Method for differentiating platelets, culture medium and application of culture medium

Abstract

The invention relates to a method for inducing pluripotent cells to proliferate and / or differentiate to platelets. The method comprises the step of using a culture medium containing specific additive factors. The invention further provides a culture medium for inducing proliferation and / or differentiation of cells to platelets and application of the culture medium.

Description

A method for differentiating platelets, culture medium and application thereof Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for differentiating platelets, a culture medium, and applications thereof. Background Art

[0002] Platelets are small, circulating, anucleated cells that differentiate from mature megakaryocytes. They stop bleeding by aggregating and forming emboli in damaged blood vessels. A single platelet has a lifespan of approximately 7-10 days in the body, so the body produces a large number of fresh platelets daily to maintain a normal platelet count. As an intrinsic component of the body, platelets can evade clearance by the immune system and are inextricably linked to physiological processes such as endothelial injury repair, immune response, atherosclerosis, neurodegeneration, and tumor growth and metastasis, offering broad application prospects.

[0003] Platelet transfusion is a life-saving procedure used to prevent or stop bleeding in patients with thrombocytopenia and functional platelet disorders. It can also be used to prevent bleeding in patients with thrombocytopenia caused by chemotherapy for malignant tumors or hematopoietic stem cell transplantation. Currently, platelets are primarily obtained from donors. Due to the short shelf life and insufficient number of donors, platelets have become a scarce resource. Furthermore, the platelet processing, preparation, and transfusion processes carry the risk of bacterial contamination and deterioration. Therefore, the development of alternative strategies is essential.

[0004] Pluripotent cells can proliferate and expand indefinitely in vitro and possess the ability to differentiate and proliferate into one or more blood cell lineages. Therefore, efficient platelet differentiation from pluripotent cells in vitro could be an effective approach to alleviate platelet shortages while also reducing the risk of platelet contamination and deterioration.

[0005] Summary of the Invention

[0006] The present application provides a method for differentiating pluripotent cells and / or hematopoietic stem / progenitor cells and / or megakaryocyte progenitor cells into platelets. The method uses a platelet differentiation medium containing N2 additive and B27 additive components, which can improve the differentiation ability of pluripotent cells, hematopoietic stem / progenitor cells, and megakaryocyte progenitor cells, and promote their differentiation into platelets.

[0007] On the one hand, the present application provides a method for inducing pluripotent cells to proliferate and / or differentiate through embryoid bodies, hematopoietic stem / progenitor cells, megakaryocyte progenitor cells, and platelet pathways, the method comprising using a platelet differentiation medium containing N2 additives and B27 additives.

[0008] On the one hand, the present application provides a method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate through megakaryocyte progenitor cells and platelet pathways, the method comprising using a platelet differentiation medium containing N2 additive and B27 additive components.

[0009] In certain embodiments, the N2 additive concentration in the method is about 0.5%.

[0010] In certain embodiments, the method comprises providing a B27 additive at a concentration of about 1%.

[0011] In certain embodiments, the platelet differentiation medium in the method may further comprise one or more of TPO, SCF, IL6, ITS, glutamine, NEAA and ascorbic acid.

[0012] In certain embodiments, the concentration of TPO in the methods is about 5-100 ng / mL.

[0013] In certain embodiments, the concentration of SCF in the methods is about 5-100 ng / mL.

[0014] In certain embodiments, the method comprises administering IL6 at a concentration of about 5-100 ng / mL.

[0015] In certain embodiments, the method comprises providing an ITS concentration of about 1%.

[0016] In certain embodiments, the method comprises providing glutamine at a concentration of about 1%.

[0017] In certain embodiments, the concentration of ascorbic acid in the method is about 50-200 μM.

[0018] In certain embodiments, the platelet differentiation medium in the method may further comprise a basal medium, the medium may comprise serum, or the medium may be serum-free.

[0019] In certain embodiments, the basal culture medium in the method can be selected from one or more of the following groups: IMDM, StemSpan TM SFEM II, APEL, and mTeSR.

[0020] In certain embodiments, the method may include adding one or more of the following substances to the platelet differentiation medium: a metalloproteinase inhibitor, a ROCK inhibitor.

[0021] In certain embodiments, the metalloproteinase inhibitor in the method can be GM6001.

[0022] In certain embodiments, the ROCK inhibitor in the method can be Y27632.

[0023] In certain embodiments, the method comprises administering Y27632 at a concentration of about 1 μM to about 50 μM.

[0024] In certain embodiments, the method can involve culturing cells at about 37-39°C.

[0025] In certain embodiments, the method can involve culturing cells under conditions of about 5% CO2.

[0026] In certain embodiments, the methods can involve culturing cells under serum-free conditions.

[0027] In certain embodiments, the methods can culture cells without co-culture conditions.

[0028] In certain embodiments, the methods can involve culturing cells on a matrix-coated surface.

[0029] In certain embodiments, the matrix in the method can be laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel. TM .

[0030] In certain embodiments, the substrate in the method can be vitronectin.

