culture medium A; and selective ATP-competitive Rho-related protein kinase inhibitors and glycogen synthase kinase 3 inhibitors;

[0009] (2) The culture medium II includes basal culture medium B; and bone morphogenetic protein 4 (BMP4), angiogenesis factor (VEGF), and fibroblast growth factor 2 (FGF2);

[0010] (3) The culture medium III includes basal culture medium C; and BMP4, VEGF, FGF2, and transforming growth factor signal transduction inhibitors;

[0011] (4) The culture medium IV includes basal culture medium D; and VEGF, FGF2, and stem cell factor SCF;

[0012] (5) The culture medium V includes basal culture medium E; and VEGF, FGF2, SCF, thrombopoietin TPO, and Fms-associated tyrosine kinase 3 ligand FLT3L;

[0013] The basal culture medium A is mTeSR™1 complete culture medium;

[0014] The basal culture media B, C, D, and E are StemPro™-34 SFM complete culture media.

[0015] According to an embodiment of the present invention, the Rho-related protein kinase inhibitor includes Y27632.

[0016] According to an embodiment of the present invention, the glycogen synthase kinase 3 inhibitor includes CHIR99021.

[0017] According to an embodiment of the present invention, the transforming growth factor signaling inhibitor includes SB431542.

[0018] According to an embodiment of the present invention, the culture medium I further comprises penicillin G sodium salt and streptomycin sulfate.

[0019] According to an embodiment of the present invention, culture media II, III, IV, and V further comprise penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1-thioglycerol, B27, and ascorbic acid.

[0020] According to an embodiment of the present invention, the concentration of Y27632 used is 5-10 μM.

[0021] According to an embodiment of the present invention, the concentration of CHIR99021 used is 5-15 μM.

[0022] According to an embodiment of the present invention, the concentration of BMP4 used is 50-100 ng / mL.

[0023] According to an embodiment of the present invention, the concentration of VEGF used is 25-75 ng / mL.

[0024] According to an embodiment of the present invention, the concentration of FGF2 used is 25-75 ng / mL.

[0025] According to an embodiment of the present invention, the concentration of SB431542 used is 6-10 μM.

[0026] According to an embodiment of the present invention, the concentration of SCF used is 25-75 ng / mL.

[0027] According to an embodiment of the present invention, the concentration of TPO used is 15-45 ng / mL.

[0028] According to an embodiment of the present invention, the concentration of FLT3L used is 5-15 ng / mL.

[0029] According to an embodiment of the present invention, the concentration of penicillin G sodium salt is 100 U / mL, the concentration of streptomycin sulfate is 0.1 mg / mL, the concentration of L-alanyl-L-glutamine is 1%, the concentration of insulin-transferrin-selenoethanolamine additive is 1%, the concentration of 1-thioglycerol is 400 μM, the concentration of B27 is 2%, and the concentration of ascorbic acid is 10 μg / mL.

[0030] In the aforementioned culture medium combination, the primary function of medium I is to maintain stem cell survival, activate Wnt signaling, and induce cell differentiation into the lateral mesoderm, thereby mimicking the initiation of hematopoietic development. The primary function of medium II is to further enhance lateral mesoderm differentiation and induce its directed specialization into endothelial precursor cells. The primary function of medium III is to induce sustained HOXA gene expression, further enhancing directed hematopoietic differentiation and inhibiting primitive hematopoiesis. The primary function of medium IV is to induce the acquired hematopoietic potential of already oriented endothelial precursor cells, promoting their specialization into hematopoietic endothelial cells, thereby initiating the endothelial-hematopoietic transition process. The primary function of medium V is to promote the efficient release of iHSCs generated from hematopoietic endothelial cells into the culture medium and maintain their stem state, thereby achieving continuous accumulation of iHSCs.

[0031] In a second aspect, the present invention provides the application of the above-mentioned culture medium combination for inducing pluripotent stem cells to differentiate into hematopoietic stem cells in the preparation of hematopoietic stem cells.

[0032] In a third aspect, the present invention provides a method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, comprising the following steps:

[0033] Human induced pluripotent stem cells were sequentially induced into protostoma, lateral mesoderm, and hematopoietic endothelial cells using the above-mentioned culture medium combination. Then, endothelial-hematopoietic transformation was carried out to obtain hematopoietic stem cells.

[0034] Specifically, the method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to the present invention includes the following steps:

[0035] a. Inoculate human induced pluripotent stem cells into culture medium I and incubate statically for 1.0-1.5 days to obtain embryoid body A. Then transfer the culture plate to a shaker at 80-120 rpm and culture dynamically for 2.0-3.0 days to obtain embryoid body B.

[0036] b. Transfer the embryoid B from step a to culture medium II and culture it dynamically on a shaker at 80-120 rpm for 1.0-1.5 days to obtain embryoid C;

[0037] c. Transfer the embryoid C from step b to culture medium III and culture it dynamically on a shaker at 80-120 rpm for 4.0-5.0 days to obtain embryoid D;

[0038] d. Transfer the embryoid body D from step c to culture medium IV and culture it dynamically on a shaker at 80-120 rpm for 3.0-3.5 days to obtain embryoid body E;

[0039] e. Transfer the embryoid body E from step d to culture medium V to obtain embryoid body F, and continuously culture it dynamically on a shaker at 80-120 rpm.

