Method for preparing hematopoietic stem cells and progenitor cells

Abstract

A method for preparing hematopoietic stem cells and progenitor cells is provided for producing hematopoietic stem / progenitor cells (HSPCs) from embryonic stem cells or iPSCs. Also provided are a hematopoietic stem / progenitor cell (HSPC) culture product generated by the method, and the use of differentiating the hematopoietic stem / progenitor cells (HSPCs) into blood cells of interest. The method successfully achieves efficient differentiation into HSPCs, and the obtained culture product does not contain heterologous substances, meets the GMP standard, and is prone to large-scale production. Additionally, the method also has the advantages of shorter differentiation time and higher efficiency.

Description

A method for preparing hematopoietic stem cells and progenitor cells

[0001] This disclosure claims priority to the Chinese patent application filed with the China Patent Office on October 11, 2023, with the invention name “A method for preparing hematopoietic stem cells and progenitor cells” and application number 202311309571.9. The entire contents of the above application are incorporated by reference into this disclosure. Technical Field

[0002] The present invention relates to a method for preparing hematopoietic stem and progenitor cells. More specifically, the present invention relates to a method for large-scale production of hematopoietic stem and progenitor cells from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells by in vitro culture, the hematopoietic stem and progenitor cell cultures thus produced, and their use for differentiating into hematopoietic lineage cells. Background Art

[0003] Hematopoietic stem and progenitor cells (HSPCs) are a subset of bone marrow (BM) cells that have the ability to self-renew and generate all cell types of the hematopoietic lineage. Hematopoietic stem cell transplantation is a common therapy for certain hematological malignancies, such as multiple myeloma, acute myeloid leukemia, and myelodysplastic syndrome. Given their potential to differentiate into any blood cell, HSPCs are also crucial in generating different blood cell types for cell therapy to treat different disease indications.

[0004] CD34 is a well-known surface marker associated with progenitor cells with hematopoietic / endothelial / mesenchymal differentiation potential, which is commonly used to isolate HSPCs from peripheral blood mononuclear cell (PBMC) pools. Another surface marker, CD43, further determines the hematopoietic orientation of CD34+ progenitor cells with hematopoietic lineage multipotency. Although PBMCs are routinely used to isolate CD34+ HSPCs, it is difficult to regularly obtain large batches of high-quality cells from different donors, thereby increasing the complexity of allogeneic cell therapy applications.

[0005] Hematopoietic stem / progenitor cells have been considered to originate from a population of CD34-positive cells. Specifically, CD34+ endothelial cells undergo endothelial-to-hematopoietic transition (EHT) to become hematopoietic progenitors, which subsequently give rise to downstream differentiated blood cell types. However, CD34 expression alone is not sufficient to distinguish hematopoietic cells from cells with committed endothelial and mesenchymal fates, which also appear among CD34+ progenitors. Previous studies have demonstrated that the addition of additional surface markers to CD34 expression can better identify progenitor cells of different lineages. Specifically, CD43 has been shown to be a marker of hematopoietic commitment, which can reliably distinguish hematopoietic CD34+ cells from CD34+ / CD43– / CD31+ / KDR+ / endothelial cells and CD34+CD43–CD31–KDR– mesenchymal cells (Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures, Blood (2006) 108(6): 2095–2105.).

[0006] Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) are self-renewing pluripotent stem cells with the potential to differentiate into all cell types of the three germ layers. These pluripotent stem cells have also been used to produce CD34+ / CD43+ HSPCs, which first appear as pre-directed erythromegakaryocytic progenitor cells and later appear as multipotent lymphohematopoietic progenitor cells. Although HSPCs derived from pluripotent stem cells have broad potential as a substitute for HSPCs derived from PBMCs, most of the differentiation protocols available so far are not easy to scale up, and are limited by the relatively low efficiency and purity of the HSPCs obtained. The prior art has a method for obtaining HSPCs using iPSC differentiation, but there is no report on the efficient differentiation method of HSPCs (such as CD34+ / CD43+ types) of immune cell lines. Therefore, there is an urgent need in the art for an improved, scalable method for generating HSPCs from pluripotent stem cells.

[0007] Summary of the Invention

[0008] Problems to be solved by the invention

[0009] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a method for efficiently differentiating HSPCs, so that the product does not contain xenobiotics, and at the same time has a shorter differentiation time and higher efficiency.

[0010] Solutions for solving problems

[0011] The present invention provides a method for preparing hematopoietic stem cells and progenitor cells, the method comprising the following steps:

[0012] a. inducing embryonic stem cells or induced pluripotent stem cells (iPSCs) to differentiate into embryoid bodies (EBs) in the presence of a GSK-3 inhibitor; and

[0013] b. Inducing the EB formed in step a to differentiate into hematopoietic stem and progenitor cells (HSPC) in a hematopoietic differentiation medium comprising one or more factors selected from the following: BMP4, SCF, SB431542;

[0014] wherein said step a comprises step a1, wherein said step a1 is induced on day 0 in the presence of a GSK-3 inhibitor;

[0015] The induction in step b comprises an induction step b1 in the presence of BMP4, a subsequent induction step b2 in the presence of BMP4, SCF and SB431542, and a further induction step b3 in the presence of SCF.

[0016] Preferably, the induction in step a1 is performed on day 0 in the presence of a GSK-3 inhibitor and a ROCK inhibitor.

[0017] Preferably, the GSK-3 inhibitor is CHIR 99021, and the ROCK inhibitor is Rki.

[0018] Preferably, the step a further comprises a step a2, wherein the step a2 is to induce again on day 1 in the presence of a GSK-3 inhibitor.

[0019] Preferably, the step a2 is to induce again on day 1 in the presence of CHIR 99021.

[0020] Preferably, the method comprises the following steps:

[0021] a1) culturing iPSCs in suspension in StemScale PSC complete suspension medium supplemented with CHIR 99021 and Rki; a2) continuing suspension culture in StemScale PSC complete suspension medium supplemented with CHIR 99021 to form EBs;

[0022] b1) culturing EBs in suspension in hematopoietic differentiation medium 1 supplemented with BMP4, FGF2, and VEGF;

[0023] b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB431542;

[0024] b3) further culturing in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, and VEGF to form HSPCs; and

[0025] c) expanding the HSPCs generated in step b) and further culturing them in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, VEGF, FLT3 ligand (FLT3L), TPO, IL-11, IGF1, IL-3, and IL-6.

[0026] Preferably, step a1 is performed on day 0, step a2 is performed on day 1, step b1 is performed on day 2-3, step b2 is performed on day 4-5, step b3 is performed on day 6-7, and step c is performed on day 8-10.

[0027] Preferably, the HSPCs have a CD34+CD43+ phenotype.

[0028] Preferably, the HSPCs have a CD235a-CD14-CD43+CD34+ phenotype.

[0029] The present invention also provides HSPCs prepared according to the method and a culture thereof.

[0030] The present invention also provides a pharmaceutical preparation comprising a therapeutically or prophylactically effective amount of the HSPC, a culture thereof, a pharmaceutically acceptable carrier or diluent, and optionally one or more other active agents.

[0031] The present invention also provides a use of the HSPC or its culture, or the pharmaceutical preparation in preparing a preparation for cell transplantation-related diseases.

[0032] Preferably, the disease is selected from the group consisting of leukemia, lymphoma, myeloma, solid tumors such as sarcoma, cancer, rheumatoid arthritis, lupus, inflammatory bowel disease, multiple sclerosis, type I diabetes, psoriasis, scleroderma, vasculitis, fibrosis and neuropathies, immune diseases, infections.

[0033] Preferably, the disease is multiple myeloma, acute myeloid leukemia or myelodysplastic syndrome.

[0034] The present invention also provides a kit for inducing iPSCs to generate EBs, wherein the kit comprises a combination of factors for implementing one or more steps.

[0035] Effects of the Invention

[0036] The present invention successfully achieves efficient differentiation into HSPCs by culturing embryonic stem cells or induced pluripotent stem cells in vitro in stepwise fashion under appropriate conditions. The resulting culture products are xenobiotic-free, meet GMP standards, and are easily scalable for production. Furthermore, the method offers the advantages of shorter differentiation times and higher efficiency, better meeting existing technical requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of differentiating induced pluripotent stem cells into HSPCs using the method of the present invention. iPSCs are cultured in rotation to generate 3D iPSC spheres, which are then guided to differentiate into mesodermal EBs, hematopoietic EBs, and HSPCs.

