Culture medium composition for differentiating pluripotent stem cells into hematopoietic stem progenitor cells as well as use method and application of culture medium composition

Abstract

The invention provides a culture medium composition for differentiating from pluripotent stem cells to hematopoietic stem progenitor cells as well as a use method and application of the culture medium composition. The method is clear in stage, repeatable in result, capable of effectively generating the hematopoietic endothelial cells and remarkably improving the differentiation efficiency and function of the hematopoietic stem cells, meanwhile, the differentiation period is shortened, operation is easy and convenient, the GMP standard is met, and the wide requirement from basic research to large-scale industrial production is met.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine. In particular, the present disclosure relates to a culture medium composition for differentiating pluripotent stem cells into hematopoietic stem and progenitor cells, a method for using the same, and an application thereof. Background Art

[0002] Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have attracted much attention in recent years in the fields of regenerative medicine, disease models, drug screening, and gene therapy due to their unlimited self-renewal ability and potential to differentiate into various somatic cell types. Generating hematopoietic stem / progenitor cells (HSPCs) from hPSCs is expected to provide new cell therapy strategies for blood system diseases such as leukemia and aplastic anemia.

[0003] Generating hematopoietic stem cells (HSCs) starting from pluripotent stem cells (hPSCs) involves the selection of multiple lineage branches. Therefore, when designing an in vitro differentiation protocol, it is necessary to fully consider the microenvironment and niche signals that regulate embryonic hematopoietic fate at specific stages to control the differentiation direction of cells. Current experimental protocols usually differentiate human pluripotent stem cells into mesoderm by using signals such as BMP, FGF, TGF-β, and / or WNT, and then add vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), and other factors promoting hematopoiesis to further guide the cells to differentiate into hematopoietic stem cells. However, these signals may also lead to the formation of cells of other lineages, thus affecting the differentiation efficiency and cell purity of hematopoietic stem cells.

[0004] More and more studies have shown that hematopoietic stem cells in the human embryo originate from hemogenic endothelial cells in the AGM region. These cells generate HSCs through endothelial-hematopoietic transition, migrate to the liver of the embryo through blood circulation for amplification, and then transfer to the bone marrow to produce bone marrow hematopoiesis. Therefore, when generating functional hematopoietic stem cells in vitro, it is necessary to first generate hemogenic endothelial cells.

[0005] There are problems and deficiencies in the current technology for differentiating hPSCs into hematopoietic stem cells in vitro:

[0006] (1) The differentiation efficiency of hematopoietic stem and progenitor cells (CD34 + CD43 + CD45 + ) is low and difficult to repeat: Existing differentiation protocols for inducing hPSCs to differentiate into CD34 + CD43 + CD45 +When differentiating into hematopoietic cells, the differentiation efficiency is usually low, and the experimental results are not easily reproducible among pluripotent stem cells from different sources or different laboratories. This inefficiency and irreproducibility not only affect the reliability of basic research but also pose great challenges to large-scale production and clinical applications.

[0007] (2) Lack of generation of hematopoietic endothelial cells (CD34 + CD144 + CD43 - CD73 - ) generation: Existing literature shows that the typical phenotype of hematopoietic endothelial cells (HECs) is CD34 + CD144 + CD43 - CD73 - . However, the endothelial cells generated by current non-transgenic techniques are mainly of the CD73 + phenotype, rather than hematopoietic endothelial cells. Hematopoietic endothelial cells play a key role in embryonic hematopoiesis. They generate functional hematopoietic stem cells through endothelial-hematopoietic transition (EHT) and promote the development of the hematopoietic system. Therefore, if hematopoietic endothelial cells cannot be effectively generated during in vitro differentiation, it will significantly limit the differentiation efficiency and function of hematopoietic stem cells, thus hindering the efficient generation of functional hematopoietic cells.

[0008] (3) Long differentiation cycle: Existing in vitro differentiation methods usually require a long culture period (usually 15 - 20 days), increasing the time cost.

[0009] Given the above problems, there is an urgent need to find a new solution. Summary of the Invention

[0010] Technical problems to be solved:

[0011] First, the present disclosure addresses the drawback in the prior art that the differentiation system of hematopoietic stem and progenitor cells lacks the generation of hematopoietic endothelial cells with the CD73 - phenotype. Secondly, aiming at the drawback of the long differentiation cycle of hematopoietic stem and progenitor cells in the prior art, a new differentiation idea and system are provided.

[0012] Technical solutions:

[0013] In the first aspect of the present disclosure, a culture medium composition for differentiating pluripotent stem cells (PSCs) into hematopoietic stem and progenitor cells (HSCs) is provided, and the composition includes:

[0014] Culture medium A: The culture medium A includes a basal medium, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (FGF2), activin A, and a GSK-3 inhibitor;

[0015] Culture medium B: The culture medium B includes a basal medium, BMP4, a PI3K inhibitor, an adenylate cyclase activator, a TGF-β inhibitor, vascular endothelial growth factor (VEGF), a Wnt / β-catenin inhibitor, and L-ascorbic acid 2-phosphate (AA2P); and

[0016] Culture medium C: The culture medium C includes a basal medium, stem cell factor (SCF), FGF2, thrombopoietin (TPO), VEGF, interleukin 3 (IL3), and AA2P.

[0017] In some embodiments, culturing pluripotent stem cells in a staged manner using the above culture medium composition can surprisingly produce hematopoietic endothelial cells with typical phenotypes of CD34 + CD144 + CD43 - CD73 - Furthermore, the object of the present disclosure is achieved. Generally, methods for differentiating hematopoietic endothelial cells into hematopoietic stem and progenitor cells are known in the art. However, to further achieve the object of the present disclosure, in some embodiments, the culture medium composition further includes: Culture medium D: The culture medium D includes a basal medium, SCF, FGF2, TPO, VEGF, FMS-like tyrosine kinase 3 ligand (FIT3L), IL3, interleukin 6 (IL6), and AA2P.

[0018] In some embodiments, any suitable GSK-3 inhibitor can be used. For example, in some embodiments, the above GSK-3 inhibitor can be at least one selected from CHIR99021, BIP-135, CP21R7, 1-Azakenpaullone, TDZD-8, SB216763, SB415286, BIO-acetoxime, Tideglusib, AR-A014418, lithium chloride, VP3.15, GSK-3 inhibitor 1, or GSK-3 inhibitor 3. Preferably, in one embodiment, the above GSK-3 inhibitor can be CHIR99021.

[0019] In some embodiments, any suitable one of the PI3K inhibitors can be used. For example, in some embodiments, the above PI3K inhibitor can be at least one selected from GDC-0941, Buparlisib, Copanlisib, GSK1059615, Alpelisib, Inalidex, Idelalisib, Duvelisib, IPI-549, Dactolisib, Omipalisib, Taselisib, Seletalisib, RP-5264, LY294002 or Wortmannin. Preferably, in one embodiment, the above PI3K inhibitor can be GDC-0941.

[0020] In some embodiments, any suitable one of the adenylate cyclase activators can be used. For example, in some embodiments, the above adenylate cyclase activator can be at least one selected from Forskolin, epinephrine, norepinephrine, dobutamine, milrinone, levosimendan, adrenocorticotropic hormone or glucagon. Preferably, in one embodiment, the above adenylate cyclase activator can be Forskolin.

[0021] In some embodiments, any suitable one of the TGF-β inhibitors can be used. For example, in some embodiments, the above TGF-β inhibitor can be at least one selected from SB431542, Galunisertib, Vactosertib, LY3200882, Fresolimumab, NIS793, AVID200, Sotatercept, Trabedersen, Decorin, BMP-7 or Cilengitide. Preferably, in one embodiment, the above TGF-β inhibitor can be SB431542.