[0040] The culture medium combination includes: culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V, wherein culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V are added in the first, second, third, fourth, and fifth stages of differentiation, respectively;

[0041] (1) The culture medium I includes basal culture medium A; and selective ATP-competitive Rho-related protein kinase inhibitors and glycogen synthase kinase 3 inhibitors;

[0042] (2) The culture medium II includes basal culture medium B; and bone morphogenetic protein 4 (BMP4), angiogenesis factor (VEGF), and fibroblast growth factor 2 (FGF2);

[0043] (3) The culture medium III includes basal culture medium C; and BMP4, VEGF, FGF2, and transforming growth factor signal transduction inhibitors;

[0044] (4) The culture medium IV includes basal culture medium D; and VEGF, FGF2, and stem cell factor SCF;

[0045] (5) The culture medium V includes basal culture medium E; and VEGF, FGF2, SCF, thrombopoietin TPO, and Fms-associated tyrosine kinase 3 ligand FLT3L;

[0046] The basal culture medium A is mTeSR™1 complete culture medium;

[0047] The basal culture media B, C, D, and E are StemPro™-34 SFM complete culture media.

[0048] According to an embodiment of the present invention, the Rho-related protein kinase inhibitor includes Y27632.

[0049] According to an embodiment of the present invention, the glycogen synthase kinase 3 inhibitor includes CHIR99021.

[0050] According to an embodiment of the present invention, the transforming growth factor signaling inhibitor includes SB431542.

[0051] According to an embodiment of the present invention, the culture medium I further comprises penicillin G sodium salt and streptomycin sulfate.

[0052] According to an embodiment of the present invention, culture media II, III, IV, and V further comprise penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1-thioglycerol, B27, and ascorbic acid.

[0053] According to an embodiment of the present invention, the concentration of Y27632 used is 5-10 μM.

[0054] According to an embodiment of the present invention, the concentration of CHIR99021 used is 5-15 μM.

[0055] According to an embodiment of the present invention, the concentration of BMP4 used is 50-100 ng / mL.

[0056] According to an embodiment of the present invention, the concentration of VEGF used is 25-75 ng / mL.

[0057] According to an embodiment of the present invention, the concentration of FGF2 used is 25-75 ng / mL.

[0058] According to an embodiment of the present invention, the concentration of SB431542 used is 6-10 μM.

[0059] According to an embodiment of the present invention, the concentration of SCF used is 25-75 ng / mL.

[0060] According to an embodiment of the present invention, the concentration of TPO used is 15-45 ng / mL.

[0061] According to an embodiment of the present invention, the concentration of FLT3L used is 5-15 ng / mL.

[0062] According to an embodiment of the present invention, the concentration of penicillin G sodium salt is 100 U / mL, the concentration of streptomycin sulfate is 0.1 mg / mL, the concentration of L-alanyl-L-glutamine is 1%, the concentration of insulin-transferrin-selenoethanolamine additive is 1%, the concentration of 1-thioglycerol is 400 μM, the concentration of B27 is 2%, and the concentration of ascorbic acid is 10 μg / mL.

[0063] In the above-mentioned method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, the culture conditions in step ae are 37°C and 5% CO2.

[0064] In the above-mentioned method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, the culture plates in step ae are all ultra-low adhesion culture plates.

[0065] The induced pluripotent stem cells mentioned above were purchased from Beijing Saibei Biotechnology Co., Ltd.

[0066] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0067] 1) This invention significantly improves the differentiation efficiency of human induced pluripotent stem cells into lateral plate mesoderm and hematopoietic stem cells, with the mesoderm induction efficiency increased to over 60%, thereby improving the subsequent differentiation of hematopoietic stem cells and solving the problem of low induction efficiency of iHSCs in the prior art;

[0068] 2) This invention uses a normal oxygen environment for cultivation, eliminating the need for hypoxia equipment, thus reducing the requirements for cultivation equipment and overcoming the dependence of existing three-dimensional cultivation systems on hypoxia environments;

[0069] 3) This invention only requires changing the culture medium throughout the entire process; CD34 is collected directly from the culture supernatant. + CD45 + iHSCs eliminate the need for cell reseeding, digestion, sorting and enrichment, enabling non-destructive collection. The operation steps are significantly simplified, reducing the experience requirements for experimental personnel.

[0070] 4) This invention enables at least three consecutive harvests of CD34 with a purity higher than 95% within a single induction cycle. + CD45 + iHSCs break through the limitations of the traditional "one-time induction, one-time harvest" culture model and improve cell harvesting efficiency;

[0071] 5) The CD34 obtained in this invention + CD45 + iHSCs possess in vitro expansion capabilities and multi-lineage differentiation potential, and their functionality is superior to that of iHSCs in the current nascent stage.

[0072] 6) This invention employs three-dimensional suspension dynamic culture, which is suitable for closed culture systems, has controllable contamination risks, facilitates large-scale scaling, and lays the foundation for clinical applications;

[0073] 7) The culture medium of the present invention is free of xenogeneic animal components, has clearly defined components, and contains few types of factors, which provides possibilities for future clinical translation in terms of biosafety and therapeutic efficacy;

[0074] 8) This invention optimizes the entire process of "induction-collection-amplification" in the large-scale preparation of iHSCs, provides a controllable in vitro model for studying the hematopoietic development mechanism in the human embryonic period, provides an unlimited supply of hematopoietic stem cells for cell therapy and drug screening, and effectively alleviates the problem of donor shortage in hematopoietic stem cell transplantation. Attached Figure Description

[0075] Figure 1 This study examines the morphological changes at various stages of human induced pluripotent stem cell differentiation into hematopoietic stem cells.

[0076] Figure 2 This represents the results of mesodermal cell differentiation efficiency assay. A represents the KDR of the control and this assay. + The proportion of mesodermal cells induced; B represents the mRNA expression of specific marker genes in EBs on day 5 of stage 3 of this protocol, as analyzed by RT-qPCR.

[0077] Figure 3 The results are from flow cytometry analysis of hematopoietic stem cell differentiation efficiency.