[0038] Figure 2 shows the changes in cell morphology during the differentiation of iPSC to HSPC. Two donor iPSC cell lines were used, of which cell line 1 corresponds to an iPS cell line derived from adult PBMCs and cell line 2 corresponds to an iPS cell line derived from placental blood CD34-positive cells. Representative images of the two cell lines were recorded on day 0, day 1, and day 7 during the culture process of the present invention. The results showed that around day 6-7 of the differentiation culture, HSPCs began to appear in the culture as round and bright suspended cells. Scale bar = 200 μm. Scale bar in the magnified image = 100 μm.

[0039] Figure 3 shows the surface marker identification of HSPCs derived from iPSCs by the method of the present invention. AB: Flow cytometry analysis of hematopoietic progenitor cell markers CD34 and CD43 on HSPCs derived from iPSCs by the method of the present invention. As can be seen from the figure, >90% of the cells obtained from the differentiation culture expressed CD43, indicating that the cell fate was directed into the hematopoietic lineage, of which the majority (60-80%) retained CD34 expression, which is a stem cell potency marker for HSPCs. C: Flow cytometric analysis of hematopoietic lineage markers CD45, CD14, CD235a and mesoderm markers CD73 / CD184 was performed on HSCPs derived from iPSCs by the method of the present invention.

[0040] Figure 4 shows cell morphological changes during iPSC to HSPC differentiation. It shows a timeline of hematopoietic stem and progenitor cell (HSPC) differentiation from induced pluripotent stem cells (iPSCs) via embryoid bodies, with arrows indicating the appearance of CD34+ hematopoietic stem cells in suspension.

[0041] Figure 5 is a flow cytometric analysis of HSPC markers. A is a representative of HSPC cell gating for flow cytometric analysis. HSPC cells were identified as (CD235a-CD14-CD43+CD34+) on day 8 of condition 6. B is a representative of HSPC cell gating for flow cytometric analysis; HSPC cells were identified as (CD235a-CD14-CD43+CD34+) on day 10 of condition #6.

[0042] Figure 6 shows the percentage of CD34+CD43+ cells in live cells. A: IBX iPSCs on day 8; B: CNTB iPSCs on day 8.

[0043] Figure 7 shows representative Brightfield images of iPSC culture stages.

[0044] Figure 8 shows the comparison results of CHIR99021 addition time of 1 day vs. 2 days.

[0045] FIG9 shows the results of the cell stability experiment.

[0046] FIG10 shows the results of downstream immune differentiation experiments of iNK cells.

[0047] Figure 11 shows qRT-PCR analysis of the expression of pluripotency markers OCT4, NANOG, SOX2, TRA-1-60, and T cell lineage markers GATA3, BCL11B, and RUNX1 in iPSC cell lines, primary T cells, and iT cells. There were no residual iPSCs in the differentiated iT cell cultures. Compared to iPSCs and primary T cells, iT cells did not express the pluripotency markers that are highly expressed in iPSCs and showed high expression of T lineage markers.

[0048] Figure 12 shows cytokine production by iT cells upon in vitro stimulation. Primary T cells and iT cells were stimulated with PMA / ionomycin for 16 hours, and production of the characteristic effector cytokines IFNγ and TNFα was assessed by intracellular staining and flow cytometric analysis. iT cells differentiated from both iPSC lines produced similar levels of IFNγ and TNFα compared to primary CD8+ T cells.

[0049] Figure 13 shows characterization of iMAC differentiation. A shows cell morphological changes during iPSC-to-iMAC differentiation. B shows flow cytometry comparison of macrophage marker expression (CD68, CD86, CD80) on iMAC (i-M0) and monocyte-derived macrophages (m-M0). The upper panel shows the percentage of marker-positive cells; the lower panel shows the median fluorescence intensity (MFI) of the corresponding markers.

[0050] Figure 14 shows representative brightfield images of iB cells during differentiation from iPSC-derived CD34+ HSPCs, and images of representative IBX cell lines and control CD34+ cells sorted from PBMCs.

[0051] Figure 15 shows representative brightfield images of iB cells during differentiation of CD34+ HSPCs derived from iPSCs. CD34+ cells were obtained from the IBX cell line on day 8 and plated in early B cell differentiation medium to induce B cell differentiation. As a control, CD34+ cells were sorted from human PBMCs. Images are magnified 4x, scale bar = 200 μm.

[0052] Figure 16 is a flow cytometric analysis of iB cell markers. Representative of the iB cell gating for flow cytometric analysis, iB cells were identified as (CD24+CD10+CD38+CD19+CD20+).

[0053] Figure 17 shows flow cytometric analysis of iB cell markers. Isotype controls for each marker used in the flow cytometric analysis, light grey histogram: iB cell isotype; dark grey histogram: iB cells. DETAILED DESCRIPTION

[0054] 1. Definition

[0055] In the context of the present invention, the relevant terms are defined in accordance with the conventional definitions in the art. Specifically, the meanings of some terms are as follows:

[0056] Pluripotent stem cells: Pluripotent stem cells are cells that have the ability to develop and differentiate into multiple tissues or cell types. They retain the potential of stem cells, namely the potential to develop and differentiate into the three germ layers. In the present invention, pluripotent stem cells include, for example, embryonic stem cells and induced pluripotent stem cells.

[0057] Embryonic stem cells: Embryonic stem cells are undifferentiated pluripotent cells originally derived from embryos, which can be, for example, established embryonic stem cell lines, embryonic stem cells made from early embryos, embryonic stem cells made by in vitro fertilization and embryos that develop no more than 14 days, or embryonic cells generated from adult cells by artificial means such as nuclear transfer that have the ability to differentiate into any cell type.

[0058] Induced pluripotent stem cells: Induced pluripotent stem cells are cells generated from somatic cells by expressing or inducing the expression of cytokines and have the potential to differentiate into various germ layers.

[0059] Hematopoietic stem and progenitor cells (HSPCs): A subset of bone marrow (BM) cells that have the capacity to self-renew and give rise to all cell types of the hematopoietic lineage. HSPCs can be identified by the expression of the cell surface markers CD34 and CD43.

[0060] The pluripotent stem cells used in the present invention include embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) and can be a variety of animal sources, including humans, and species such as mice, pigs, and non-human primates that can obtain embryonic stem cells and induced pluripotent stem cell types. Other animal sources are also suitable, as long as their embryonic stem cells and / or induced pluripotent stem cells can be used. Pluripotent stem cells can proliferate in vitro for a long time while retaining the potential to differentiate into all cell types of the body, including, for example, hematopoietic stem cells and precursor cells of the present invention. In the present invention, by culturing embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) in vitro under appropriate culture conditions, hematopoietic stem cells and precursor cells are provided at low cost and high efficiency, which lays a solid foundation for the prevention and treatment of some blood diseases or tumors.

[0061] Subject: The term "subject" as used in the present invention refers to an individual suitable for prevention, treatment and / or alleviation by administering the HSPCs of the present invention, including, for example, various mammals, such as humans, other non-human primates, mice, rats, pigs, etc.

[0062] The present invention relates to some factors, which have conventional meanings in the art. For example, some of these factors are explained as follows:

[0063] Y-27632 is a selective, ATP-competitive inhibitor of Rho-associated protein kinases (ROCKs), including p160Rock (Ki=140nM) and Rock-II. Treatment with 10uM Y-27632 prevents cell apoptosis and increases cell survival.

[0064] CHIR 99021 is a potent and highly selective glycogen synthase kinase 3 (GSK-3) inhibitor that acts as an activator of the Wnt signaling pathway.

[0065] ITS-G (Insulin-Transferrin-Selenium). ITS-G is used as a basal culture medium supplement to reduce the amount of fetal bovine serum (FBS) required to culture cells. Insulin promotes glucose and amino acid uptake, lipogenesis, intracellular transport, and protein and nucleic acid synthesis. Transferrin, an iron carrier, also helps reduce toxic levels of oxygen free radicals and peroxides. Selenium (provided as sodium selenite) is a cofactor for glutathione peroxidase and other proteins and acts as an antioxidant in culture media.

[0066] SB431542 is a potent small molecule inhibitor of transforming growth factor (TGF)-β signaling.

[0067] Bone morphogenetic protein 4 (BMP4) is a member of the bone morphogenetic protein family, which is part of the transforming growth factor beta superfamily.

[0068] Fibroblast growth factor 2 (FGF2), also known as basic fibroblast growth factor (bFGF) and FGF-β, is a growth factor and signaling protein encoded by the FGF2 gene. It binds and exerts its effects through specific fibroblast growth factor receptor (FGFR) proteins, which themselves are a family of closely related molecules.

[0069] Vascular endothelial growth factor (VEGF) is an important signaling protein involved in angiogenesis and vasculogenesis.

[0070] Stem cell factor (SCF) is a cytokine that binds to the c-KIT receptor (CD117). This cytokine plays an important role in hematopoiesis, spermatogenesis, and melanogenesis.