[0022] In some embodiments, any suitable one of the Wnt / β-catenin inhibitors can be used. For example, in some embodiments, the above Wnt / β-catenin inhibitor can be at least one selected from XAV939, IWR-1-endo, WIKI4, iCRT3, CWP232228, PRI-724, ICG-001, Nitazoxanide, BC2059, Dictamnine, Longdaysin, Fz7-21 or Zamaporvint. Preferably, in one embodiment, the above Wnt / β-catenin inhibitor can be XAV939.

[0023] The second aspect of the present disclosure is to provide a method for generating hematopoietic endothelial cells (HECs), including:

[0024] Step a) contacting pluripotent stem cells with the above-mentioned culture medium A for about 1 to 2 days to obtain mesoderm progenitor cells;

[0025] Step b) replacing the culture medium to contact the above-mentioned mesoderm progenitor cells with the above-mentioned culture medium B for about 1 to 2 days to obtain lateral plate mesoderm cells;

[0026] Step c) replacing the culture medium to contact the above-mentioned lateral plate mesoderm cells with the above-mentioned culture medium C for about 3 to 6 days to obtain hematopoietic endothelial cells.

[0027] In some embodiments, the above-mentioned pluripotent stem cells can be human pluripotent stem cells; the human pluripotent stem cells can be human embryonic stem cells or human induced pluripotent stem cells (iPSCs).

[0028] The third aspect of the present disclosure is to provide hematopoietic endothelial cells or a cell population thereof generated by or directly from the above method, and the phenotype of the hematopoietic endothelial cells is CD34 + CD144 + CD43 - CD73 - .

[0029] The fourth aspect of the present disclosure is to provide a method for generating hematopoietic stem and progenitor cells, including:

[0030] Step a) contacting pluripotent stem cells with the above-mentioned culture medium A for about 1 to 2 days to obtain mesoderm progenitor cells;

[0031] Step b) replacing the culture medium to contact the above-mentioned mesoderm progenitor cells with the above-mentioned culture medium B for about 1 to 2 days to obtain lateral plate mesoderm cells;

[0032] Step c) replacing the culture medium to contact the above-mentioned lateral plate mesoderm cells with the above-mentioned culture medium C for about 3 to 6 days to obtain the hematopoietic endothelial cells;

[0033] Step d) replacing the culture medium to contact the above-mentioned hematopoietic endothelial cells with a hematopoietic stem and progenitor cell differentiation medium to obtain the hematopoietic stem and progenitor cells.

[0034] Generally, the above-mentioned hematopoietic stem and progenitor cell differentiation medium is well-known in the art. However, in order to further achieve the purpose of the present disclosure, in some embodiments, the hematopoietic stem and progenitor cell differentiation medium is the above-mentioned culture medium D.

[0035] In some embodiments, the above-mentioned pluripotent stem cells can be human pluripotent stem cells; the human pluripotent stem cells can be human embryonic stem cells or human induced pluripotent stem cells (iPSCs).

[0036] A fourth aspect of the present disclosure is to provide hematopoietic stem and progenitor cells or a cell population thereof produced by or directly from the above-described method.

[0037] A fifth aspect of the present disclosure is to provide a composition for cell therapy, which composition comprises the above-described hemogenic endothelial cells or a cell population thereof, or the above-described hematopoietic stem and progenitor cells or a cell population thereof.

[0038] A sixth aspect of the present disclosure is to provide an in vitro cell differentiation system, comprising:

[0039] a) pluripotent stem cells;

[0040] b) a culture system for pluripotent stem cells;

[0041] c) a plurality of culture medium storage devices, which at least store the above-described culture medium A, culture medium B, and culture medium C, and optionally store the above-described culture medium D; and

[0042] d) a system or device for replacing the culture medium A, culture medium B, culture medium C, or culture medium D.

[0043] A seventh aspect of the present disclosure is to provide the above-described culture medium composition, the above-described hemogenic endothelial cells or a cell population thereof, the above-described hematopoietic stem and progenitor cells or a cell population thereof, the above-described composition for cell therapy, or the above-described in vitro cell differentiation system for use in the in vitro preparation of mesoderm progenitor cells, lateral plate mesoderm cells, hemogenic endothelial cells, hematopoietic stem cells, hematopoietic progenitor cells, erythroid progenitor cells, proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatic erythroblasts, reticulocytes, erythrocytes, megakaryocyte progenitor cells, megakaryocytes, platelets, granulocyte-macrophage progenitor cells (GMPs), neutrophils, eosinophils, basophils, tissue macrophages, dendritic cells (DCs), mast cells, T lymphocytes (T Cells), B lymphocytes (B Cells), natural killer cells (NK Cells), or innate lymphoid cells (ILCs).

[0044] Effects of the present disclosure:

[0045] The protocol for the in vitro differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells in the present disclosure has a clear and repeatable process, can generate hemogenic endothelial cells, improve the differentiation efficiency and function of hematopoietic stem cells, and shorten the differentiation time. Specifically:

[0046] (1) Improve the differentiation efficiency of hematopoietic stem and progenitor cells (CD34 + CD43 + CD45 + ) and ensure high repeatability: This protocol gradually differentiates induced pluripotent stem cells into mesoderm progenitor cells, lateral plate mesoderm cells, hemogenic endothelial cells, and hematopoietic stem cells by precisely controlling the composition of the culture medium and the timing of growth factor addition. The entire differentiation process is clear and applicable to human pluripotent stem cells from multiple sources. The final proportion of CD34 + CD43 + CD45 + cells can reach 65% - 95%, and has good repeatability and stability.

[0047] (2) Can generate hemogenic endothelial cells (CD34 + CD144 + CD43 - CD73 - ): This protocol can effectively generate CD73 - hemogenic endothelial cells, which play a key role in embryonic hematopoiesis, can generate functional hematopoietic stem cells through endothelial-hematopoietic transition (EHT), and promote the development of the hematopoietic system.

[0048] (3) The obtained hematopoietic stem and progenitor cells have complete functions: This protocol further analyzes the phenotype (CD34 + CD38 - CD45RA - CD90 + CD49f + ) of the differentiated hematopoietic stem and progenitor cells, and verifies their functionality. The obtained cells have strong colony-forming ability and in vitro expansion ability, and can efficiently differentiate into enucleated red blood cells and natural killer (NK) cells, proving their functional hematopoietic potential.

[0049] (4) Shorten the differentiation cycle: This protocol can obtain high-purity hematopoietic stem cells in about 10 days. Compared with existing methods, the differentiation time is significantly shortened, the operation is more convenient, and the differentiation efficiency and operability are greatly improved.

[0050] By optimizing the culture conditions and differentiation process, the technical solution disclosed herein provides an efficient, stable and simple method for differentiating human pluripotent stem cells into hematopoietic stem cells, which is applicable to clinical applications and large-scale production in the field of regenerative medicine and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the protocol for inducing pluripotent stem cells to differentiate into hematopoietic stem / progenitor cells by monolayer differentiation method in the embodiments of the present disclosure;

[0052] Figure 2 Photographs of cell morphology during the differentiation process in Example 2 of the present disclosure, scale bar, 200 μm;

[0053] Figure 3 Graph showing the results of qPCR gene expression analysis of cells harvested on day 0, day 1 and day 2 in Examples 1, 2 and 3 of the present disclosure;

[0054] Figure 4 Graph showing the results of qPCR gene expression analysis of cells harvested on day 2 in Examples 1, 2 and 3 of the present disclosure and on day 4 in Comparative Example 1, wherein, A is the graph of results of genes related to lateral plate mesoderm cells, B is the graph of results of genes related to paraxial mesoderm cells, C is the graph of results of genes related to endoderm cells, and D is the graph of results of genes related to ectoderm cells;