[0078] Figure 4 This refers to the in vitro proliferation of hematopoietic stem cells. A represents CD34 cells expanded in vitro within 15 days. + CD201 + A representative flow cytometry plot of iHSCs percentage; B and C are CD34 values ​​for the corresponding time periods. + iHSC and CD34 + CD201 + Statistical results of the proportion of iHSCs; D represents CD34 induced within 15 days and derived from umbilical cord blood. + Amplification of HSCs.

[0079] Figure 5 This image shows the results of in vitro differentiation and colony formation of hematopoietic stem cells. In this image, A shows representative images of various colonies; B shows statistical data for each type of colony.

[0080] Figure 6 The results of scRNA-Seq for hematopoietic stem cells are shown. A represents the visualization of UMAP dimensionality reduction and clustering results for 13,884 cells after quality control screening; B shows the expression levels of representative marker genes in each subpopulation of suspended cells; C shows the proportion analysis of each subpopulation of suspended cells; and D shows the expression of Ery / Mega, HSC / MPP, Mye / Lyn, and characteristic genes of human HSCs in suspended cells. Detailed Implementation

[0081] This invention provides a culture medium combination for inducing pluripotent stem cells to differentiate into hematopoietic stem cells. The culture medium combination includes: culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V; culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V are added at the first, second, third, fourth, and fifth stages of differentiation, respectively.

[0082] (1) The culture medium I includes basal culture medium A; and selective ATP-competitive Rho-related protein kinase inhibitors and glycogen synthase kinase 3 inhibitors;

[0083] (2) The culture medium II includes basal culture medium B; and bone morphogenetic protein 4 (BMP4), angiogenesis factor (VEGF), and fibroblast growth factor 2 (FGF2);

[0084] (3) The culture medium III includes basal culture medium C; and BMP4, VEGF, FGF2, and transforming growth factor signal transduction inhibitors;

[0085] (4) The culture medium IV includes basal culture medium D; and VEGF, FGF2, and stem cell factor SCF;

[0086] (5) The culture medium V includes basal culture medium E; and VEGF, FGF2, SCF, thrombopoietin TPO, and Fms-associated tyrosine kinase 3 ligand FLT3L.

[0087] According to an embodiment of the present invention, the basal culture medium A is mTeSR™1 complete culture medium.

[0088] According to an embodiment of the present invention, the basal culture medium B, basal culture medium C, basal culture medium D, and basal culture medium E are StemPro™-34 SFM complete culture medium.

[0089] According to an embodiment of the present invention, the Rho-related protein kinase inhibitor includes Y27632.

[0090] According to an embodiment of the present invention, the glycogen synthase kinase 3 inhibitor includes CHIR99021.

[0091] According to an embodiment of the present invention, the transforming growth factor signaling inhibitor includes SB431542.

[0092] According to an embodiment of the present invention, the culture medium I further comprises penicillin G sodium salt and streptomycin sulfate.

[0093] According to an embodiment of the present invention, culture media II, III, IV, and V further comprise penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1-thioglycerol, B27, and ascorbic acid.

[0094] According to an embodiment of the present invention, the concentration of Y27632 used is 5-10 μM.

[0095] According to an embodiment of the present invention, the concentration of CHIR99021 used is 5-15 μM.

[0096] According to an embodiment of the present invention, the concentration of BMP4 used is 50-100 ng / mL.

[0097] According to an embodiment of the present invention, the concentration of VEGF used is 25-75 ng / mL.

[0098] According to an embodiment of the present invention, the concentration of FGF2 used is 25-75 ng / mL.

[0099] According to an embodiment of the present invention, the concentration of SB431542 used is 6-10 μM.

[0100] According to an embodiment of the present invention, the concentration of SCF used is 25-75 ng / mL.

[0101] According to an embodiment of the present invention, the concentration of TPO used is 15-45 ng / mL.

[0102] According to an embodiment of the present invention, the concentration of FLT3L used is 5-15 ng / mL.

[0103] According to an embodiment of the present invention, the concentration of penicillin G sodium salt is 100 U / mL, the concentration of streptomycin sulfate is 0.1 mg / mL, the concentration of L-alanyl-L-glutamine is 1%, the concentration of insulin-transferrin-selenoethanolamine additive is 1%, the concentration of 1-thioglycerol is 400 μM, the concentration of B27 is 2%, and the concentration of ascorbic acid is 10 μg / mL.

[0104] The culture medium combination of this invention is specifically designed for the directed differentiation of pluripotent stem cells into hematopoietic stem cells. Specific culture media are used for different differentiation stages to obtain hematopoietic stem cells more easily and efficiently. Crucially, CHIR99021 is added to culture medium I, which can activate Wnt / β-catenin signaling and initiate mesoderm differentiation. SB431542 is also added to culture medium III, which can inhibit Activin / Nodal / TGF-β signaling, suppress the generation of primitive hematopoietic progenitor cells, and confine differentiation to directed hematopoiesis. By using different culture media from this invention at different differentiation stages, combined with the culture method of this invention, the mesoderm induction efficiency can be increased to over 60%, and purified iHSCs can be continuously released into the culture medium. This breaks through the limitations of the traditional "one-time induction, one-time harvest" culture model, enabling a highly efficient "one-time induction, multiple harvests" culture method.

[0105] Specifically, the key parameters of the culture medium combination and induction culture method of the present invention are shown in Table 1 below.

[0106] Table 1. Culture medium composition and methods

[0107]

[0108] This invention also provides a method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells. The entire culture process utilizes a shaker, better simulating the blood flow environment; it operates under normoxic conditions throughout, eliminating the need for a hypoxic environment, resulting in milder experimental conditions. By using different culture media at different stages, the efficiency of mesodermal induction is improved, and the induced and purified iHSCs are continuously released into the culture medium, significantly enhancing culture efficiency.