[0031] In certain embodiments, the matrix in the method can be Matrigel TM .

[0032] In certain embodiments, the method may comprise the step of culturing and / or maintaining the cells in a stem cell culture medium.

[0033] In certain embodiments, the method may comprise the step of digesting cells that have proliferated to a sufficient number into single cells.

[0034] In certain embodiments, the digestion step in the method may use Accutase digestion solution.

[0035] In certain embodiments, the digestion step in the method may utilize Trypsin and EDTA.

[0036] In certain embodiments, the pluripotent cells in the method can be derived from a mammal.

[0037] In certain embodiments, the pluripotent cells in the methods can be derived from non-mammalian sources.

[0038] In certain embodiments, the pluripotent cells in the method can be human embryonic stem cells or human induced pluripotent stem cells.

[0039] In certain embodiments, the hematopoietic stem / progenitor cells in the method can be derived from human embryonic stem cells, human induced pluripotent stem cells, ex vivo human blood, umbilical cord blood, or bone marrow.

[0040] In certain embodiments, the hematopoietic stem / progenitor cells in the method may be CD34 + Hematopoietic stem / progenitor cells.

[0041] In certain embodiments, the megakaryocyte progenitor cells in the method can be derived from human embryonic stem cells, human induced pluripotent stem cells, ex vivo human blood, umbilical cord blood, bone marrow, CD34 + Hematopoietic stem / progenitor cells.

[0042] On the other hand, the present application provides a platelet differentiation culture medium, which contains N2 additive and B27 additive components and can promote cell differentiation into platelets.

[0043] In certain embodiments, the concentration of the N2 additive in the culture medium is about 0.5%.

[0044] In certain embodiments, the concentration of B27 supplement in the culture medium is about 1%.

[0045] In certain embodiments, the culture medium may comprise Platelet Differentiation Medium I. In certain embodiments, the Platelet Differentiation Medium I may comprise a basal medium, and the basal medium may be mTeSR.

[0046] In certain embodiments, the platelet differentiation medium I may include an inhibitor. In certain embodiments, the inhibitor may be a ROCK inhibitor. In certain embodiments, the ROCK inhibitor may be Y27632, and the Y27632 concentration may be about 1-50 μM.

[0047] In certain embodiments, the culture medium may comprise platelet differentiation medium II. In certain embodiments, the platelet differentiation medium II may comprise a basal medium, and the basal medium may be APEL.

[0048] In certain embodiments, the platelet differentiation medium II may contain one or more of BMP4, VEGF, and SCF.

[0049] In certain embodiments, the concentration of BMP4 in the platelet differentiation medium II is about 5-100 ng / mL.

[0050] In certain embodiments, the concentration of VEGF in the platelet differentiation medium II is about 5-100 ng / mL.

[0051] In certain embodiments, the concentration of SCF in the platelet differentiation medium II is about 5-100 ng / mL.

[0052] In certain embodiments, the culture medium may comprise platelet differentiation medium III. In certain embodiments, the platelet differentiation medium III may comprise a basal medium, which may be StemSpan TM SFEM II.

[0053] In certain embodiments, the platelet differentiation medium III may contain one or more of IL3, IL6, SCF, TPO, and FLt3.

[0054] In certain embodiments, the concentration of IL3 in the platelet differentiation medium III is about 5-100 ng / mL.

[0055] In certain embodiments, the concentration of IL6 in the platelet differentiation medium III is about 5-100 ng / mL.

[0056] In certain embodiments, the concentration of SCF in the platelet differentiation medium III is about 5-100 ng / mL.

[0057] In certain embodiments, the concentration of TPO in the platelet differentiation medium III is about 5-100 ng / mL.

[0058] In certain embodiments, the concentration of FLt3 in the platelet differentiation medium III is about 5-100 ng / mL.

[0059] In certain embodiments, the culture medium may comprise platelet differentiation medium IV. In certain embodiments, the platelet differentiation medium IV may comprise a basal medium, and the basal medium may be IMDM.

[0060] In certain embodiments, the platelet differentiation medium IV may contain one or more of N2 supplement, B27 supplement, NEAA, glutamine, ITS, ascorbic acid, IL6, SCF, and TPO.

[0061] In certain embodiments, the concentration of the N2 supplement in the platelet differentiation medium IV is about 0.5%.

[0062] In certain embodiments, the concentration of B27 supplement in the Platelet Differentiation Medium IV is about 1%.

[0063] In certain embodiments, the concentration of glutamine in the platelet differentiation medium IV is about 1%.

[0064] In certain embodiments, the concentration of ITS in the platelet differentiation medium IV is about 1%.

[0065] In certain embodiments, the concentration of ascorbic acid in the platelet differentiation medium IV is about 50-200 μM.

[0066] In certain embodiments, the concentration of IL6 in the platelet differentiation medium IV is about 5-100 ng / mL.

[0067] In certain embodiments, the concentration of SCF in the platelet differentiation medium IV is about 5-100 ng / mL.