[0071] FMS-like tyrosine kinase 3 ligand (FLT3L) is an endogenous small molecule that acts as a cytokine and growth factor to increase the number of immune cells by activating hematopoietic progenitor cells. It works by binding to and activating FLT3 (CD135).

[0072] Thrombopoietin (TPO) is a key cytokine for megakaryocyte and platelet production and is also reported to be crucial for the maintenance of hematopoietic stem cells.

[0073] Interlukin11 (IL-11) is a cytokine that is important for hematopoiesis and binds to its transmembrane IL-11Rα receptor.

[0074] Insulin-like growth factor 1 (IGF-1), also known as somatropin C, is a hormone with a molecular structure similar to insulin. IGF-1 is a protein that stimulates the proliferation and survival of various cell types in vitro, including muscle, bone, and cartilage tissue. It plays an important role in childhood growth and continues to have anabolic effects in adults. IGF-1 belongs to the family of insulin-like growth factors. It binds to the IGF-1 receptor and activates the PI3K / AKT pathway as well as the ERK1 / 2 pathway. It is primarily produced in the liver and, together with IL-3, stimulates the differentiation and proliferation of bone marrow cells.

[0075] Interleukin-3 (IL-3) is a hematopoietic growth factor with a broad range of biological activities. These include stimulation of the proliferation and differentiation of immature multipotent hematopoietic stem cells and various lineage-committed progenitor cells, leading to the generation of most major blood cell types.

[0076] Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses, hematopoiesis, acute phase responses, and inflammatory responses.

[0077] 2.ES cells

[0078] ES cells have proliferation potential while maintaining their pluripotency. Specifically, embryonic stem cells: (i) can differentiate into cells of all three germ layers; (ii) express at least Oct4 and alkaline phosphatase; and (iii) can produce teratomas when transplanted into immunodeficient animals. Embryonic stem cells include: cells derived from the inner cell mass (ICM) of a mammalian blastocyst or morula, including those that have been passaged as a cell line; cells derived from a fertilized egg, a blastomere (e.g., a blastomere of a cleavage-stage or morula-stage embryo) or a mammalian embryo at the blastocyst stage, cells produced by artificial fusion of a sperm and an egg cell, cells obtained by fertilizing an egg cell using a sperm, cells obtained by nuclear transfer (e.g., somatic cell nuclear transfer (SCNT)), cells of parthenogenesis, cells of male parthenogenesis, or cells obtained by chromatin reprogramming and subsequently introducing reprogrammed chromatin into cells.

[0079] It should be noted that embryonic stem cells do not refer to, and should not refer to, cells generated by destroying an embryo. On the contrary, there are a variety of methods that can be used to generate ES cells without destroying the embryo, such as a human embryo. For example, ES cells can be generated from a single blastomere derived from an embryo in a manner similar to the extraction of blastomeres for preimplantation genetic diagnosis (PGD). Examples of such cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. An exemplary human embryonic stem cell line that can be used is MA09 cells. The isolation and preparation of MA09 cells were previously described in Klimanskaya, et al. (2006) "Human Embryonic Stem Cell lines Derived from Single Blastomeres." Nature 444:481-485, and Chung et al. (2008) "Human Embryonic Stem Cell Lines Generated without Embryo Destruction, Cell Stem Cell, 2(2):113-117. All of these cell lines were generated without embryo destruction.

[0080] Other ES cells that can be used include, but are not limited to, human ES cells, such as MA01, MA09, ACT-4, MA142, MA143, MA144, MA145, MA146, MA126 (NED1), MA127 (NED2), MA128 (NED3), MA129 (NED4), NED7, No. 3, H1, H7, H9, H14, and ACT30 ES cells.

[0081] Reference may also be made to the NIH Human Embryonic Stem Cell Registry.Human ES cells used in accordance with the present disclosure may be derived and maintained according to GMP standards known in the art.

[0082] As mentioned herein, ES cells are derived from a variety of species, including mouse, pig, various species of non-human primates, and humans, and embryo-derived stem cells have been generated from a variety of other species.

[0083] 3. Induced Pluripotent Stem Cells

[0084] As previously mentioned, induced pluripotent stem cells are cells generated from somatic cells by expressing or inducing the expression of cytokines, and capable of differentiating into various germ layers. Differentiated somatic cells, including cells from fetuses, newborns, young or adult mammals, such as primates, including humans, pigs, mice, etc., are somatic cells suitable for the methods of the present invention. Such somatic cells can be reprogrammed into induced pluripotent stem cells using reprogramming methods known in the art. Suitable somatic cells include, but are not limited to, bone marrow cells, epithelial cells, endothelial cells, fibroblasts, peripheral blood mononuclear cells, hematopoietic cells, keratinocytes, hepatocytes, intestinal cells, mesenchymal cells, bone marrow precursor cells, and spleen cells. Alternatively, somatic cells can be cells that can themselves proliferate and differentiate into other cell types, including blood stem cells, muscle / bone stem cells, brain stem cells, and liver stem cells. Suitable somatic cells have receptive properties, or can be made to have receptive properties using methods known in the art, so that they can take up transcription factors, including genetic material encoding transcription factors, thereby being induced into the desired cell type. Preferably, the pluripotent stem cells are derived from humans, mice, pigs, or other primates.

[0085] It is known in the art how to produce induced pluripotent stem cells. The induction of pluripotency was initially achieved by reprogramming somatic cells with the introduction of transcription factors related to pluripotency using mouse cells (Yamanaka et al., 2006) and human cells (Yu et al., 2007; Takahashi et al., 2007) in 2006. Most of the ethical and practical issues associated with the large-scale clinical use of ES cells can be avoided using iPSC, and patients with iPSC-derived autologous transplants may not need lifelong immunosuppressive therapy to prevent transplant rejection. Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSC) using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells, see, for example, disclosed U.S. Patent Application No. 20090246875, disclosed U.S. Patent Application No. 2010 / 0210014; Published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,268,620; PCT Publication No. WO 2007 / 069666 A1 and U.S. Patent No. 8,268,620, which are incorporated herein by reference. Typically, nuclear reprogramming factors are used to produce pluripotent stem cells from somatic cells. Somatic cells are reprogrammed to a dedifferentiation or pluripotent state generally involving the expression of reprogramming factors (including transcription factors). The relevant transcription factors may comprise one or more of the following, or consist of or consist essentially of one or more of the following factors: OCT4, SOX2, KLF4 and MYC (OSKM); SOX2, KLF4 and OCT4 (SKO); OCT4, SOX2, KLF4 and GLIS1 (OSKG); OCT4, SOX2, NANOG and LIN28 (OSNL); or OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28 (OKSMNL).

[0086] In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0087] The mouse and human cDNA sequences of these nuclear reprogramming materials can refer to the NCBI accession number mentioned in WO 2007 / 069666 and U.S. Patent No. 8,183,038, which are incorporated herein by reference. The method for introducing one or more reprogramming materials or encoding the nucleic acid of these reprogramming materials is known in the art, and is disclosed in, for example, U.S. Patent No. 8,268,620, 8,691,574, 8,741,648, 8,546,140, ​​disclosed U.S. Patent No. 8,900,871 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference. They can be delivered in the form of integrated (such as slow virus) or non-integrated (such as Sendai virus) virus, mRNA (non-modified or modified mRNA, self-replicating mRNA), free plasmid or protein. Another method for generating iPSC relates to the use of somatic cell nuclear transplantation technology (SCNT).

[0088] 4. Maintenance and Culture of Pluripotent Stem Cells

[0089] Pluripotent stem cells to be differentiated into hematopoietic cells and their precursors can be cultured in a medium sufficient to maintain pluripotency, for example, various culture media and techniques developed for culturing primate pluripotent stem cells, more particularly embryonic stem cells, as described in U.S. Patent Application No. 20070238170 and U.S. Patent Application No. 20030211603 can be used.

[0090] As is known in the art, pluripotent stem cells can be maintained in culture on feeder cells. Alternatively, pluripotent stem cells can also be maintained in a feeder-free system and thus proliferate. Preferably, this is done on a matrix. Suitable matrices include, but are not limited to, laminin, fibronectin, vitronectin, proteoglycans, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel, TM Or any combination thereof. Matrigel TM Available from commercial sources, such as BD Biosciences. Laminin (including recombinant human laminin) can be used as Matrigel TM alternatives.

[0091] For example, pluripotent stem cells can be maintained in their undifferentiated state in Matrigel without feeder layers. TMOr laminin. When confluency reaches approximately 80-90%, cells are typically passaged or frozen. Passaging can be performed using enzymatic (e.g., dispase) or non-enzymatic (e.g., PBS-based dissociation buffer, Invitrogen) means. In some embodiments, pluripotent stem cells can be cultured under xeno-free conditions and, in some cases, can be cultured in the presence of a serum supplement (e.g., but not limited to, N2 serum supplement).