[0055] Figure 5 Graph showing the results of qPCR gene expression analysis of cells harvested on day 6 in Examples 1, 2 and 3 of the present disclosure and on day 8 in Comparative Example 1;

[0056] Figure 6 Graph showing the flow cytometry detection results of the phenotypes (CD34, CD43, CD144, CD73, CD184) of cells harvested on day 6 in Examples 1, 2 and 3 of the present disclosure and on day 8 in Comparative Example 1;

[0057] Figure 7 Graph showing the flow cytometry detection results of the phenotypes CD34 / CD43 / CD45 / CD144 of suspended cells harvested on day 10 in Examples 1, 2 and 3 of the present disclosure and on day 12 in Comparative Example 1;

[0058] Figure 8 Graph showing the flow cytometry detection results of the phenotypes CD34 / CD38 / CD45RA / CD90 / CD49f of suspended cells harvested on day 10 in Examples 1, 2 and 3 of the present disclosure and on day 12 in Comparative Example 1;

[0059] Figure 9 Graph showing the results of hematopoietic-related gene expression analysis of suspended cells harvested on day 10 in Examples 1, 2 and 3 of the present disclosure and CD34+ cells derived from human umbilical cord blood;

[0060] Figure 10 Hematopoietic colony formation results of the suspension cells harvested on the 10th day in Examples 1, 2, and 3 of the present disclosure and on the 12th day in Comparative Example 1. Scale bar, 100 μm;

[0061] Figure 11 Graph showing the analysis results of hematopoietic-related gene expression of the suspension cells harvested on the 10th day in Examples 1, 2, and 3 of the present disclosure and CD34+ cells derived from human umbilical cord blood;

[0062] Figure 12 Graph showing the results of differentiation into red blood cells in Examples 1, 2, and 3 of the present disclosure;

[0063] Figure 13 Graph showing the results of differentiation into NK cells in Example 1 of the present disclosure. Detailed implementation manners

[0064] The present disclosure provides a culture medium composition for differentiating pluripotent stem cells into hematopoietic stem and progenitor cells, its usage method and application. Those skilled in the art can draw on the content herein and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. And those skilled in the art can obviously make changes or appropriate alterations and combinations to the content described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0065] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise clearly indicated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components. The term "a" ( "an" and "the") includes plural referents. The term "plural" means two or more. Terms such as "such as", "for example", etc. are intended to indicate exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0066] In the present disclosure, the term "and / or" means and encompasses any and all possible combinations of one or more of the related listed items.

[0067] In the present disclosure, when a value range is provided, it should be understood that unless the context clearly indicates otherwise, the endpoints are included in the range and each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the specified range and any value within the smaller range between the specified values are all encompassed.

[0068] In the present disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, for example, within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0069] In the present disclosure, "an embodiment", "an example", "some embodiments", "a specific example", "related examples", "a certain example", "certain examples", "additional examples", or "further examples", "further implementation", or "another example", "other examples", means that at least one feature or characteristic description is included in the relevance of the example. Therefore, throughout the present disclosure, the above phrases do not necessarily refer to the same example. In addition, specific features can be combined in any suitable manner in one or more embodiments.

[0070] In the present disclosure, unless otherwise specified, the terms "comprising" and "including" and their variants will be understood to include the stated components, features, elements or steps, or combinations of components, features, elements or steps.

[0071] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common terms in molecular biology can be found in Lewin’s GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, published by Jones & Bartlett Learning. Definitions of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, published by W. H. Freeman. Definitions of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, published by Garland Science. Definitions of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, published by Jones & Bartlett Learning.

[0072] Unless otherwise specified, experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0073] Definition:

[0074] The term "pluripotent stem cell" or "PSCs" refers to cells that have the ability to differentiate into more than one differentiated cell type under different conditions, and preferably into cell types that exhibit the characteristics of all three germ cell layers. The main characteristic of pluripotent stem cells is their ability to differentiate into multiple cell types, preferably into all three germ layers, using, for example, the nude mouse teratoma formation assay. Such cells include human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), human embryo-derived cells (hEDC), and adult-derived stem cells. In some embodiments, the pluripotent stem cells are human embryonic stem cells, which can be autologous embryonic stem cells or commercially available embryonic stem cells, such as, for example, the H1, H7, H9, H13, or H14 cell lines. Pluripotent stem cells can be genetically modified. In some embodiments, the pluripotent stem cells are not genetically modified. Genetically modified cells can include markers, such as fluorescent proteins, to facilitate their identification. Pluripotency is also demonstrated by the expression of embryonic stem cell markers.

[0075] The term "iPSC" or "induced pluripotent stem cell" is used interchangeably and refers to pluripotent stem cells that are artificially derived (e.g., induced or by complete reprogramming), for example, from non-pluripotent stem cells (usually adult somatic cells) by inducing the forced expression of one or more genes.

[0076] The term "hematopoietic stem and progenitor cells" (HSPCs), namely hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC), are a type of pluripotent stem cells that exist in bone marrow, peripheral blood, and umbilical cord blood, have the ability of self-renewal, and can differentiate into all types of mature blood cells (such as red blood cells, white blood cells, platelets) and immune cells, and are the core cell population maintaining the lifelong hematopoietic function of the body. They form progenitor cells of each lineage (such as myeloid progenitor cells, lymphoid progenitor cells) through staged and multi-level proliferation and differentiation, and finally generate blood cells with specific functions. Among them, hematopoietic progenitor cells are progenitor cells that are formed by the proliferation and differentiation of hematopoietic stem cells under the regulation of a certain microenvironment and certain factors. It is also a rather primitive cell with the ability of proliferation, but has lost the ability of multi-directional differentiation and can only proliferate and differentiate in a directed manner into one or several blood cell lineages, so it is also called committed stem cell. Human hematopoietic stem cells and hematopoietic stem and progenitor cells usually express the CD34 surface marker.

[0077] The term "culture medium" or "media" or "medium" generally refers to a cell culture medium used to maintain cells or allow cell growth, which may include salts, amino acids, vitamins, lipids, buffers, growth factors, hormones, cytokines, trace elements, and / or carbohydrates. Examples of salts include magnesium salts, iron salts, potassium salts, sodium salts, and calcium salts. Examples of amino acids include all 20 known protein amino acids, such as histidine, glutamine, threonine, serine, and methionine. Examples of vitamins include ascorbic acid, biotin, choline, inositol, and d-benzoate, riboflavin. Examples of lipids include fatty acids, such as linoleic acid and oleic acid, as well as soy peptides and ethanolamine. Examples of buffers include Hepes. Examples of growth factors / hormones / cytokines include IGF, hydrocortisone, and recombinant insulin. Examples of trace elements include Zn, Mg, and Se. Examples of carbohydrates include glucose, fructose, galactose, and pyruvate. In some embodiments of the present disclosure, the provided culture medium can be liquid or solid. As a solid, it can be in powder form and dissolved during use. In some embodiments, the provided culture medium can be packaged as a basal medium already mixed with the components of the culture medium described in the present disclosure; it can also be packaged as a combination of a basal medium and the components of the culture medium, and the basal medium and the components of the culture medium are mixed in proportion during use. In some embodiments, the culture medium can be a culture medium containing serum (e.g., fetal bovine serum, calf serum, etc.), or it can be a serum-free medium (SFM), preferably a serum-free medium. Any suitable culture medium can be selected to achieve the purpose of the present disclosure, for example, including but not limited to, Stemfit ® Basic04, mTesR TM 1, Stempro TM 34, STEMdiff ® APEL2 Medium. Generally, the culture medium can be stored at room temperature or under refrigeration conditions.