[0109] The induction method of this invention is simple to operate and allows for long-term, non-invasive acquisition of hematopoietic stem cells. Human induced pluripotent stem cells spontaneously assemble to form embryoid bodies without the involvement of stromal cells or serum. Hematopoietic stem cells are obtained by simulating the in vivo hematopoietic microenvironment through cell-cell interactions and the addition of exogenous cytokines. These hematopoietic stem cells can be continuously and automatically released into the culture medium without digestion, dissociation, sorting, or enrichment. Furthermore, the hematopoietic stem cells obtained by this method possess the ability to expand in vitro over a long period, significantly upregulating CD201 expression during expansion and achieving multi-lineage differentiation in vitro. Single-cell transcriptome sequencing confirmed the presence of RUNX1 in suspension cells. + HOXA9 + MLLT3 + MECOM + HLF + SPINK2 +The HSCs population exhibited a strong tendency to differentiate into erythrocytes, granulocytes, macrophages, and megakaryocytes, while the T cell differentiation tendency was relatively weak. This invention represents a significant advancement.

[0110] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0111] Example 1: Differentiation of human induced pluripotent stem cells into hematopoietic stem cells

[0112] (1) Human induced pluripotent stem cells of passages 30 to 50 were seeded in aggregate form into Matrigel coated 6-well plates for 1 hour and cultured in an incubator at 37°C and 5% CO2. The mTeSR™1 complete medium was changed daily until the cell confluence reached 80%.

[0113] (2) After completing step (1), discard the old culture medium, rinse with 1.0 mL / well D-PBS and discard; rinse with 0.5 mL / well Accutase and discard; add 0.5 mL / well Accutase and incubate at 37°C for 5 min; add 3 mL / well mTeSR complete culture medium to stop digestion; centrifuge at 1500 rpm for 3 min, carefully discard the supernatant, and resuspend the cell pellet in 1 mL mTeSR™1 complete culture medium. Take a small amount of cell suspension and mix it with 0.4% trypan blue staining solution at a ratio of 1:1 (v / v), and count the viable cells using a hemocytometer.

[0114] (3) After completing step (2), use 1.0×10 6 Calculate the number of cells / well, extract the required volume of iPSC single-cell suspension, centrifuge at 1500 rpm for 3 min, discard the supernatant, add culture medium I, gently resuspend the cells, and obtain a single-cell suspension. 6 Cells were seeded per 2 mL of medium I per well into an ultra-low adhesion culture plate. On day 1, the plates were statically cultured in an incubator to obtain spherical embryoids A. From day 2 onwards, the plates were transferred to a horizontal shaker at 100 rpm. On days 2 and 3, 1 mL of medium I was added to obtain embryoids B. The entire process was carried out in 5% CO2. 2、 Incubate at 37°C.

[0115] (4) After completing step (3), take the culture plate, gently rotate the culture plate to allow the embryoids B to gather in the center of the well plate, let it stand for 3 minutes, tilt the well plate, completely discard culture medium I, add culture medium II at a rate of 2 mL / well, and then add it to a 5% CO2 solution. 2、 Cultured on a shaker at 100 rpm in a 37℃ incubator for 1 day to obtain embryoid C.

[0116] (5) After completing step (4), take the culture plate, gently rotate the culture plate to allow the embryonic bodies C to gather in the center of the well plate, let it stand for 3 minutes, tilt the well plate, completely discard culture medium II, add culture medium III at a rate of 2 mL / well, add 1 mL of culture medium III to each well the next day, and then replace 0.5 mL of fresh culture medium III in each well every day thereafter, in 5% CO2 2、 The embryoids (D) were cultured for 4.5 days on a shaker at 100 rpm in a 37°C incubator to obtain embryoid bodies (D).

[0117] (6) After completing step (5), take the culture plate, gently rotate the culture plate to allow the embryoids D to gather in the center of the well plate, let it stand for 3 minutes, tilt the well plate, completely discard culture medium III, add culture medium IV at a rate of 3 mL / well, and add it to 5% CO2. 2、 Embryomorphs E were obtained by culturing them for 3 days on a shaker at 100 rpm in a 37℃ incubator.

[0118] (7) After completing step (6), take the culture plate, gently rotate the culture plate to allow the embryoids E to gather in the center of the well plate, let it stand for 3 minutes, tilt the well plate, completely discard the culture medium IV, add culture medium V at a rate of 4 mL / well, obtain the embryoids F, and then incubate them in 5% CO2. 2、 Cultured on a shaker at 100 rpm in a 37°C incubator for 4–5 days, single cells or "rice flower cluster"-like cell-rich clusters A are released from embryoid bodies F into the culture medium. Cell-rich clusters A are CD34. + CD45 + Hematopoietic stem cells.

[0119] (8) After completing step (7), take the culture plate, gently pipette the culture medium, let it stand for 1 min, allowing single cells to float to the supernatant of the culture medium, and embryoid bodies F to settle to the bottom of the culture medium. Collect the supernatant of the culture medium as much as possible (avoid aspirating embryoid bodies F), centrifuge at 1200 rpm for 3 min, and the cell-rich cluster A precipitate is hematopoietic stem cells. Keep the old culture medium V, mix the old culture medium V with the fresh culture medium V at a volume ratio of 1:1, add 4 mL / well of the mixed culture medium V, and incubate at 5% CO2. 2、 Cultured on a shaker at 100 rpm in a 37℃ incubator for 4–5 days, single cells or "rice flower" shaped cell clusters A are continuously released into the culture medium by embryoid bodies F.

[0120] (9) Step (8) can be repeated over a long period of time.