[0092] Culture media for the maintenance of pluripotent stem cells are known in the art. For example, pluripotent stem cells can be maintained in 80% DMEM / F12 (Gibco #11330032 or #11320082), 20% KnockOut serum replacement, 1% non-essential amino acids, 1mM L-glutamine, 0.1mM β-mercaptoethanol and bFGF (4-100ng / mL) (PCT application WO99 / 20741). Alternatively, human pluripotent stem cells can be maintained in a chemically defined serum-free medium such as mTeSR1. In one embodiment, the pluripotent stem cells of the present invention can be cultured in Essential 8TM medium. In further embodiments, the pluripotent stem cells of the present invention can be cultured in a culture medium selected from the following: StemFit, mTeSR TM 1. mTeSR TM 2. TeSR TM -E8 TM 、mTeSR TM PLUS, PluriSTEM TM 、ExCellerate TM iPSC Expansion Medium, StemScale PSC Suspension Medium, StemFlex TM Culture medium, mTeSR TM 3D culture medium or StemMACS TM PSC-Brew XF Medium.

[0093] Preferably, suitable cytokines are added to the culture medium to culture the PSC cells to maintain their pluripotency. Suitable cytokines that can be added include, for example, leukocyte inhibitory factor. Alternatively, the PSC cells can be maintained on an embryonic fibroblast feeder layer. Such embryonic fibroblasts can be primary or cultured fibroblasts from mice, or established embryonic fibroblast cell lines that have been used in the culture of pluripotent stem cells such as embryonic stem cells.

[0094] In order to maintain the culture of pluripotent stem cells, various suitable auxiliary components may be added to the culture medium, such as serum albumin, glutamine, antibiotics, etc. The ranges and dosage levels thereof are well known in the art.

[0095] In the present invention, in addition to being cultured as a monolayer culture, pluripotent stem cells can also be cultured in the form of a three-dimensional culture. Equipment for three-dimensional culture includes, for example, spinner flasks, shaking bioreactors, or cell culture bags.

[0096] 5. Induction of pluripotent stem cells into hematopoietic stem and progenitor cells

[0097] Under appropriate conditions, pluripotent stem cells can differentiate into appropriate cell types.

[0098] The present invention unexpectedly discovered that pluripotent stem cells can be directed to differentiate into desired cell types in the presence of appropriate cytokines.

[0099] Therefore, in one aspect, the present invention provides a method for preparing hematopoietic stem and progenitor cells, wherein the method comprises the following steps:

[0100] a. inducing embryonic stem cells or induced pluripotent stem cells (iPSCs) to differentiate into embryoid bodies (EBs) in the presence of a GSK-3 inhibitor; and

[0101] b. Inducing the EB formed in step a to differentiate into hematopoietic stem and progenitor cells (HSPC) in a hematopoietic differentiation medium comprising one or more factors selected from the following: BMP4, SCF, SB431542;

[0102] wherein said step a comprises step a1, wherein said step a1 is induced on day 0 in the presence of a GSK-3 inhibitor;

[0103] The induction in step b comprises an induction step b1 in the presence of BMP4, a subsequent induction step b2 in the presence of BMP4, SCF and SB431542, and a further induction step b3 in the presence of SCF.

[0104] Optionally, the hematopoietic stem cells and progenitor cells generated in step b) can be cultured and expanded, for example, by a hematopoietic stem cell and progenitor cell expansion method or expansion medium known in the art. Preferably, the expansion can be carried out in a hematopoietic stem cell expansion medium supplemented with P / S and GlutaMax to which one or more of ITS-G, SCF, FGF2, VEGF, FLT3 ligand (FLT3L), TPO, IL-11, IGF1, IL-3, and IL-6 are added.

[0105] In one embodiment, embryonic stem cells can be adopted to induce differentiation into hematopoietic stem cells and progenitor cells. The embryonic stem cell can be a cell produced by the artificial fusion of sperm and egg cell, a cell obtained by fertilizing the egg cell, preferably no more than 14 days after fertilization, an embryonic stem cell obtained by nuclear transfer (such as somatic cell nuclear transplantation (SCNT)), a cell of parthenogenesis, a cell of male parthenogenesis, or a stem cell obtained by chromatin reprogramming and subsequently introducing the reprogrammed chromatin into the cell. Alternatively, the embryonic stem cell adopted in the present invention can be an established embryonic stem cell line, preferably selected from the embryonic stem cell lines of MAOI, MA09, ACT-4, HI, H7, H9\H13, H14 and ACT30.

[0106] In another embodiment, induced pluripotent stem cells can be used to induce differentiation into hematopoietic stem cells and progenitor cells. The induced pluripotent stem cells can be generated by reprogramming somatic cells from an animal under conditions in which at least three or at least four factors among Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are added.

[0107] There are a variety of cytokines that can promote embryoid body (EB) formation from pluripotent stem cells, such as one or more of BMP4, FGF2, VEGF, SB431542, and SCF. Therefore, one or more of these cytokines can be used in step b). These cytokines are commonly available to those skilled in the art. For example, BMP4 can be obtained from Miltenyi Biotec, catalog number 130-111-167; FGF2 can also be obtained from Miltenyi Biotec, catalog number 130-093-564; VEGF and SCF can also be obtained from Miltenyi Biotec, catalog numbers 130-109-396 and 130-096-695, respectively; and SB431542 can be obtained from Tocris, catalog number 1614 / TB1614-GMP.

[0108] There are also a variety of cytokines suitable for use in step b), including but not limited to one or more of IL11, IL-6, TPO, IGF-1, IL-3, and FLT3L. These cytokines are routinely available to those skilled in the art, for example, from Miltenyi Biotec, with catalog numbers #130-103-439, #130-093-932, #130-095-752, #130-093-886, #130-095-070, and #130-096-477, respectively. The levels of these factors used can also be determined through routine experimentation.

[0109] In certain embodiments, the induction in step b can be performed in a single step or in multiple steps.

[0110] In certain embodiments, the induction in step b can be performed in 2 steps, 3 steps or more.

[0111] In certain embodiments, the induction in step b can be performed in 4 or more steps.

[0112] In certain embodiments, the inducing step comprises performing the inducing step bl in the presence of BMP4.

[0113] In certain embodiments, the inducing step comprises performing the inducing step b2 in the presence of BMP4, SB431542, and SCF.

[0114] In certain embodiments, the inducing step comprises performing the inducing step b3 in the presence of SCF.

[0115] In certain embodiments, step a comprises step a1.

[0116] In certain embodiments, step a1 is induced on day 0 in the presence of a GSK-3 inhibitor.

[0117] In certain embodiments, the step a1 is induced on day 0 in the presence of a GSK-3 inhibitor and a ROCK inhibitor.

[0118] In certain embodiments, the GSK-3 inhibitor is CHIR 99021.

[0119] In certain embodiments, the ROCK inhibitor is Rki.

[0120] In certain embodiments, the step a further comprises step a2.

[0121] In certain embodiments, step a2 is performed again on day 1 by inducing in the presence of a GSK-3 inhibitor.

[0122] In certain embodiments, step a2 further comprises inducing again on day 1 in the presence of CHIR 99021.

[0123] In certain embodiments, the day 0 refers to the 0th to 24th hour of preparing hematopoietic stem cells and progenitor cells according to the method of the present invention.

[0124] In certain embodiments, the first day refers to the 24th to 48th hour of preparing hematopoietic stem and progenitor cells according to the method of the present invention.

[0125] In certain embodiments, step b1 uses a cytokine combination of BMP4, FGF2, and VEGF for induction.

[0126] In certain embodiments, step b2 uses a combination of cytokines BMP4, SCF, FGF2, VEGF, and SB431542 for induction.

[0127] In certain embodiments, step b3 uses a combination of SCF, FGF2, and VEGF for induction.

[0128] In certain embodiments, the method comprises the steps of:

[0129] a1) iPSCs were cultured in suspension in StemScale PSC complete suspension medium supplemented with CHIR 99021 and RKI.

[0130] a2) Continue suspension culture in StemScale PSC complete suspension medium supplemented with CHIR 99021 to allow EB formation;

[0131] b1) culturing EBs in suspension in hematopoietic differentiation medium 1 supplemented with BMP4, FGF2, and VEGF;

[0132] b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB431542;

[0133] b3) further culturing in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, and VEGF to form HSPCs; and

[0134] c) expanding the HSPCs generated in step b) and further culturing them in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, VEGF, FLT3 ligand (FLT3L), TPO, IL-11, IGF1, IL-3, and IL-6.

[0135] In certain embodiments, step c can use a hematopoietic stem cell expansion method that is commercially available or reported in the literature.