[0078] The term "Mesodermal Progenitor Cells" refers to a cell population capable of differentiating into various mesodermal lineages, such as vascular cells and heart cells. For another example, the mesodermal progenitor cell population may be capable of differentiating into endothelial cells and / or cardiomyocytes. Another aspect of the present disclosure is to provide mesodermal progenitor cells or a mesodermal progenitor cell population obtained by the method of the present disclosure. Generally, mesodermal progenitor cells express MIXL1, TBXT, and / or Eomes marker genes.

[0079] The term "Lateral Plate Mesoderm Cells" refers to a type of mesodermal cells found in the periphery of the embryo. The lateral plate mesoderm is a precursor tissue of the vascular lineage and can give rise to the heart, blood vessels, the hematopoietic system of the circulatory system, and the mesodermal part of the limbs. The lateral plate mesoderm is divided into two layers. The somatic lateral plate mesoderm forms the future body wall, and the visceral lateral plate mesoderm forms the circulatory system. Generally, lateral plate mesoderm cells that can differentiate into endothelial cells and hematopoietic cells express at least the FLI1, KDR, LMO2, and SCL marker genes. (See Prummel, K.D., Nieuwenhuize, S., and Mosimann, C. (2020). The lateral plate mesoderm. Development 147.10.1242 / dev.175059.)

[0080] The term "hemogenic endothelial cells" refers to a group of endothelial cells or their precursor cells with hemogenic potential. Under specific conditions, hemogenic endothelial cells can gradually transform into hematopoietic cells. Hemogenic endothelial cells or hemogenic endothelial progenitor cells have both endothelial cell-related molecular characteristics and hematopoietic cell-related molecular characteristics. Hemogenic endothelial cells usually express the CD34 and CD144 surface markers and do not express CD43 and / or CD73. Hemogenic endothelial cells express at least one of the CD34, CDH5, PECAM-1, CXCR4, DLL4, and SOX17 genes.

[0081] Examples of the culture medium composition for differentiating pluripotent stem cells into hematopoietic stem and progenitor cells in the present disclosure :

[0082] Medium A, comprising:

[0083] Activin A, the effective final concentration thereof in Medium A can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the Activin A in Medium A can be about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the Activin A in Medium A can be about 30 ng / mL.

[0084] BMP4, the effective final concentration thereof in Medium A can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the BMP4 in Medium A can be about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the BMP4 in Medium A can be about 40 ng / mL.

[0085] GSK-3 inhibitors, GSK3 inhibitors specifically inhibit GSK3 and substantially do not inhibit most other mammalian kinases. Any GSK3 inhibitor can be used in the culture medium compositions described in this disclosure. Exemplary examples include, CHIR99021, BIP-135, CP21R7, 1-Azakenpaullone, TDZD-8, SB216763, SB415286, BIO-acetoxime, Tideglusib, AR-A014418, lithium chloride, VP3.15, GSK-3 inhibitor 1 or GSK-3 inhibitor 3, and combinations of the above molecules. In some embodiments, the GSK-3 inhibitor is CHIR99021, and its effective final concentration in medium A can be about 2-10 μM. In some embodiments, the effective final concentration of CHIR99021 in medium A can be about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM. Preferably, in one embodiment, the effective final concentration of CHIR99021 in medium A can be about 6 μM.

[0086] bFGF, whose effective final concentration in medium A can be about 10-100 ng / mL. In some embodiments, the effective final concentration of bFGF in medium A can be about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. Preferably, in one embodiment, the effective final concentration of bFGF in medium A can be about 20 ng / mL.

[0087] Basal medium: The basal medium of Medium A can be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is an SFM medium. The formulation of the SFM medium can be 40 - 60% (v / v) Iscove's Modified Dulbecco's Medium (IMDM), 40 - 60% (v / v) Ham's F-12K (Kaighn's) medium, polyvinyl alcohol (PVA) with a final concentration of 0.5 - 2 mg / ml, N-2 supplement at 0.5 - 2% (v / v), B-27 supplement at 0.5 - 2% (v / v), mercaptoethanol (MTG) with a final concentration of 300 - 600 μM, and Glutmax at 0.5 - 2% (v / v).

[0088] In the embodiments of the present disclosure, any components can also be added to Medium A as needed. For example, but not limited to, buffers (such as Hepes, sodium bicarbonate), antibiotics (such as penicillin-streptomycin, gentamicin), antifungal agents (such as amphotericin B), stabilizers, amino acids (such as L-glutamine, NEAA), vitamins and trace elements (such as vitamin B12, biotin, folic acid, Se, Zn, Cu, Fe), anticoagulants (such as heparin, EDTA), antioxidants (such as β-mercaptoethanol, vitamin C), pH indicators (such as phenol red), osmotic pressure regulators (such as glucose, sodium chloride), cell protectants (such as DMSO, albumin), lipid mixtures, transferrin, insulin, etc.

[0089] Medium B, comprising:

[0090] BMP4, whose effective final concentration in Medium B can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of BMP4 in Medium B can be about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 31 ng / mL, about 32 ng / mL, about 33 ng / mL, about 34 ng / mL, about 35 ng / mL, about 36 ng / mL, about 37 ng / mL, about 38 ng / mL, about 39 ng / mL, about 40 ng / mL, about 41 ng / mL, about 42 ng / mL, about 43 ng / mL, about 44 ng / mL, about 45 ng / mL, about 46 ng / mL, about 47 ng / mL, about 48 ng / mL, about 49 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of BMP4 in Medium B can be about 40 ng / mL.

[0091] PI3K inhibitors, any PI3K inhibitor can be used in the culture medium composition described in the present disclosure. Exemplary examples include, GDC-0941, Buparlisib, Copanlisib, GSK1059615, Alpelisib, Inalidex, Idelalisib, Duvelisib, IPI-549, Dactolisib, Omipalisib, Taselisib, Seletalisib, RP-5264, LY294002 or Wortmannin, and combinations of the above molecules. In some embodiments, the PI3K inhibitor is GDC-0941, and its effective final concentration in medium B can be about 1-5 μM. In some embodiments, the effective final concentration of GDC-0941 in medium B can be about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM. Preferably, in one embodiment, the effective final concentration of GDC-0941 in medium B can be about 2.5 μM.

[0092] Adenylate cyclase activators, any adenylate cyclase activator can be used in the culture medium composition described in the present disclosure. Exemplary examples include, Forskolin, epinephrine, norepinephrine, dobutamine, milrinone, levosimendan, adrenocorticotropic hormone or glucagon, and combinations of the above molecules. In some embodiments, the adenylate cyclase activator is Forskolin, and its effective final concentration in medium B can be about 5-15 μM. In some embodiments, the effective final concentration of Forskolin in medium B can be about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM. Preferably, in one embodiment, the effective final concentration of Forskolin in medium B can be about 10 μM.

[0093] TGF-β inhibitors, any TGF-β inhibitor can be used in the culture medium compositions described in this disclosure. Exemplary examples include SB431542, Galunisertib, Vactosertib, LY3200882, Fresolimumab, NIS793, AVID200, Sotatercept, Trabedersen, Decorin, BMP-7 or Cilengitide, and combinations of the above molecules. In some embodiments, the TGF-β inhibitor is SB431542, and its effective final concentration in medium B can be about 2-10 μM. In some embodiments, the effective final concentration of SB431542 in medium B can be about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM. Preferably, in one embodiment, the effective final concentration of SB431542 in medium B can be about 6 μM.

[0094] VEGF, whose effective final concentration in medium B can be about 10-200 ng / mL. In some embodiments, the effective final concentration of VEGF in medium B can be about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 82 ng / mL, about 84 ng / mL, about 86 ng / mL, about 88 ng / mL, about 90 ng / mL, about 92 ng / mL, about 94 ng / mL, about 96 ng / mL, about 98 ng / mL, about 100 ng / mL, about 102 ng / mL, about 104 ng / mL, about 106 ng / mL, about 108 ng / mL, about 110 ng / mL, about 112 ng / mL, about 114 ng / mL, about 116 ng / mL, about 118 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 160 ng / mL, about 170 ng / mL, about 180 ng / mL, about 190 ng / mL, about 200 ng / mL. Preferably, in one embodiment, the effective final concentration of VEGF in medium B can be about 100 ng / mL.