[0121] Through the treatment in steps (1) to (3), we significantly increased the proportion of mesodermal cells. After the mesodermal cells fully developed, they provided an excellent growth microenvironment for subsequent hematopoietic stem cells. At the same time, because the shaking of the culture medium in the shaker simulated blood flow, the blood flow in the body promoted the growth of endothelial cells into hematopoietic stem cells, thus achieving "one-time induction, multiple harvests" of hematopoietic stem cells.

[0122] The composition of culture medium I in the example includes: mTeSR™1 complete culture medium with the addition of penicillin G sodium salt at a final concentration of 100 U / mL, streptomycin sulfate at 0.1 mg / mL, ROCKi Y-27632, and 10 μM CHIR99021.

[0123] The culture medium II described in the example comprises: StemPro™-34 SFM, with the addition of penicillin G sodium salt at a final concentration of 100 U / mL, streptomycin sulfate at 0.1 mg / mL, 1% L-alanyl-L-glutamine, 1% insulin-transferrin-selenoethanolamine additive, 400 μM 1-thioglycerol, 2% B27, 10 μg / mL ascorbic acid, 50 ng / mL bone morphogenetic protein 4 (BMP4), 50 ng / mL angiogenesis factor VEGF, and 50 ng / mL fibroblast growth factor 2 (FGF2).

[0124] The culture medium III described in the examples comprises: StemPro™-34 SFM, with the addition of penicillin G sodium salt at a final concentration of 100 U / mL, 0.1 mg / mL streptomycin sulfate, 1% L-alanyl-L-glutamine, 1% insulin-transferrin-selenoethanolamine additive, 400 μM 1-thioglycerol, 2% B27, 10 μg / mL ascorbic acid, 50 ng / mL BMP4, 50 ng / mL VEGF, 50 ng / mL FGF2, and 6 μM SB431542.

[0125] The culture medium IV described in the examples comprises: StemPro™-34 SFM, with the addition of penicillin G sodium salt at a final concentration of 100 U / mL, streptomycin sulfate at 0.1 mg / mL, 1% L-alanyl-L-glutamine, 1% insulin-transferrin-selenoethanolamine additive, 400 μM 1-thioglycerol, 2% B27, 10 μg / mL ascorbic acid, 50 ng / mL VEGF, 50 ng / mL FGF2, and 50 ng / mL stem cell factor SCF.

[0126] The culture medium V described in the examples comprises: StemPro™-34 SFM, with the addition of penicillin G sodium salt at a final concentration of 100 U / mL, 0.1 mg / mL streptomycin sulfate, 1% L-alanyl-L-glutamine, 1% insulin-transferrin-selenoethanolamine additive, 400 μM 1-thioglycerol, 2% B27, 10 μg / mL ascorbic acid, 50 ng / mL VEGF, 50 ng / mL FGF2, 50 ng / mL SCF, 15 ng / mL thrombopoietin TPO, and 10 ng / mL Fms-associated tyrosine kinase 3 ligand FLT3L.

[0127] Morphological changes at various stages of differentiation of human induced pluripotent stem cells into hematopoietic stem cells, such as... Figure 1 As shown. After the first day of step (1), a globular embryoid body is formed. As subsequent induction proceeds, the volume of the embryoid body continues to increase, and cystic or transparent areas appear. After step (7) is completed, single cells or "rice flower cluster"-shaped cell-rich clusters A are released from embryoid body F into the culture medium. As steps (8) and (9) proceed, single cells or "rice flower cluster"-shaped cell-rich clusters A are continuously released from embryoid body F into the culture medium over a long period of time.

[0128] Cell cluster A in step (7) is high-purity CD34 + CD45 + For hematopoietic stem cells, steps (8) and (9) are repetitions of step (7). In the first three repetitions, cell-rich cluster A is still high-purity CD34. + CD45 + Hematopoietic stem cells, but with increasing number of repetitions, CD11b in cluster A becomes more abundant. + The proportion of cells gradually increased, CD34 + The gradual decrease in the proportion of CD34 cells means that CD34 + CD45 + Hematopoietic stem cells differentiate into myeloid progenitor cells and myeloid cells.

[0129] Example 2: Detection of human induced pluripotent stem cells during differentiation into hematopoietic stem cells

[0130] 1. Detection of embryoid body D (mesoderm cell detection)

[0131] (1) Take the embryoid body D obtained in step (5) of Example 1, wash it twice with DPBS, resuspend the embryoid body D with TrypLE Express, and digest it on a shaker at 37°C. The embryoid body D is completely dissociated into single cells. The digestion is terminated with DPBS buffer containing 10% (v / v) fetal bovine serum, centrifuged, and resuspended to obtain two single-cell suspensions (each with a volume of 100 μL and containing 3 × 10⁻⁶ cells). 5 (cells).

[0132] (2) Set the above single-cell suspensions as BLANK and SAMPLE respectively. Add Alexa Fluor488 labeled CD309 (VEGFR2 / KDR) antibody to the SAMPLE tube, incubate at 4°C in the dark for 30 min, wash twice with DPBS buffer containing 2% FBS, resuspend in 100 μL of DPBS buffer containing 2% FBS, and continue to stain with PerCP labeled 7-AAD antibody to distinguish between dead and live cells.

[0133] (3) Go to the computer, use FlowJo 10.8.1 to analyze the collected data, draw a cell population distribution map, calculate the proportion of different cell subpopulations, and obtain the experimental results.

[0134] (4) Take the embryoid body D obtained in step (5) of Example 1 and undifferentiated pluripotent stem cells, extract total RNA, and reverse transcribe it into cDNA. Using cDNA as a template, add specific primers and fluorescent dye, and amplify and detect it in a real-time quantitative PCR instrument. After normalization by internal reference genes, the relative expression level of the target gene is analyzed by the 2⁻ΔΔCt method.