[0136] In certain embodiments, steps b1, b2, b3 and c further comprise conventional factors for inducing cell expansion, P / S, GlutaMax, ascorbic acid and ITS-G.

[0137] Each step of the method of the present invention can be carried out at a suitable temperature for an appropriate time. Suitable temperature and time are routinely determined by those skilled in the art. For example, step a, step b and / or step c can be carried out in the temperature range of 25-42° C., preferably 30-40° C., more preferably about 35-37° C. Step a, step b (e.g., step b1, step b2 and / or step b3) and / or step c can be carried out for about 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours or 24 hours to about 28 days, to about 21 days, to about 14 days, to about 7 days, to about 3 days, to about 48 hours or to about 24 hours, for example, from about 6 hours to about 3 days, from about 12-48 hours or from about 15-24 hours; or for about 48 hours to 5 days, more preferably from about 3 days or 4 days.

[0138] In certain embodiments, step a1 is performed on day 0, step a2 is performed on day 1, step b1 is performed on day 2-3, step b2 is performed on day 4-5, step b3 is performed on day 6-7, and step c is performed on day 8-10.

[0139] In certain embodiments, the cytokines can be directly added exogenously to the culture medium to promote the directed differentiation of pluripotent stem cells.

[0140] In certain embodiments, the expression of the cytokine in the cells can be enhanced by transgenic means. Specifically, the gene encoding the additional cytokine is introduced into pluripotent stem cells in an integrating or non-integrating manner, and the gene is induced to express appropriately, thereby promoting the directed differentiation of the pluripotent stem cells. The gene can be expressed under the control of a constitutive or inducible promoter to achieve an appropriate level of expression.

[0141] In certain embodiments, endogenous expression of the cytokine can be increased by introducing suitable transcriptional or translational regulatory sequences, such as promoters. The transcriptional or translational regulatory sequences can be introduced into the target pluripotent stem cells via a suitable gene delivery method, such as by homologous recombination or gene editing. Methods for modifying endogenous genes are well known in the art, and there are numerous scientific and technological documents available for reference.

[0142] In certain embodiments, the expression level of the cytokine in pluripotent stem cells can be increased by introducing mRNA. Those skilled in the art are aware of suitable methods for delivering mRNA, including, for example, by lipofection. The mRNA used in the present invention can be in an unmodified form. Alternatively, the mRNA used in the present invention can be in a modified form to improve its stability in the cell.

[0143] In the above method of the present invention, the culture medium used in step a) can be DMEM culture medium, MEM culture medium, Essential 8 culture medium, StemScale PSC culture medium, etc. The additional cytokine added can be, for example, Y27632, and its concentration can be 0.1-200 μM, 0.5-150 μM, 1-100 μM, 2-80 μM, 5-50 μM or 10-20 μM. By culturing in an incubator under suitable culture conditions, cells can form embryoid bodies (EB). Alternatively, 0.1-100 μM, 0.2-50 μM, 0.5-25 μM or 1-20 μM CHIR99021 can be supplemented in the culture from day -1 to day 1 of the culture to promote the formation of embryoid bodies by pluripotent stem cells. As described above, the additional cytokine can be exogenously added to the culture medium, or its expression in the cell can be increased by transgenic means, or additional expression can be achieved by introducing mRNA.

[0144] The embryoid bodies obtained in step a) can be induced to form hematopoietic stem cells and progenitor cells by culturing them in the presence of additional cytokines. Suitable culture media include, for example, Stemline II hematopoietic stem cell expansion medium, StemPro TM-34SFM, XVIVO15 serum-free medium, StemSpan-AOF medium, StemSpan-SFEM medium, StemSpan SFEMII medium, APE II medium, BPEL medium (EP2841563A1), etc. The formulations of each medium are well known to those skilled in the art. For example, one composition of BPEL medium is as follows: Iscove's modified Dulbecco's medium (IMDM, 86 ml, Invitrogen), etc. F12 nutrient mix containing Glutamx I (86 mL, Invitrogen), 10% deionized bovine serum albumin (BSA, 5 mL, Sigma), 5% polyvinyl alcohol (10 mL, Sigma), linoleic acid (20 uL of a 1 mg / mL solution, Sigma), linolenic acid (20 uL of a 1 mg / mL solution, Sigma), Synthecol 500X solution (Sigma), α-thioglycerol (Sigma), Protein-free hybridoma mix II (Invitrogen), ascorbic acid (5 mg / mL, Sigma), Glutamax I (Invitrogen), insulin-transferrin-selenium 100x solution (Invitrogen), and penicillin / streptomycin (Invitrogen). Cytokines that can be added include, for example, ITS-G (see https: / / www.thermofisher.com / order / catalog / product / 41400045), BMP4, FGF2, or VEGF, or any combination thereof.

[0145] In certain embodiments, the concentration of ITS-G added to the culture medium is 0.1-10%, 0.2-5%, 0.5-2.5%, or 1-2%, based on the total volume of the culture medium.

[0146] BMP4 can be used at various suitable concentrations, for example, 0.1-2000 ng / ml, 0.2-1000 ng / ml, 0.5-500 ng / ml, 1-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, or 5-20 ng / ml based on the total volume of the culture medium.

[0147] FGF2 can also be used in various suitable concentrations. For example, based on the total volume of the culture medium, the concentration of FGF2 can be 0.1-2000 ng / ml, 0.2-1000 ng / ml, 0.5-500 ng / ml, 1-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, or 5-20 ng / ml.

[0148] VEGF can also be used at various suitable concentrations, such as 0.1-2000 ng / ml, 0.2-1000 ng / ml, 0.5-500 ng / ml, 1-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, or 5-20 ng / ml based on the total volume of the culture medium.

[0149] In certain embodiments, the embryoid bodies are suspended and further cultured in Stemline II hematopoietic stem cell expansion medium supplemented with 1% ITS-G, 1-100 ng / ml BMP4, 1-100 ng / ml FGF2, and 1-100 ng / ml VEGF. Optionally, the medium can also be supplemented with a suitable concentration of P / S, GlutaMax, and / or ascorbic acid, such as 0.1-5%, 0.5-2%, or about 1% P / S, 0.1-10 mM, 0.2-7.5 mM, 0.5-5 mM, 1-2.5 mM, or about 2 mM GlutaMax, and / or 5-500 μg / ml, 10-250 μg / ml, 25-125 μg / ml, 50-100 μg / ml, or about 50 μg / ml ascorbic acid.

[0150] SB431542 may also be added to the culture medium in step b), and its concentration may be 0.1-200 μM, 0.2-100 μM, 0.5-50 μM, 1-20 μM, 2-10 μM, or about 5 μM.

[0151] In certain embodiments, ITS-G, SCF, FGF2 and / or VEGF or any combination thereof may be added to the culture medium in step b) to further culture EBs to promote differentiation of EBs into hematopoietic stem and progenitor cells. Based on the total volume of the culture medium, the level of ITS-G added may be 0.1-5%, 0.2-2.5%, 0.5-2%, or approximately 1%; the level of SCF added may be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or approximately 10 ng / ml; the level of FGF2 added may be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or approximately 10 ng / ml. In some embodiments, the EBs are cultured in a culture medium at a concentration of 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; and / or the level of added VEGF can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml. After a period of culture in the culture medium, the EBs can be harvested for further culture.

[0152] The embryoid bodies obtained by the aforementioned culture steps can be further cultured in a hematopoietic stem cell expansion medium to differentiate into hematopoietic stem and progenitor cells and expand them. In the culture medium, inhibitory or multiple suitable factors can be added, such as one or more of ITS-G, SCF, FGF2, VEGF, FLT3 ligand (FLT3L), TPO, IL-11, IGF1, IL-3, and / or IL-6.Based on the total volume of the culture medium, the level of ITS-G added can be 0.1-5%, 0.2-2.5%, 0.5-2%, or about 1%; the level of SCF added can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; the level of FGF2 added can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; the level of VEGF added can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, The level of added FLT3L can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; the level of added TPO can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; the level of added IL-11 can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml. -20 ng / ml, or about 10 ng / ml; the level of added IG1F can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; the level of added IL-3 can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml; and / or the level of added IL-6 can be 0.1-500 ng / ml, 0.2-250 ng / ml, 0.5-200 ng / ml, 1-100 ng / ml, 2-50 ng / ml, 5-20 ng / ml, or about 10 ng / ml. After culture in hematopoietic stem cell expansion medium, EBs and suspension cells can be harvested for downstream differentiation into HSPCs or further expansion.