[0095] Wnt / β-catenin inhibitors. Any Wnt / β-catenin inhibitor can be used in the culture medium composition described in the present disclosure. Exemplary examples include XAV939, IWR-1-endo, WIKI4, iCRT3, CWP232228, PRI-724, ICG-001, Nitazoxanide, BC2059, Dictamnine, Longdaysin, Fz7-21 or Zamaporvint, and combinations of the above molecules. In some embodiments, the Wnt / β-catenin inhibitor is XAV939, and its effective final concentration in Medium B can be about 0.5 to 2 μM. In some embodiments, the effective final concentration of XAV939 in Medium B can be about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM. Preferably, in one embodiment, the effective final concentration of XAV939 in Medium B can be about 1 μM.

[0096] AA2P, and its effective final concentration in Medium B can be about 50 to 5000 μg / mL. In some embodiments, the effective final concentration of AA2P in Medium B can be about 50 μg / mL, about 100 μg / mL, about 120 μg / mL, about 130 μg / mL, about 140 μg / mL, about 150 μg / mL, about 160 μg / mL, about 170 μg / mL, about 180 μg / mL, about 190 μg / mL, about 200 μg / mL, about 210 μg / mL, about 220 μg / mL, about 230 μg / mL, about 240 μg / mL, about 250 μg / mL, about 300 μg / mL, about 400 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, about 2000 μg / mL, about 3000 μg / mL, about 4000 μg / mL, about 5000 μg / mL. Preferably, in one embodiment, the effective final concentration of AA2P in Medium B can be about 100 μg / mL.

[0097] Basal medium: The basal medium of medium B can be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is an SFM medium. The formulation of the SFM medium can be 40-60% (v / v) Iscove's Modified Dulbecco's Medium (IMDM), 40-60% (v / v) Ham's F-12K (Kaighn's) medium, polyvinyl alcohol (PVA) with a final concentration of 0.5-2 mg / ml, N-2 supplement at 0.5-2% (v / v), B-27 supplement at 0.5-2% (v / v), mercaptoethanol (MTG) with a final concentration of 300-600 μM, and Glutmax at 0.5-2% (v / v).

[0098] In the embodiments of the present disclosure, any component can also be added to the above medium B as needed. For example, but not limited to, buffers (such as Hepes, sodium bicarbonate), antibiotics (such as penicillin-streptomycin, gentamicin), antifungal agents (such as amphotericin B), stabilizers, amino acids (such as L-glutamine, NEAA), vitamins and trace elements (such as vitamin B12, biotin, folic acid, Se, Zn, Cu, Fe), anticoagulants (such as heparin, EDTA), antioxidants (such as β-mercaptoethanol, vitamin C), pH indicators (such as phenol red), osmotic pressure regulators (such as glucose, sodium chloride), cell protectants (such as DMSO, albumin), lipid mixtures, transferrin, insulin, etc.

[0099] Medium C, comprising:

[0100] SCF, whose effective final concentration in medium C can be about 10 - 200 ng / mL. In some embodiments, the effective final concentration of the SCF in medium C can be about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 82 ng / mL, about 84 ng / mL, about 86 ng / mL, about 88 ng / mL, about 90 ng / mL, about 92 ng / mL, about 94 ng / mL, about 96 ng / mL, about 98 ng / mL, about 100 ng / mL, about 102 ng / mL, about 104 ng / mL, about 106 ng / mL, about 108 ng / mL, about 110 ng / mL, about 112 ng / mL, about 114 ng / mL, about 116 ng / mL, about 118 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 160 ng / mL, about 170 ng / mL, about 180 ng / mL, about 190 ng / mL, about 200 ng / mL. As a preference, in one embodiment, the effective final concentration of the SCF in medium C can be about 100 ng / mL.

[0101] bFGF, whose effective final concentration in medium C can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the bFGF in medium C can be about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the bFGF in medium C can be about 20 ng / mL.

[0102] TPO, the effective final concentration thereof in culture medium C can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the TPO in culture medium C can be about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the TPO in culture medium C can be about 50 ng / mL.

[0103] VEGF, the effective final concentration thereof in culture medium C can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the VEGF in culture medium C can be about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the VEGF in culture medium C can be about 50 ng / mL.

[0104] IL-3, the effective final concentration thereof in culture medium C can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the IL-3 in culture medium C can be about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 25 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 32 ng / mL, about 34 ng / mL, about 36 ng / mL, about 38 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the IL-3 in culture medium C can be about 25 ng / mL.

[0105] AA2P, the effective final concentration thereof in medium C can be about 10 - 200 μg / mL. In some embodiments, the effective final concentration of the AA2P in medium C can be about 10 ng / mL, about 15 ng / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL. Preferably, in one embodiment, the effective final concentration of the AA2P in medium C can be about 50 μg / mL.

[0106] Basal medium: The basal medium of medium C can be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is Stempro TM 34 medium.

[0107] In the embodiments of the present disclosure, any components can also be added to the above medium C as needed. For example, including but not limited to, buffers (such as Hepes, sodium bicarbonate), antibiotics (such as penicillin-streptomycin, gentamicin), antifungal agents (such as amphotericin B), stabilizers, amino acids (such as L-glutamine, NEAA), vitamins and trace elements (such as vitamin B12, biotin, folic acid, Se, Zn, Cu, Fe), anticoagulants (such as heparin, EDTA), antioxidants (such as β-mercaptoethanol, vitamin C), pH indicators (such as phenol red), osmotic pressure regulators (such as glucose, sodium chloride), cell protectants (such as DMSO, albumin), lipid mixtures, transferrin, insulin, etc.

[0108] Optional medium D includes:

[0109] SCF, whose effective final concentration in medium D can be about 10 - 200 ng / mL. In some embodiments, the effective final concentration of the SCF in medium C can be about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 82 ng / mL, about 84 ng / mL, about 86 ng / mL, about 88 ng / mL, about 90 ng / mL, about 92 ng / mL, about 94 ng / mL, about 96 ng / mL, about 98 ng / mL, about 100 ng / mL, about 102 ng / mL, about 104 ng / mL, about 106 ng / mL, about 108 ng / mL, about 110 ng / mL, about 112 ng / mL, about 114 ng / mL, about 116 ng / mL, about 118 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 160 ng / mL, about 170 ng / mL, about 180 ng / mL, about 190 ng / mL, about 200 ng / mL. As a preference, in one embodiment, the effective final concentration of the SCF in medium C can be about 100 ng / mL.

[0110] bFGF, whose effective final concentration in medium D can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the bFGF in medium D can be about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the bFGF in medium D can be about 20 ng / mL.

[0111] TPO, the effective final concentration thereof in medium D can be about 10 - 100 ng / mL. In some embodiments, the effective final concentration of the TPO in medium D can be about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the TPO in medium D can be about 50 ng / mL.

[0112] VEGF, the effective final concentration thereof in medium D can be about 5 - 100 ng / mL. In some embodiments, the effective final concentration of the VEGF in medium D can be about 5 ng / mL, about 8 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, about 20 ng / mL, about 21 ng / mL, about 22 ng / mL, about 23 ng / mL, about 24 ng / mL, about 25 ng / mL, about 26 ng / mL, about 27 ng / mL, about 28 ng / mL, about 29 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. As a preference, in one embodiment, the effective final concentration of the VEGF in medium D can be about 20 ng / mL.