[0135] The results of the detection of lateral mesodermal cells obtained by differentiation of human induced pluripotent stem cells are shown in the figure. Figure 2 . Figure 2 A indicates that, using culture media I, II, and III for induction, the proportion of mesoderm cells in the lateral plate of embryoid bodies D obtained on days 4-5 of stage 3 was >60%, which was much higher than that of the control group. Figure 2 B indicates that, compared with undifferentiated induced pluripotent stem cells, the pluripotency marker genes (NANOG, SOX2) were significantly downregulated in the embryoid bodies obtained on day 5 of stage 3, while the posterior primitive streak-related marker genes (MIXL1, BRACHYURY) and the lateral plate mesoderm-related marker genes (PDGFRα, KDR, ETV2, APLNR) were significantly upregulated. This protocol used culture media I, II, and III for induction, and lateral plate mesoderm cells were successfully and stably obtained from the embryoid bodies D obtained on days 4-5 of stage 3.

[0136] 2. Continuous flow cytometry results of cell cluster A (iHSC assay)

[0137] (1) After each step (8) or (9), collect cell-rich cluster A, wash twice with DPBS buffer, and resuspend to obtain two single-cell suspensions (each with a volume of 100 μL and containing 3 × 10⁻⁶ cells). 5 (cells).

[0138] (2) Set the above single-cell suspensions as BLANK and SAMPLE respectively. Add Fvs620 (PE-CF594) live and dead dye to the SAMPLE tube at a ratio of 1:20000. Incubate for 10 min at room temperature in the dark. Wash twice with DPBS buffer containing 2% FBS. Resuspend in 100 μL of DPBS buffer containing 2% FBS. Continue to stain with PE-Cy7 labeled CD34 antibody and FITC labeled CD45 antibody at a volume ratio of 0.5:100. Incubate at 4℃ in the dark for 30 min. Wash twice with DPBS buffer containing 2% FBS. Resuspend in 150 μL of DPBS buffer containing 2% FBS.

[0139] (3) Go to the computer, use FlowJo 10.8.1 to analyze the collected data, draw a cell population distribution map, calculate the proportion of different cell subpopulations, and obtain the experimental results.

[0140] The results of the A-cell cluster enrichment assay obtained using human induced pluripotent stem cells are shown below. Figure 3 The results showed that in the first three rounds of collection, CD34 was abundant in cell cluster A. + CD45 + The proportion of hematopoietic stem cells was >95%. As the collection time increased, the proportion of CD34 in cell-rich cluster A decreased, while CD45 remained >95%. The above experimental results reflect that in our experiment, after culturing embryoid bodies E, hematopoietic stem cells can be repeatedly generated, and the yield of hematopoietic stem cells in the early stage does not decrease due to repeated culturing.

[0141] 3. Detection of in vitro proliferation capacity of cell cluster A (iHSC assay)

[0142] (1) After completing step (8) for the first time, collect cell-rich cluster A and wash twice with DPBS buffer. Use StemSpan containing 100 U / mL penicillin G sodium, 0.1 mg / mL streptomycin sulfate, 20 μM UM171, 25 ng / mL TPO, 25 ng / mL SCF, and 25 ng / mL FLT3L. TM Resuspend in SFEMII medium and adjust the concentration to 2×10⁻⁶. 5 cells / mL.

[0143] (2) Seed the cell suspension into a non-Treated 12-well plate, 1.0 mL per well. Add D-PBS solution to the empty wells. Place the cell plate in a 37℃ 5% CO2 incubator and culture for 15 days. Inoculate with 2×10⁻⁶ cells per well on days 4, 7, and 11. 5 Subculture at a density of cells / mL / well, completely replacing the culture medium during subculturing.

[0144] (3) Flow cytometry was performed on days 0, 4, 7, 11, and 15. The single-cell suspensions were designated as BLANK and SAMPLE, respectively. Fvs620 (PE-CF594) live / dead stain was added to the SAMPLE tube at a ratio of 1:20000. The tubes were incubated at room temperature in the dark for 10 min, washed twice with DPBS buffer containing 2% FBS, and resuspended in 100 μL of DPBS buffer containing 2% FBS. The tubes were then stained with PE-Cy7-labeled CD34 antibody and APC-labeled CD201 antibody at a volume ratio of 0.5:100. The tubes were incubated at 4°C in the dark for 30 min, washed twice with DPBS buffer containing 2% FBS, and resuspended in 150 μL of DPBS buffer containing 2% FBS. The collected data were then analyzed using FlowJo 10.8.1 to plot the cell population distribution, calculate the proportion of different cell subpopulations, and obtain the experimental results.

[0145] The results of the amplification capacity assay for cell cluster A obtained using human induced pluripotent stem cells are shown in the figure. Figure 4 . Figure 4 A, Figure 4 B indicates that the cell population exhibited high homogeneity (CD34) from the early stages of culture. + With a cell percentage >95% and this dominant phenotype lasting for 7 days, its main components exhibit good in vitro stability. Notably, CD34, which exhibits stronger stem cell activity... + CD201 + Although the initial proportion of the subpopulation was extremely low (<5%), it rapidly expanded to over 70% by day 4 of culture, exhibiting a strong explosive proliferative capacity. Figure 4 C). In addition, Figure 4 D indicates that during the peak amplification period in the first 4 days, CD34 + The proliferation rate of iHSCs was comparable to that of HSCs derived from umbilical cord blood. In summary, the iHSCs obtained using this method not only maintained the key phenotypes but also exhibited excellent in vitro expansion capabilities.