[0153] 6. Culture of Hematopoietic Stem and Progenitor Cells

[0154] The present invention thus relates to cultures of hematopoietic stem and progenitor cells (HSPCs) obtained by the methods of the present invention. The hematopoietic stem and progenitor cells of the present invention can be cultured under a variety of culture conditions suitable for hematopoietic stem cell culture. The culture medium employed contains nutrients required for the growth of hematopoietic stem and progenitor cells, such as serum albumin, glutamine, antibiotics, etc. The culture medium may also contain nutrients required for the maintenance and differentiation of hematopoietic stem and progenitor cells, such as various cytokines.

[0155] In one embodiment, the cells of the present invention can be suspended in any suitable culture medium, including, for example, DMEM, Stemline II hematopoietic stem cell expansion medium, F12 culture medium, etc. In another embodiment, the hematopoietic stem cells and progenitor cells of the present invention can also be cultured adherently, for example, as a monolayer cell culture. In yet another embodiment, the hematopoietic stem cells and progenitor cells of the present invention can also be cultured in three dimensions, including, for example, in a shake flask, a rotary flask, a shaking bioreactor, or a cell culture bag. After the culture is complete, the hematopoietic stem cells and progenitor cells formed are harvested mechanically or enzymatically for confirmation, preservation, and further use.

[0156] Preferably, the cell culture comprises 10 3 -10 9 More preferably, the cell culture comprises 10 4 -10 8 Most preferably, the cell culture comprises 10 5 -10 7 cells / ml, for example approximately 10 6 cells / ml.

[0157] 7. Characterization of Hematopoietic Stem and Progenitor Cells

[0158] The hematopoietic stem and progenitor cells obtained by the method of the present invention can be characterized by their cell surface markers, cytokine expression, and other characteristics. For example, hematopoietic stem and progenitor cells characteristically express the cell markers CD43 and CD34. In addition to CD34 and CD43, HSPCs also express the cell markers Sca-1, CD27, CD38, CD48, CD117, and CD150. Therefore, the hematopoietic stem and progenitor cells obtained by the method of the present invention can be identified by their protein or mRNA expression levels. For example, cells obtained by the method of the present invention can be assayed by Western blotting. Alternatively, the expression levels of the aforementioned cell markers in cells obtained by the method of the present invention can be assayed by Northern blotting or RT-PCR. In addition, the presence and levels of hematopoietic stem and progenitor cells can also be detected by flow cytometry. Optionally, the measured protein or mRNA expression levels can be compared with the corresponding expression levels of negative controls such as fibroblasts and / or known hematopoietic stem and progenitor cell positive controls to determine whether the cells of the present invention have achieved the desired phenotype.

[0159] Hematopoietic stem cells and progenitor cells have the important characteristics of self-renewal and differentiation. Therefore, the hematopoietic stem cells and progenitor cells obtained by the present invention can also be characterized by the ability of differentiating into desired blood cell types in vitro or in vivo. In one embodiment, the cell obtained by the method of the present invention is cultivated under the in vitro conditions suitable for differentiating into cells such as T cells, B cells, NK cells and myeloid cells in blood, as megakaryocytes, erythrocytes, granulocytes and macrophages, and the type of differentiated cells is determined, thus the cell of the present invention is characterized. In another embodiment, the cell culture obtained by the method of the present invention can be transplanted into an animal such as a mouse body, and determine that the cells are differentiated into cells such as T cells, B cells, NK cells and myeloid cells in blood, as the level of megakaryocytes, erythrocytes, granulocytes and macrophages. Optionally, the cell obtained by the method of the present invention is labeled, and its differentiation type and whereabouts in animal body can be easily tracked thus.

[0160] 8. Application of Hematopoietic Stem Cells and Progenitor Cells

[0161] The hematopoietic stem and progenitor cells obtained by the methods of the present invention can have a variety of in vitro and in vivo uses.

[0162] In one aspect, the hematopoietic stem cells and progenitor cells of the present invention can be used to induce differentiation into desired blood cell types in vitro or in vivo, including, for example, T cells, B cells, NK cells and myeloid lineage cells, including megakaryocytes, platelets, red blood cells, granulocytes, mast cells, monocytes, dendritic cells, macrophages and microglia, etc.

[0163] In another aspect, the hematopoietic stem and progenitor cells of the present invention can be directly transplanted into patients suffering from various diseases, where they differentiate and develop into the body's blood cell components. For example, the hematopoietic stem and progenitor cells of the present invention can be directly used in bone marrow transplantation to treat or alleviate hematologic malignancies such as leukemias and lymphomas, solid tumors, aplastic anemia / bone marrow failure states, and an ever-expanding list of autoimmune and inherited metabolic and immunodeficiency diseases. Therefore, the present invention encompasses the use of the hematopoietic stem and progenitor cells for treating the various diseases mentioned above.

[0164] HSPC-derived cell types can also be used for the immunotherapy of cancer, including but not limited to various leukemias, lymphomas, sarcomas and carcinomas. They can also be used to treat autoimmune diseases with immunomodulatory effects, such as rheumatoid arthritis, lupus, inflammatory bowel disease, multiple sclerosis, type I diabetes, psoriasis, scleroderma, vasculitis, fibrosis and neuropathy (e.g., regulatory T cells).

[0165] Other potential applications of HSPC-derived cell types include, but are not limited to, targeting bacterial / viral infections and vaccination (e.g., dendritic cells and B cells), and transfusion (e.g., red blood cells and platelets).

[0166] The present invention relates to the prevention, treatment, or alleviation of various blood diseases, tumors, immune diseases, infections, and the like, such as delaying their progression. In some embodiments, the blood disease is a malignant blood disease, such as leukemia, lymphoma, or myeloma, and is preferably selected from multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, and the like.

[0167] The cells used in the present invention can be hematopoietic stem and progenitor cells (HSPCs) directly differentiated from pluripotent stem cells, expanded HSPCs, thawed or further expanded HSPCs that have been cryopreserved, and / or in vitro differentiated HSPCs. Therefore, the present invention also relates to hematopoietic stem and progenitor cells generated by the methods of the present invention, as well as differentiated cells derived from further culture or induced differentiation, and cultures thereof.

[0168] The HSPC cells of the present invention may also be genetically modified to improve their function and / or activity.

[0169] The cells of the present invention can also be used in combination with one or more other drugs to improve their therapeutic effects. The other drugs can be selected from, for example, chemotherapeutic agents, radiotherapeutic agents, cytokines, anti-infective agents, immunopotentiators, etc.

[0170] In certain embodiments, the present invention also provides a kit for inducing pluripotent stem cells to generate embryoid bodies, which comprises factors for implementing one or more steps included in the method of the present invention, preferably comprising a combination of factors for implementing steps a and b (for example, including steps b1, b2 and / or b3) and optionally step c.

[0171] The present invention provides a method for preparing hematopoietic stem cells and progenitor cells, the method comprising the following steps:

[0172] a. inducing embryonic stem cells or induced pluripotent stem cells (iPSCs) to differentiate into embryoid bodies (EBs) in the presence of a GSK-3 inhibitor; and

[0173] b. Inducing the EBs formed in step a to differentiate into hematopoietic stem and progenitor cells (HSPCs) in a hematopoietic differentiation medium comprising one or more factors selected from the group consisting of BMP4, SCF, and SB431542.

[0174] In certain embodiments, the inducing in step b comprises performing the inducing step b1 in the presence of BMP4.

[0175] In certain embodiments, the inducing in step b comprises performing the inducing step b2 in the presence of BMP4, SCF, and SB431542.

[0176] In certain embodiments, the inducing in step b comprises performing the inducing step b3 in the presence of SCF.

[0177] In certain embodiments, step a comprises step a1.

[0178] In certain embodiments, step a1 is induced on day 0 in the presence of a GSK-3 inhibitor.

[0179] In certain embodiments, the step a1 is induced on day 0 in the presence of a GSK-3 inhibitor and a ROCK inhibitor.

[0180] In certain embodiments, the GSK-3 inhibitor is CHIR 99021 and the ROCK inhibitor is Rki.

[0181] In certain embodiments, the step a further comprises step a2.

[0182] In certain embodiments, step a2 is performed again on day 1 by inducing in the presence of a GSK-3 inhibitor.

[0183] In certain embodiments, step a2 is performed again on day 1 in the presence of CHIR 99021.

[0184] In certain embodiments, the method comprises the steps of:

[0185] a1) iPSCs were cultured in suspension in StemScale PSC complete suspension medium supplemented with CHIR 99021 and Rki.

[0186] a2) Continue suspension culture in StemScale PSC complete suspension medium supplemented with CHIR 99021 to allow EB formation;

[0187] b1) culturing EBs in suspension in hematopoietic differentiation medium 1 supplemented with BMP4, FGF2, and VEGF;

[0188] b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF, and SB431542;

[0189] b3) further culturing in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, and VEGF to form HSPCs; and

[0190] c) expanding the HSPCs generated in step b) and further culturing them in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, VEGF, FLT3 ligand (FLT3L), TPO, IL-11, IGF1, IL-3, and IL-6.