[0113] IL-6, the effective final concentration thereof in Medium D can be about 10-100 ng / mL. In some embodiments, the effective final concentration of the IL-6 in Medium D can be about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 25 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 32 ng / mL, about 34 ng / mL, about 36 ng / mL, about 38 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL. Preferably, in one embodiment, the effective final concentration of the IL-6 in Medium D can be about 25 ng / mL.

[0114] AA2P, the effective final concentration thereof in Medium D can be about 10-200 μg / mL. In some embodiments, the effective final concentration of the AA2P in Medium D can be about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL. Preferably, in one embodiment, the effective final concentration of the AA2P in Medium D can be about 50 μg / mL.

[0115] Basal medium: The basal medium of Medium D can be any suitable serum-free medium for cell differentiation. In some embodiments, the basal medium is STEMdiff ® APEL2 Medium.

[0116] In an embodiment of the present disclosure, any components can also be added to the above-mentioned culture medium D as needed. For example, but not limited to, buffers (such as Hepes, sodium bicarbonate), antibiotics (such as penicillin-streptomycin, gentamicin), antifungal agents (such as amphotericin B), stabilizers, amino acids (such as L-glutamine, non-essential amino acids (NEAA)), vitamins and trace elements (such as vitamin B12, biotin, folic acid, Se, Zn, Cu, Fe), anticoagulants (such as heparin, EDTA), antioxidants (such as β-mercaptoethanol, vitamin C), pH indicators (such as phenol red), osmotic pressure regulators (such as glucose, sodium chloride), cytoprotective agents (such as DMSO, albumin), lipid mixtures, transferrin, insulin, etc.

[0117] The above-mentioned "effective amount", "effective dose" or "effective final concentration" refers to the amount that effectively provides at least one desired biological result.

[0118] Examples of the methods in the present disclosure :

[0119] Culture of undifferentiated pluripotent stem cells:

[0120] Any suitable pluripotent stem cells can be used as the starting cells for the method in the present disclosure. For example, human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), human embryo-derived cells (hEDC), and adult-derived stem cells as described above. In some embodiments, the pluripotent stem cells are human embryonic stem cells, which can be autologous embryonic stem cells or commercially available embryonic stem cells, such as H1, H7, H9, H13, or H14 cell lines. Human induced pluripotent stem cells (hiPSC) can be prepared using methods and techniques well known in the art.

[0121] Any suitable method can be used to culture and expand the above-mentioned pluripotent stem cells. For reference, see, for example, Paulo A. Marinho, et al. Systematic optimization of human pluripotent stem cells media using Design of Experiments. Scientific Reports volume 5, Article number: 9834 (2015). / Robert T. Schinzel, et al. Efficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells. PLoS ONE 6(12): e27495. / The records in 《Human Pluripotent Stem Cells: Methods and Protocols》.

[0122] Pluripotent stem cells can be cultured in either a serum-containing or serum-free culture system. In some embodiments, the serum-containing culture system is a basal medium supplemented with stem cell-grade serum, along with the addition of growth factors such as bFGF, and the assistance of feeder cells is also required. Currently, the commonly used serum-free stem cell culture systems are Gibco Essential8 medium and the mTesR system of Stemcell Technologies. Both systems are serum-free, do not require the additional addition of growth factors, and are free from the limitation of feeder cells. Cell adhesion can be achieved by coating the culture plate. Therefore, in some other embodiments, a serum-free pluripotent stem cell culture system is used. In some embodiments, the above-mentioned serum-free pluripotent stem cell culture system can also be added with, for example, including but not limited to, a Rock inhibitor (such as Y27632) with a final concentration of 10 μM, Accutase or EDTA (for cell dissociation), and PBS (without Ca²⁺ / Mg²⁺). In the present disclosure, whether a serum-containing or serum-free pluripotent stem cell culture system is adopted, it will not affect the substantial contribution of the present invention. In some other embodiments, the method of 3D suspension culture can also be used to culture undifferentiated pluripotent stem cells.

[0123] Compared with the traditional differentiation methods of embryoid body (EB) formation, co-culture with feeder cells, or 3D micro-scaffold culture, in some embodiments of the present disclosure, the method of monolayer culture of pluripotent stem cells is adopted, that is, the pluripotent stem cells are digested into single cells, at a density of 500 - 5000 cells / cm 2are inoculated at the above density onto a solid surface for differentiation culture. The above density can be, for example, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 cells / cm 2 . The digestion can be carried out using TrypLE to obtain single cells. In some embodiments, the above-mentioned monolayer culture of pluripotent stem cells can be carried out on a blank solid surface or on a solid surface pre-coated with Matrigel ® , recombinant laminin (such as Laminin-521), vitronectin, or fibronectin. In some embodiments, the above solid surface includes but is not limited to standard commercial tissue culture flasks or cell culture plates, such as 6-well, 24-well, 96-well, or 144-well plates. Other solid surfaces include but are not limited to microcarriers and dishes. The solid surface suitable for growing undifferentiated pluripotent stem cells can be made of various substances, including but not limited to glass or plastic, such as polystyrene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, polyester film (melinex), thermanox, or a combination thereof. The suitable solid surface can also contain one or more polymers, such as one or more acrylates. The solid surface can also be in a three-dimensional shape. In some embodiments, the culture conditions for pluripotent stem cells can be 37 °C, 5% CO2, and 95% humidity.

[0124] Replacement of the culture medium at different stages during the differentiation process:

[0125] In some embodiments, the differentiation of pluripotent stem cells into hematopoietic stem and progenitor cells is completed most rapidly within about 10 days. Compared with the prior art, the cycle is greatly shortened and the production efficiency is improved. The differentiation process is divided into four stages.

[0126] The first stage is about the 1st day or about the 2nd day after replacing the culture medium A. In some embodiments, the end point of the culture can be determined according to the proportion of cells expressing genes related to mesoderm progenitor cells (for example, MIXL1, TBXT). For example, the proportion of cells expressing genes related to mesoderm progenitor cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.

[0127] The second stage is about day 1 (about day 2 after the start of differentiation) or about day 2 (about day 3 after the start of differentiation) after changing to medium B. In some embodiments, the end point of the culture can be determined according to the proportion of cells expressing genes related to lateral plate mesoderm cells (e.g., FLI1, KDR, LMO2, SCL). For example, the proportion of cells expressing genes related to lateral plate mesoderm cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.

[0128] The third stage is about day 4 (about day 6 after the start of differentiation) or about day 5 (about day 7 after the start of differentiation) after changing to medium C. In some embodiments, the end point of the culture can be determined according to the proportion of cells expressing genes related to hematopoietic endothelial cells (e.g., CD34, CDH5, PECAM-1, CXCR4, DLL4, SOX17). For example, the proportion of cells expressing genes related to lateral plate mesoderm cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%.

[0129] The fourth stage is about day 4 (about day 10 after the start of differentiation) or about day 5 (about day 11 after the start of differentiation) after changing to medium D. Harvest and detect the number of generated hematopoietic progenitor cells (CD34 + CD43 + CD45 + ), and hematopoietic stem cells (CD34 + CD38 - CD90 + CD45RA - CD49f + ).

[0130] The degree of cell differentiation in each of the above stages can be determined by methods in the prior art, such as flow cytometry, immunofluorescence, RT-PCR and other techniques to detect the expression of cell markers.

[0131] In vitro cell differentiation system in the present disclosure :

[0132] In some embodiments, the in vitro cell differentiation system includes:

[0133] a) Pluripotent stem cells;

[0134] b) A culture system for pluripotent stem cells;

[0135] c) A plurality of culture medium storage devices, which store at least the above-mentioned medium A, medium B and medium C, and optionally store the above-mentioned medium D; and

[0136] d) A system or device for replacing the medium A, medium B, medium C or medium D.