[0146] 4. In vitro multi-lineage differentiation capacity assay of cell cluster A (iHSC assay)

[0147] (1) After completing step (8) for the first time, collect cell-rich cluster A and wash twice with DPBS buffer. Resuspend cell-rich cluster A in IMDM basal medium containing 25 mM HEPES, 100 U / mL penicillin G sodium salt, and 0.1 mg / mL streptomycin sulfate, and adjust the concentration to 1 × 10⁻⁶. 5 Cells / mL, obtain cell suspension A.

[0148] (2) Mix 400 μL of cell suspension A with 4 mL of MethoCult™ H4034 Optimum methylcellulose complete medium, and immediately use a vortex mixer to mix at 2000 rpm for 30 seconds. Then place the mixture on ice and let it stand for 30 minutes to promote the dissipation of bubbles and obtain mixture A.

[0149] (3) Draw 1.3 mL of mixture A into a 2 mL syringe, and slowly add 1.1 mL of mixture A dropwise into a culture dish; cover the culture dish, tilt and rotate it slightly to ensure the mixture evenly covers the bottom. Place it in an incubator with humidity >95%, 37℃, and 5% CO2, and incubate for 15 days. During the incubation period, observe the colony formation regularly and replenish sterile water as needed to prevent drying.

[0150] (4) After completing step (3), use a phase contrast microscope to count the morphology and number of colonies and complete image acquisition.

[0151] The in vitro multilineage differentiation capacity of cell clusters A obtained from human induced pluripotent stem cells is shown in the figure. Figure 5 The results showed that cell-rich cluster A could form erythrocyte colonies, granulocyte colonies, macrophage colonies, and mixed colonies of granulocytes and macrophages with granulocytes, erythrocytes, macrophages, and megakaryocytes. In other words, cell-rich cluster A has multi-lineage differentiation capacity, similar to natural hematopoietic stem cells, but exhibits a more pronounced myeloid bias.

[0152] 5. scRNA-Seq detection (iHSC detection) of cell clusters rich in A

[0153] (1) After completing step (8) for the first time, collect cell-rich cluster A and wash twice with DPBS buffer. Resuspend the cells in DPBS buffer and adjust the cell concentration to >1×10⁻⁶. 6 cells / mL, obtain cell suspension B.

[0154] (2) Take 10 μL of cell suspension B into a centrifuge tube, add an equal volume of AO / PI double staining reagent, gently mix with a pipette, take 10 μL of the mixed suspension, and load it onto a cell counting chamber for analysis. Ensure that the sample is in a single-cell state, with a particle size <40 μm and a live cell ratio >90%.

[0155] (3) Based on a cell count of 1.0 × 10 5Cell suspension B was loaded, and single-cell capture and barcoding were performed using a 10× Genomics Chromium system. Reverse transcription, cDNA amplification, and library construction were then performed according to the manufacturer's standard procedures. After the library passed quality control, high-throughput sequencing was performed on the NovaSeq 6000 platform (PE150 strategy), with single-cell sequencing depth normalized to 50,000-100,000 reads / cell.

[0156] (4) Using the 10× Genomics CellRanger software (9.0.1) and referencing the Human Genome GRCh38 (2024-A), the original sequencing files were converted into cell-gene expression matrix files. Then, the converted sequencing matrix data were analyzed and visualized using the computer programming language R (4.4.2) and software packages developed based on R.

[0157] Single-cell RNA sequencing results of cell clusters A obtained from human induced pluripotent stem cells are shown in the figure. Figure 6 The results showed that detailed analysis of 13,884 high-quality single cells identified 11 cell subpopulations with significant transcriptomic characteristics based on cell population marker genes: hematopoietic stem cell / multipotent progenitor cells (HSC / MPP) (including three subpopulations: HSC / MPP_IKZF2, HSC / MPP_EMCN, and HSC / MPP_SPINK2), myeloid progenitor cells (Mye Pro), neutrophils (Neu), eosinophils (Eos), mast cells (Mast), erythrocytes / megakaryocytes (Ery / Mega), megakaryocytes (Mega), macrophages (Mac), and T-like cells (T like). Figure 6 (A~B). Furthermore, the sequencing results clearly showed the proportion of each cell subpopulation, with the HSC / MPP population accounting for 25.12% (3,489 / 13,884) of the total cell count. Figure 6 C), higher than recently published induction protocols. Based on the expression of various cell marker genes widely used in the literature across different cell clusters, cell types of the main cell clusters were summarized and annotated. Figure 6(D) Based on the hematopoietic endothelial cell / hematopoietic stem cell marker gene CD34, the HSC / MPP_IKZF2, HSC / MPP_EMCN, and HSC / MPP_SPINK2 cell clusters were defined as hematopoietic stem cells / pluripotent progenitor cells. Based on the erythrocyte / megakaryocyte marker gene GATA1, the Ery / Mega and Mega cell clusters were defined as erythrocytes and megakaryocytes, respectively; and based on the inflammatory marker gene ANXA, the Mye Pro, Neu, Eos, Mast, and T like cell clusters were defined as myeloid cells and lymphoid cells, respectively. Human embryonic-like HSCs containing RUNX1⁺HOXA9⁺MLLT3⁺MECOM⁺HLF⁺SPINK2⁺ were distinguished from other hematopoietic progenitor cells. The above results indicate that the present invention can generate a hematopoietic cell population with a high proportion of HSCs / MPPs, and some cells already exhibit lineage bias.