[0191] In certain embodiments, step a1 is performed on day 0.

[0192] In certain embodiments, step a2 is performed on day 1.

[0193] In certain embodiments, step b1 is performed on day 2-3.

[0194] In certain embodiments, step b2 is performed on day 4-5.

[0195] In certain embodiments, step b3 is performed on day 6-7.

[0196] In certain embodiments, step c is performed on day 8-10.

[0197] In certain embodiments, the HSPCs have a CD34+CD43+ phenotype.

[0198] In certain embodiments, the HSPCs have a CD235a-CD14-CD43+CD34+ phenotype.

[0199] The present invention also provides HSPCs prepared according to the method and cultures thereof.

[0200] The present invention also provides a pharmaceutical preparation comprising a therapeutically or prophylactically effective amount of the HSPC, a culture thereof, a pharmaceutically acceptable carrier or diluent, and optionally one or more other active agents.

[0201] The present invention also provides a use of the HSPC or its culture, or the pharmaceutical preparation in preparing a preparation for cell transplantation-related diseases.

[0202] In certain embodiments, the disease is selected from the group consisting of leukemia, lymphoma, myeloma, solid tumors such as sarcoma, carcinoma, rheumatoid arthritis, lupus, inflammatory bowel disease, multiple sclerosis, type I diabetes, psoriasis, scleroderma, vasculitis, fibrosis and neuropathies, immune diseases, infections.

[0203] In certain embodiments, the disease is multiple myeloma, acute myeloid leukemia, or myelodysplastic syndrome.

[0204] The present invention also provides a kit for inducing iPSCs to generate EBs, comprising a combination of factors for implementing one or more steps.

[0205] The present invention is illustrated below by way of examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention in any way. The scope of protection of the present invention shall be subject to the appended claims.

[0206] Example

[0207] The following reagents were used in this example:

[0208] StemScale PSC Suspension Medium: ThermoFisher, #A4965001

[0209] Y27632:Tocris,#1254 / #TB1254-GMP

[0210] CHIR99021:Sigma,SML1046

[0211] Stemline II Hematopoietic Stem Cell Expansion Medium: Sigma, #S0192

[0212] Penicillin-Streptomycin (P / S): ThermoFisher, #15140122

[0213] GlutaMAX Supplement: ThermoFisher, #35050061

[0214] L - Ascorbic acid: Sigma - Aldrich, #A4544

[0215] ITS - G: ThermoFisher, #41400045

[0216] BMP4: Miltenyi Biotec, #130 - 111 - 167

[0217] FGF2: Miltenyi Biotec, #130 - 093 - 564

[0218] VEGF: Miltenyi Biotec, #130 - 109 - 396

[0219] SB431542: Tocris, #1614 / TB1614 - GMP

[0220] SCF: Miltenyi Biotec, #130 - 096 - 695

[0221] DPBS: ThermoFisher, #14190 - 144

[0222] IL - 11: Miltenyi Biotec, #130 - 103 - 439

[0223] IL - 6: Miltenyi Biotec, #130 - 093 - 932

[0224] TPO: Miltenyi Biotec, #130 - 095 - 752

[0225] IGF - 1: Miltenyi Biotec, #130 - 093 - 886

[0226] IL - 3: Miltenyi Biotec, #130 - 095 - 070

[0227] FLT3L: Miltenyi Biotec, #130 - 096 - 477

[0228] Example 1 (Condition #8)

[0229] Human iPSCs are typically maintained in commercially available Essential 8 Medium. When iPSCs reach 70% confluence, they are checked for healthy morphology and absence of spontaneous differentiation. iPSCs are dissociated into single cells using Accutase. Collected single cells are counted and plated in StemScale PSC Suspension Complete Medium supplemented with 5-50 μM Y27632.

[0230] The cells were incubated in a 37°C, 5% CO2 incubator on a CO2-resistant orbital shaker with continuous rotation at 30-100 rpm. Within 24 hours (day 0), a half-fluid change was performed using StemScale PSC suspension complete medium with the addition of the ROCK inhibitor Rki and the GSK-3 inhibitor CHIR 99021.

[0231] After 24 hours (day 1), CHIR 99021 was added.

[0232] After 24 hours (day 2), the formed embryoid bodies (EBs) were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 1.

[0233] After 24-48 hours (days 3-4), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, 1-100ng / ml VEGF, 1-100ng / ml SCF and 1-20uM SB431542 as hematopoietic differentiation medium 2.

[0234] After 24-72 hours (days 5-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml SCF, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 3.

[0235] After 24 hours (day 8), EBs and suspension cells were harvested in Stemline II hematopoietic stem cell expansion medium containing 50 ug / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml SCF, 1-100 ng / ml FGF2, 1-100 ng / ml VEGF, 1-100 ng / ml FLT3 ligand (FLT3L), 1-100 ng / ml TPO, 1-100 ng / ml IL-11, 1-100 ng / ml IGF1, 1-100 ng / ml IL-3, 1-100 ng / ml IL-6 and supplemented with 1% P / S, 2 mM GlutaMax as hematopoietic differentiation medium 4 for downstream differentiation or further expansion. On days 8-10, HSPCs were harvested and assayed for cell surface markers. The results are shown in FIG3 . As can be seen from the results in FIG3 , the HSPCs generated by the method of the present invention can express the cell markers CD34 and CD43 of hematopoietic stem cells and progenitor cells, and the main cell type is CD34+ / CD43+. It can be seen that this method obtains HPCs of the hematopoietic lineage.

[0236] Example 2 (Condition #4)

[0237] Human iPSCs are typically maintained in commercially available Essential 8 Medium. When iPSCs reach 70% confluence, they are checked for healthy morphology and absence of spontaneous differentiation. iPSCs are dissociated into single cells using Accutase. Collected single cells are counted and plated in StemScale PSC Suspension Complete Medium supplemented with 5-50 μM Y27632.

[0238] The cells were incubated in a 37°C, 5% CO2 incubator on a CO2-resistant orbital shaker with continuous rotation at 30-100 rpm. Within 24 hours (day 0), a half-fluid change was performed with StemScale PSC suspension complete medium supplemented with the ROCK inhibitor Rki.

[0239] After 24 hours (day 1), CHIR 99021 (2X) was added.

[0240] After 24 hours (day 2), the formed embryoid bodies (EBs) were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 1.

[0241] After 24-48 hours (days 3-4), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, 1-100ng / ml VEGF, 1-100ng / ml SCF and 1-20uM SB431542 as hematopoietic differentiation medium 2.

[0242] After 24-72 hours (days 5-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml SCF, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 3.

[0243] After 24 hours (day 8), EBs and suspension cells were harvested in Stemline II hematopoietic stem cell expansion medium containing 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml SCF, 1-100 ng / ml FGF2, 1-100 ng / ml VEGF, 1-100 ng / ml FLT3 ligand (FLT3L), 1-100 ng / ml TPO, 1-100 ng / ml IL-11, 1-100 ng / ml IGF1, 1-100 ng / ml IL-3, 1-100 ng / ml IL-6 and supplemented with 1% P / S, 2 mM GlutaMax as hematopoietic differentiation medium 4 for downstream differentiation or further expansion. On days 8-10, HSPCs were harvested and assayed for cell surface markers.

[0244] Example 3 (Condition #6)

[0245] Human iPSCs are typically maintained in commercially available Essential 8 Medium. When iPSCs reach 70% confluence, they are checked for healthy morphology and absence of spontaneous differentiation. iPSCs are dissociated into single cells using Accutase. Collected single cells are counted and plated in StemScale PSC Suspension Complete Medium supplemented with 5-50 μM Y27632.

[0246] The cells were incubated in a 37°C, 5% CO2 incubator on a CO2-resistant orbital shaker with continuous rotation at 30-100 rpm. Within 24 hours (day 0), a half-fluid change was performed using StemScale PSC suspension complete medium with the addition of the ROCK inhibitor Rki and the GSK-3 inhibitor CHIR 99021.

[0247] After 24 hours (day 1), CHIR 99021 was added.

[0248] After 24-48 hours (day 2-3), the formed embryoid bodies (EBs) were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 1.

[0249] After 24-48 hours (days 4-5), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml BMP4, 1-100ng / ml FGF2, 1-100ng / ml VEGF, 1-100ng / ml SCF and 1-20uM SB431542 as hematopoietic differentiation medium 2.