[0137] In some embodiments, the above in vitro cell differentiation system can be industrial automated or manual. For an industrial automated in vitro cell differentiation system, the automated cell culture and automatic medium replacement can be achieved by using the systems or devices in the prior art. For example, US20170037357A1 discloses an automated cell culture and harvesting device, and for another example, CN119020163A discloses an automatic medium replacement device and method.

[0138] Examples :

[0139] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0140] Example 1:

[0141] This example uses the Figure 1 shown scheme to induce the differentiation of induced pluripotent stem cells hiPSC-WTC into hematopoietic stem / progenitor cells. The culture conditions are: 37 °C, 5% CO2.

[0142] Specifically:

[0143] (1) Preparation stage (-1 to 0 days): Inoculate hiPSC-WTC in the form of single cells into a culture container coated with extracellular matrix protein, and use the medium of the zero stage to promote the adhesion of hPSCs and maintain cell viability and pluripotency, laying a foundation for subsequent differentiation steps.

[0144] (2) The first stage (0 to 1 day): Replace the medium of the zero stage with the medium of the first stage to induce the differentiation of hPSCs into mesoderm progenitor cells.

[0145] (3) The second stage (1 to 2 days): Replace the medium of the first stage with the medium of the second stage to promote the differentiation of mesoderm progenitor cells into lateral plate mesoderm cells, and the cells at this stage have the potential for hematopoietic differentiation.

[0146] (4) The third stage (2 to 6 days): Replace the medium of the second stage with the medium of the third stage to induce the differentiation of lateral plate mesoderm cells into hemogenic endothelial cells, which is a key intermediate step in the generation of HSPCs.

[0147] (5) The fourth stage (6 to 10 days): Digest the hemogenic endothelial cells obtained in the third stage into single cells, then inoculate them into a culture dish coated with extracellular matrix protein, and use the medium of the fourth stage to promote their differentiation into HSPCs. Harvest the generated hematopoietic stem progenitor cells on the 10th day for functional determination.

[0148] The degree of differentiation of cells at each stage can be determined by detecting the expression of cell markers using techniques such as flow cytometry, immunofluorescence, and RT-PCR.

[0149] Among them, the specific formula of the medium in the zero stage is a human pluripotent stem cell commercial medium (such as Stemfit TM Basic04, mTesR TM 1) Add a Rock inhibitor (such as Y27632) with a final concentration of 10 μM.

[0150] The specific formula of the medium in the first stage is to add Activin A with a final concentration of 30 ng / mL, bone morphogenetic protein 4 (BMP4) with a final concentration of 40 ng / mL, GSK-3 inhibitor CHIR99021 with a final concentration of 6 μM, and basic fibroblast growth factor with a final concentration of 20 ng / mL to serum-free medium SFM. Among them, the formula of serum-free medium SFM is 50% (v / v) Iscove's Modified Dulbecco's Medium (IMDM), 50% (v / v) Ham's F-12K (Kaighn's) medium, polyvinyl alcohol (PVA) with a final concentration of 1 mg / ml, N-2 supplement with 1% (v / v), B-27 supplement with 1% (v / v), thioglycerol (MTG) with a final concentration of 450 μM, and Glutmax with 1% (v / v).

[0151] The specific formula of the medium in the second stage is to add BMP4 with a final concentration of 40 ng / mL, PI3K inhibitor GDC-0941 with a final concentration of 2.5 μM, adenylyl cyclase activator Forskolin with a final concentration of 10 μM, TGF-β inhibitor SB431542 with a final concentration of 6 μM, vascular endothelial growth factor (VEGF) with a final concentration of 100 ng / mL, Wnt / β-catenin inhibitor XAV939 with a final concentration of 1 μM, and L-ascorbic acid 2-phosphate (AA2P) with a final concentration of 200 μg / mL to serum-free medium SFM.

[0152] The specific formula of the medium in the third stage is to add stem cell factor (SCF) with a final concentration of 100 ng / mL, bFGF with a final concentration of 20 ng / mL, thrombopoietin (TPO) with a final concentration of 50 ng / mL, VEGF with a final concentration of 50 ng / mL, interleukin 3 (IL3) with a final concentration of 25 ng / mL, and AA2P with 50 μg / mL to Stempro34 commercial medium.

[0153] The specific formulation of the fourth-stage culture medium is to add SCF with a final concentration of 100 ng / mL, bFGF with a final concentration of 20 ng / mL, TPO with a final concentration of 50 ng / mL, VEGF with a final concentration of 20 ng / mL, FMS-like tyrosine kinase 3 ligand (FIT3L) with a final concentration of 100 ng / mL, IL3 with a final concentration of 25 ng / mL, interleukin 6 (IL6) with a final concentration of 25 ng / mL, and AA2P with a final concentration of 50 μg / mL to the APEL2 commercial culture medium.

[0154] The differentiation degree of cells at each stage can be determined by detecting the expression of cell markers through techniques such as flow cytometry, immunofluorescence, and RT-PCR.

[0155] Example 2:

[0156] The same method as in Example 1 was used, except that the type of pluripotent stem cells selected was human induced pluripotent stem cells hiPSC-SPA.

[0157] Example 3:

[0158] The same method as in Example 1 was used, except that the type of pluripotent stem cells selected was human embryonic pluripotent stem cells H9.

[0159] Comparative Example 1:

[0160] In this comparative example, the method reported by (Tursky et al. 2020) was used (see Melinda L. Tursky et al. Direct Comparison of Four Hematopoietic Differentiation Methods from Human Induced Pluripotent Stem Cells. Stem Cell Reports Vol. 15 735–748 September 8, 2020), and the type of pluripotent stem cells selected was induced pluripotent stem cells hiPSC-WTC, serving as a comparative example to Example 1.

[0161] Cell samples were collected on the 1st day of differentiation (Examples 1, 2, 3) and the 2nd day (Comparative Example 1) for real-time fluorescence quantitative PCR analysis. As Figure 3 shown, compared with Comparative Example 1, the expression levels of mesoderm progenitor cell marker genes (MIXL1, TBXT) in Examples 1, 2, and 3 were significantly upregulated. Further detection of cell samples on the 2nd day (Examples 1, 2, 3) and the 4th day (Comparative Example 1) found that (see Figure 4A, B, C, D), the expression levels of lateral plate mesoderm characteristic genes (FLI1, KDR, LMO2, SCL) in the example group were significantly higher than those in comparative example 1. In addition, the expression levels of paraxial mesoderm genes (MSGN1, TBX6) and ectoderm marker genes (SOX2) in Example 1 were significantly lower than those in comparative example 1, indicating that this solution can significantly improve differentiation specificity.

[0162] To achieve functional hematopoietic stem / progenitor cell differentiation, the directional induction of hemogenic endothelial cells (HECs) is essential. + CD43 - CD144 + CD73 - Phenotypic identification criteria, flow cytometry assay (see Figure 6 ), the proportion of hemogenic endothelial cells and arterial endothelial cells obtained in Example 1 on the 6th day was significantly increased compared with that in Comparative Example 1. qPCR analysis (see Figure 5 ) further confirmed that the expression levels of endothelial-related genes (CD34, CDH5, PECAM-1) and arterial characteristic genes (CXCR4, DLL4, SOX17) in Example 1 were significantly higher than those in Control Example 1, which fully verified the advantages of this scheme in hemogenic endothelial induction.