Claims

1. A culture medium composition for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, characterized in that: The culture medium combination includes: culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V, wherein culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V are added in the first, second, third, fourth, and fifth stages of differentiation, respectively; (1) The culture medium I includes basal culture medium A; and selective ATP-competitive Rho-related protein kinase inhibitors and glycogen synthase kinase 3 inhibitors; (2) The culture medium II includes basal culture medium B; and bone morphogenetic protein 4 (BMP4), angiogenesis factor (VEGF), and fibroblast growth factor 2 (FGF2); (3) The culture medium III includes basal culture medium C; and BMP4, VEGF, FGF2, and transforming growth factor signal transduction inhibitors; (4) The culture medium IV includes basal culture medium D; and VEGF, FGF2, and stem cell factor SCF; (5) The culture medium V includes basal culture medium E; and VEGF, FGF2, SCF, thrombopoietin TPO, and Fms-associated tyrosine kinase 3 ligand FLT3L; The basal culture medium A is mTeSR™1 complete culture medium; The basal culture media B, C, D, and E are StemPro™-34 SFM complete culture media.

2. The culture medium composition for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 1, characterized in that, The Rho-related protein kinase inhibitor must satisfy at least one of the following: Y27632; The glycogen synthase kinase 3 inhibitor includes CHIR99021; The transforming growth factor signaling inhibitors include SB431542.

3. The culture medium composition for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 1, characterized in that, The culture medium I further comprises at least one of the following: penicillin G sodium salt and streptomycin sulfate; Culture media II, III, IV, and V further include penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1-thioglycerol, vitamin B27, and ascorbic acid.

4. The culture medium composition for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 1, characterized in that, The following condition must be met: the concentration of Y27632 used is 5-10 μM; The concentration of CHIR99021 used is 5-15 μM; The concentration of BMP4 used is 50-100 ng / mL; The concentration of VEGF used is 25-75 ng / mL; The concentration of FGF2 used is 25-75 ng / mL; The concentration of SB431542 used is 6-10 μM; The concentration of SCF used is 25-75 ng / mL; The concentration of TPO used is 15-45 ng / mL; The concentration of FLT3L used is 5-15 ng / mL; The concentrations of penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1%, 1-thioglycerol, B27, and ascorbic acid are all specified.

5. The use of the culture medium combination for induced pluripotent stem cells to differentiate into hematopoietic stem cells as described in any one of claims 1-4 in the preparation of hematopoietic stem cells.

6. A method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, characterized in that, Includes the following steps: Human induced pluripotent stem cells were sequentially induced into protostoma, lateral mesoderm, and hematopoietic endothelial cells using the above-mentioned culture medium combination. Then, endothelial-hematopoietic transformation was carried out to obtain hematopoietic stem cells.

7. The method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 6, characterized in that, Includes the following steps: a. Inoculate human induced pluripotent stem cells into culture medium I and incubate statically for 1.0-1.5 days to obtain embryoid body A. Then transfer the culture plate to a shaker at 80-120 rpm and culture dynamically for 2.0-3.0 days to obtain embryoid body B. b. Transfer the embryoid B from step a to culture medium II and culture it dynamically on a shaker at 80-120 rpm for 1.0-1.5 days to obtain embryoid C; c. Transfer the embryoid C from step b to culture medium III and culture it dynamically on a shaker at 80-120 rpm for 4.0-5.0 days to obtain embryoid D; d. Transfer the embryoid body D from step c to culture medium IV and culture it dynamically on a shaker at 80-120 rpm for 3.0-3.5 days to obtain embryoid body E; e. Transfer the embryoid body E from step d to culture medium V to obtain embryoid body F, and continuously culture it dynamically on a shaker at 80-120 rpm. The culture medium combination includes: culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V, wherein culture medium I, culture medium II, culture medium III, culture medium IV, and culture medium V are added in the first, second, third, fourth, and fifth stages of differentiation, respectively; (1) The culture medium I includes basal culture medium A; And selective ATP-competitive Rho-related protein kinase inhibitors and glycogen synthase kinase 3 inhibitors; (2) The culture medium II includes basal culture medium B; and bone morphogenetic protein 4 (BMP4), angiogenesis factor (VEGF), and fibroblast growth factor 2 (FGF2); (3) The culture medium III includes basal culture medium C; and BMP4, VEGF, FGF2, and transforming growth factor signal transduction inhibitors; (4) The culture medium IV includes basal culture medium D; and VEGF, FGF2, and stem cell factor SCF; (5) The culture medium V includes basal culture medium E; and VEGF, FGF2, SCF, thrombopoietin TPO, and Fms-associated tyrosine kinase 3 ligand FLT3L; The basal culture medium A is mTeSR™1 complete culture medium; The basal culture media B, C, D, and E are StemPro™-34 SFM complete culture media.

8. The method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 7, characterized in that, The Rho-related protein kinase inhibitor must satisfy at least one of the following: Y27632; The glycogen synthase kinase 3 inhibitor includes CHIR99021; The transforming growth factor signaling inhibitor includes SB431542; The culture medium I further includes penicillin G sodium salt and streptomycin sulfate; The culture media II, III, IV, and V further include penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1-thioglycerol, vitamin B27, and ascorbic acid; The concentration of Y27632 used is 5-10 μM; The concentration of CHIR99021 used is 5-15 μM; The concentration of BMP4 used is 50-100 ng / mL; The concentration of VEGF used is 25-75 ng / mL; The concentration of FGF2 used is 25-75 ng / mL; The concentration of SB431542 used is 6-10 μM; The concentration of SCF used is 25-75 ng / mL; The concentration of TPO used is 15-45 ng / mL; The concentration of FLT3L used is 5-15 ng / mL; The concentrations of penicillin G sodium salt, streptomycin sulfate, L-alanyl-L-glutamine, insulin-transferrin-selenoethanolamine additive, 1%, 1-thioglycerol, B27, and ascorbic acid are all specified.

9. The method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 7 or 8, characterized in that: The culture conditions in step ae were all 37℃ and 5% CO2.

10. The method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells according to claim 7 or 8, characterized in that: All culture plates used in step ae are ultra-low adhesion culture plates.

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