[0250] After 24-48 hours (day 6-7), EBs were harvested and resuspended in Stemline II hematopoietic stem cell expansion medium supplemented with 1% P / S, 2mM GlutaMax, 50ug / ml ascorbic acid, 1% ITS-G, 1-100ng / ml SCF, 1-100ng / ml FGF2, and 1-100ng / ml VEGF as hematopoietic differentiation medium 3.

[0251] After 24 hours (day 8), EBs and suspension cells were harvested in Stemline II Hematopoietic Stem Cell Expansion Medium containing 50 μg / ml ascorbic acid, 1% ITS-G, 1-100 ng / ml SCF, 1-100 ng / ml FGF2, 1-100 ng / ml VEGF, 1-100 ng / ml FLT3 ligand (FLT3L), 1-100 ng / ml TPO, 1-100 ng / ml IL-11, 1-100 ng / ml IGF1, 1-100 ng / ml IL-3, 1-100 ng / ml IL-6 and supplemented with 1% P / S and 2 mM GlutaMax as hematopoietic differentiation medium 4 for downstream differentiation or further expansion. On days 8-10, HSPCs were harvested and cell surface markers were measured. The results of the cell morphology and surface marker detection are shown in Figures 4-5.

[0252] Example 4-8 (Conditions #1-3, 5, 7)

[0253] The culture methods of Examples 4-8 (Conditions #1-3, 5, and 7) were basically the same as those of Example 1. For specific conditions, see Table 1. The ratio of CD34+ / CD43+ cells on day 8 of differentiation induced by different conditions was tested, and the results are shown in FIG6 .

[0254] Table 1 Specific conditions for generating HSPCs from iPSCs

[0255] Example 9

[0256] From a morphological perspective, the addition of CHIR (condition 6) during the iPSC culture stage (i.e., day 0 in Table 1) was observed. The results are shown in Figure 7. It can be seen that the addition of CHIR allows the formation of iPSC spheres during the iPSC culture stage before the differentiation begins, thereby achieving a larger number of spheres with better uniformity for downstream differentiation.

[0257] We further attempted to extend the induction time of CHIR99021 to 2 days during the stage of iPSC induction to form mesoderm (i.e., days 1-3 of condition 6). The results are shown in Figure 8. It can be seen that adding CHIR99021 for only 1 day is more conducive to the release of HPC cells from HE EBs than adding it for 2 days, and is also more beneficial to the overall cell activity / growth.

[0258] Four batches of stability experiments were conducted under condition 6. The results are shown in Figure 9. The CD34 cells in the hematopoietic EBs are mostly early progenitor cells (CD43-), while the suspended cells released from the EB spheres can reach >50% CD34 positivity and >90% CD43 positivity, indicating that the suspended cells released from the EB spheres are mostly hematopoietic lineage progenitor cells.

[0259] Example 11 Downstream NK cell differentiation of HSPC prepared by the present invention

[0260] Harvested HSPCs were suspended in NK differentiation medium for iNK differentiation. On day 28 of iNK cell differentiation, the cultures were monitored for the emergence of spindle-shaped iNK-like cells and assessed for CD45 / CD56 expression.

[0261] iNK cell expansion was performed on day 28. iNK cells were harvested in NK expansion medium. On day 12 of iNK cell expansion, iNK cells were harvested and subjected to downstream immune differentiation experiments. The experiment was repeated four times to verify the stability and reproducibility of differentiation. The results are shown in Figure 10.

[0262] Example 12 Downstream T cell differentiation of HSPC prepared by the present invention

[0263] The harvested HSPCs were suspended in T cell differentiation medium for differentiation. iT cell expansion was performed on day 22 of T cell differentiation. On day 14 of iT cell expansion, iT cells were harvested and subjected to iPSC residual and cytokine release experiments. The experiment was repeated four times to verify the stability and reproducibility of differentiation. The results are shown in Figures 11-12.

[0264] Example 13 Downstream Macrophage Cell Differentiation of HSPC Prepared by the Present Invention

[0265] The harvested HSPCs were suspended in macrophage differentiation medium for differentiation. Phase contrast images were acquired on days 0, 1, 3, 10, 12, 18, 19, and 24 of differentiation. The cell morphology changes are shown in Figure 13A.

[0266] After iMAC differentiation (day 24), the cells were digested and harvested for purity testing. The expression of major immune cell markers on iMAC cells was determined by flow cytometry. The purity of the differentiated iMACs reached 98.80%.

[0267] Flow cytometry was used to compare the expression of macrophage-associated markers (CD68, CD86, and CD80) on unpolarized iMACs (i-M0) and monocyte-derived macrophages (m-M0). The percentage of marker-positive cells in the tested cell populations and the median fluorescence intensity (MFI) of the corresponding markers were determined. The results, shown in Figure 13B, show that iMACs differentiated using the present method exhibit similar expression of key markers to monocyte-derived macrophages.

[0268] Example 14 Downstream B cell differentiation of HSPC prepared by the present invention

[0269] The harvested HSPCs were suspended in B differentiation medium for differentiation. iB cells were harvested on day 14 of B cell differentiation and subjected to morphological analysis and marker detection. The experimental results are shown in Figures 14-17.

[0270] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A method for preparing hematopoietic stem cells and progenitor cells, wherein the method comprises the following steps: a. inducing embryonic stem cells or induced pluripotent stem cells to differentiate into embryoid bodies in the presence of a GSK-3 inhibitor; and b. inducing the EB formed in step a to differentiate into hematopoietic stem cells and progenitor cells in a hematopoietic differentiation medium comprising one or more factors selected from the following: BMP4, SCF, SB431542; Wherein the step a comprises step a1, wherein the step a1 is induced on day 0 in the presence of a GSK-3 inhibitor; The induction in step b comprises performing induction step b1 in the presence of BMP4, performing induction step b2 in the presence of BMP4, SCF and SB431542, and performing induction step b3 in the presence of SCF.

2. The method according to claim 1, wherein the step a1 is induced on day 0 in the presence of a GSK-3 inhibitor and a ROCK inhibitor.

3. The method according to claim 2, wherein the GSK-3 inhibitor is CHIR 99021 and the ROCK inhibitor is Rki.

4. The method according to claim 1, wherein the step a further comprises a step a2, wherein the step a2 is to induce again on day 1 in the presence of a GSK-3 inhibitor.

5. The method according to claim 4, wherein the step a2 is to induce again on day 1 in the presence of CHIR 99021.

6. The method according to any one of claims 1 to 5, wherein the method comprises the following steps: a1) iPSCs were cultured in suspension in StemScale PSC complete suspension medium supplemented with CHIR 99021 and Rki; a2) Continue suspension culture in StemScale PSC suspension complete medium supplemented with CHIR 99021 to form EBs; b1) EBs were cultured in suspension in hematopoietic differentiation medium 1 supplemented with BMP4, FGF2, and VEGF; b2) continuing suspension culture in hematopoietic stem cell expansion medium supplemented with BMP4, SCF, FGF2, VEGF and SB431542; b3) further culturing in suspension in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2 and VEGF to form HSPCs; and c) The HSPCs generated in the expansion step b) are further suspended and cultured in a hematopoietic stem cell expansion medium supplemented with SCF, FGF2, VEGF, FLT3 ligand, TPO, IL-11, IGF1, IL-3, and IL-6.

7. The method according to claim 6, wherein the step a1 is performed on day 0, the step a2 is performed on day 1, the step b1 is performed on day 2-3, the step b2 is performed on day 4-5, the step b3 is performed on day 6-7, and the step c is performed on day 8-10.

8. The method of claim 6, wherein the HSPCs have a CD34+CD43+ phenotype.

9. The method of claim 8, wherein the HSPCs have a CD235a-CD14-CD43+CD34+ phenotype.

10. A HSPC prepared according to the method of any one of claims 1-9 and a culture thereof.

11. A pharmaceutical preparation comprising a therapeutically or prophylactically effective amount of the HSPC according to claim 10, a culture thereof, a pharmaceutically acceptable carrier or diluent, and optionally one or more other active agents.

12. Use of the HSPC or culture thereof according to claim 10, or the pharmaceutical preparation according to claim 11, in preparing a preparation for cell transplantation-related diseases.

13. The use according to claim 12, wherein the disease is selected from the group consisting of leukemia, lymphoma, myeloma, solid tumors such as sarcoma, cancer, rheumatoid arthritis, lupus, inflammatory bowel disease, multiple sclerosis, type I diabetes, psoriasis, scleroderma, vasculitis, fibrosis and neuropathy, immune diseases, infections.

14. The use according to claim 13, wherein the disease is multiple myeloma, acute myeloid leukemia or myelodysplastic syndrome.

15. A kit for inducing iPSCs to generate EBs, wherein the kit comprises a combination of factors for implementing one or more steps described in claims 1-6.

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