[0163] For suspension cells differentiated on day 10 (see Figure 2 ), flow analysis (see Figure 7 , Figure 8 ) shows that the example successfully obtained hematopoietic progenitor cells (CD34 + CD43 + CD45 + ) and hematopoietic stem cells (CD34 + CD38 - CD90 + CD45RA - CD49f + ), of which hematopoietic progenitor cells accounted for 65%-90%, significantly better than control example 1. qPCR detection (see Figure 9 ) and bulk RNA sequencing (see Figure 11 ) further showed that the example cells and human umbilical cord blood CD34 + Hematopoietic stem / progenitor cells have highly similar gene expression profiles. Through the H4434 culture medium colony formation experiment (inoculation amount 10,000 cells, cultured for 14 days), the hematopoietic stem cells of Example 1 not only formed larger colonies, but also the number of colonies generated was significantly higher than that of Comparative Example 1 (see Figure 10 ), suggesting that the cells differentiated by this protocol have stronger in vitro expansion ability.

[0164] Functional verification showed that, under the induction of EPO, the cells of Examples 1, 2, and 3 could differentiate into enucleated red blood cells within 12 days, express adult hemoglobin genes (HBA, HBB), present a characteristic brick-red precipitate after centrifugation, and achieve an amplification of 20- to 70-fold (see Figure 12 ). In addition, the addition of IL7 / IL15 could induce directed differentiation into natural killer cells (NK) (see Figure 13 ), demonstrating that the hematopoietic stem / progenitor cells obtained by this protocol possess multi-lineage differentiation potential.

[0165] In summary, this protocol has successfully achieved the efficient directed differentiation of pluripotent stem cells into functional hematopoietic stem / progenitor cells.

[0166] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A culture medium composition for differentiating pluripotent stem cells (PSCs) into hematopoietic stem cells (HSCs), characterized in that: The composition comprises: Culture medium A: the culture medium A comprises basal culture medium, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (FGF2), activin A (Activin A) and GSK-3 inhibitor; Culture medium B: the culture medium B comprises a basal culture medium, BMP4, a PI3K inhibitor, an adenylate cyclase activator, a TGF-β inhibitor, a vascular endothelial growth factor (VEGF), a Wnt / β-catenin inhibitor and L-ascorbic acid 2-phosphate (AA2P); and Culture medium C: The culture medium C includes basal medium, stem cell factor (SCF), FGF2, angiopoietin (TPO), VEGF, interleukin 3 (IL3) and AA2P.

2. The culture medium composition according to claim 1, characterized in that The culture medium composition also includes: Culture medium D: The culture medium D includes basal medium, SCF, FGF2, TPO, VEGF, FMS-like tyrosine kinase 3 ligand (FIT3L), IL3, interleukin 6 (IL6) and AA2P.

3. The culture medium composition according to claim 1 or 2, characterized in that The GSK-3 inhibitor is at least one selected from CHIR99021, BIP-135, CP21R7, 1-Azakenpaullone, TDZD-8, SB216763, SB415286, BIO-acetoxime, Tideglusib, AR-A014418, lithium chloride, VP3.15, GSK-3 inhibitor 1 or GSK-3 inhibitor 3.

4. The culture medium composition according to claim 1 or 2, characterized in that The PI3K inhibitor is at least one selected from GDC-0941, Buparlisib, Copanlisib, GSK1059615, Alpelisib, Inarlisib, Idelalisib, Duvelisib, IPI-549, Dactolisib, Omipalisib, Taselisib, Seletalisib, RP-5264, LY294002 or Wortmannin.

5. The culture medium composition according to claim 1 or 2, characterized in that The adenylate cyclase activator is at least one selected from Forskolin, epinephrine, norepinephrine, dobutamine, milrinone, levosimendan, adrenocorticotropic hormone or glucagon.

6. The culture medium composition according to claim 1 or 2, characterized in that The TGF-β inhibitor is at least one selected from SB431542, Galunisertib, Vactosertib, LY3200882, Fresolimumab, NIS793, AVID200, Sotatercept, Trabedersen, Decorin, BMP-7 or Cilengitide.

7. The culture medium composition according to claim 1 or 2, characterized in that The Wnt / β-catenin inhibitor is at least one selected from XAV939, IWR-1-endo, WIKI4, iCRT3, CWP232228, PRI-724, ICG-001, Nitazoxanide, BC2059, Dictamnine, Longdaysin, Fz7-21 or Zamaporvint.

8. A method for producing hemogenic endothelial cells (HECs), characterized in that: include: Step a) contacting pluripotent stem cells with the culture medium A described in any one of claims 1 to 7 for 1 to 2 days to obtain mesodermal progenitor cells; Step b) replacing the culture medium so that the mesodermal progenitor cells are in contact with the culture medium B described in any one of claims 1 to 7 for 1 to 2 days to obtain lateral plate mesodermal cells; Step c) replacing the culture medium so that the lateral plate mesoderm cells are in contact with the culture medium C according to any one of claims 1 to 7 for 3 to 6 days to obtain the hemogenic endothelial cells.

9. The method according to claim 8, characterized in that The pluripotent stem cells are human pluripotent stem cells; the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells (iPSCs).

10. A hemogenic endothelial cell or cell population thereof produced by or directly from the method of claim 8 or 9, characterized in that: The phenotype of the hemogenic endothelial cells is CD34 + CD144 + CD43 - CD73 - .

11. A method for producing hematopoietic stem and progenitor cells, characterized in that: include: Step a) contacting pluripotent stem cells with the culture medium A described in any one of claims 1 to 7 for 1 to 2 days to obtain mesodermal progenitor cells; Step b) replacing the culture medium so that the mesodermal progenitor cells are in contact with the culture medium B described in any one of claims 1 to 7 for 1 to 2 days to obtain lateral plate mesodermal cells; Step c) replacing the culture medium so that the lateral plate mesoderm cells are in contact with the culture medium C described in any one of claims 1 to 7 for 3 to 6 days to obtain hemogenic endothelial cells; Step d) replacing the culture medium so that the hemogenic endothelial cells are contacted with a hematopoietic stem and progenitor cell differentiation medium to obtain the hematopoietic stem and progenitor cells.

12. The method according to claim 11, characterized in that The hematopoietic stem and progenitor cell differentiation medium is the medium D as described in claim 2.

13. The method according to claim 11 or 12, characterized in that: The pluripotent stem cells are human pluripotent stem cells; the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

14. A hematopoietic stem cell or cell population thereof produced by or directly from the method of any one of claims 11, 12 or 13.

15. A composition for cell therapy, characterized in that: The composition comprises the hemogenic endothelial cells or cell populations thereof as claimed in claim 10, or the hemogenic stem and progenitor cells or cell populations thereof as claimed in claim 14.

16. An in vitro cell differentiation system, characterized in that: include: a) Pluripotent stem cells; b) Culture system of pluripotent stem cells; c) a plurality of culture medium storage devices, wherein the culture medium storage devices store at least the culture medium A, culture medium B and culture medium C as described in any one of claims 1 to 7, and optionally store the culture medium D as described in claim 2; and d) a system or device for replacing the culture medium A, culture medium B, culture medium C or culture medium D.

17. The culture medium composition according to any one of claims 1 to 7, the hemogenic endothelial cells or cell populations thereof according to claim 10, the hematopoietic stem and progenitor cells or cell populations thereof according to claim 14, the composition for cell therapy according to claim 15, or the in vitro cell differentiation system according to claim 16 for preparing in vitro mesodermal progenitor cells, lateral plate mesoderm cells, hemogenic endothelial cells, hematopoietic stem cells, hematopoietic progenitor cells, erythroid progenitor cells, proerythroblasts, basic erythroblasts, polychromatophilic erythroblasts, orthochromatic Erythroblast), reticulocyte, mature erythrocyte, megakaryocyte, platelet, granulocyte-monocyte progenitor (GMP), neutrophil, eosinophil, basophil, tissue macrophage, dendritic cell (DC), mast cell, T lymphocyte, B lymphocyte, natural killer cell (NK Cell) or innate lymphoid cell (ILCs).

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