[0068] In certain embodiments, the concentration of TPO in the platelet differentiation medium IV is about 5-100 ng / mL.

[0069] In certain embodiments, the culture medium is serum-containing.

[0070] In certain embodiments, the culture medium is serum-containing.

[0071] On the other hand, the present application also provides the use of the culture medium in inducing the proliferation of pluripotent cells and / or differentiating platelets.

[0072] On the other hand, the present application also provides the use of the culture medium in inducing the proliferation of hematopoietic stem / progenitor cells and / or differentiation of platelets.

[0073] On the other hand, the present application also provides the use of the culture medium in inducing megakaryocyte progenitor cell proliferation and / or platelet differentiation.

[0074] On the other hand, the present application also provides a composition comprising pluripotent cells and the culture medium.

[0075] On the other hand, the present application also provides a composition comprising hematopoietic stem / progenitor cells and the culture medium.

[0076] On the other hand, the present application also provides a composition comprising megakaryocyte progenitor cells and the culture medium.

[0077] On the other hand, the present application also provides a culture platform for obtaining platelets, which comprises the method and the culture medium.

[0078] On the other hand, the present application also provides applications of the culture platform. In certain embodiments, the application comprises screening other compounds.

[0079] On the other hand, the present application also provides the method, culture medium, composition and / or culture platform, and its use for preparing platelets.

[0080] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0082] FIG1 shows the flow cytometry analysis results when the platelet differentiation medium IV described in the present application is S1.

[0083] FIG2 shows the flow cytometry analysis results when the platelet differentiation medium IV described in the present application is S2.

[0084] FIG3 shows the flow cytometry analysis results when the platelet differentiation medium IV described in the present application is S3.

[0085] FIG4 shows the flow cytometry analysis results when the platelet differentiation medium IV described in the present application is S4.

[0086] FIG5 shows the flow cytometry analysis results when the platelet differentiation culture medium IV described in the present application is S5.

[0087] FIG6 shows the flow cytometry analysis results when the platelet differentiation medium IV described in the present application is S6. DETAILED DESCRIPTION

[0088] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0089] Definition of terms

[0090] In this application, the term "pluripotent cell" generally refers to a cell that has the potential to proliferate and differentiate. Pluripotent cells can be functionally defined as cells that: (1) have the ability to differentiate into different cells, and in some cases, only produce one specialized cell type; (2) can undergo long-term self-renewal, producing one or more cells that are the same or different from the original cell type. The source and preparation method of pluripotent cells are not limited. For example, the pluripotent cells can be naturally obtained or artificially modified. For example, the pluripotent cells can include embryonic stem cells, induced pluripotent stem cells, etc.

[0091] In this application, the term "proliferation" generally refers to the generation of multiple individual cells by division of a starting cell. The multiple individual cells can be of the same type or of different types. The starting cells used for proliferation need not be the same as the cells produced by proliferation. For example, the proliferated cells can be generated from the growth and differentiation of a starting cell population.

[0092] In this application, the term "differentiation" generally refers to the process by which a non-specific or less specific cell acquires specific cell characteristics. A differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.

[0093] In this application, the term "megakaryocyte progenitor cells," also known as "megakaryocytes," generally refers to cells that can produce platelets. Megakaryocyte progenitor cells are characterized by polyploidy, large nuclei, large cell size, and abundant cytoplasm, enabling each cell to produce thousands of platelets. The source and preparation methods of megakaryocyte progenitor cells are not limited; for example, the megakaryocyte progenitor cells can be differentiated from pluripotent cells or isolated from the body.

[0094] In this application, the term "embryonic stem cells," also known as "embryonic stem cells" (abbreviated as "ESCs"), generally refers to cells that possess the properties of unlimited proliferation, self-renewal, and multidirectional differentiation. Embryonic stem cells are derived from the undifferentiated inner cell mass of the blastocyst (early embryonic stage). Their source and preparation methods are not limited. Embryonic stem cells can be induced to differentiate into nearly any cell type in the body, whether in vitro or in vivo. For example, these cell types can include hematopoietic stem cells, neural cells, and cardiomyocytes.

[0095] In this application, the term "induced pluripotent stem cells," abbreviated as "iPS" cells or "iPSCs," generally refers to a type of pluripotent stem cell artificially derived from non-pluripotent cells. Induced pluripotent stem cells can be obtained by introducing specific transcription factors to reprogram terminally differentiated somatic cells. For example, the terminally differentiated somatic cells can be fibroblasts, hematopoietic stem cells, muscle cells, neurons, epidermal cells, and the like.

[0096] In this application, the term "hematopoietic stem cell" generally refers to cells with long-term self-renewal ability and the potential to differentiate into various types of mature blood cells. The source and preparation method of hematopoietic stem cells are not limited. For example, the hematopoietic stem cell can be differentiated from pluripotent cells and can be isolated from bone marrow or blood. Hematopoietic stem cells can differentiate into a variety of cells, for example, myeloid lineage cells (for example, monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineage cells (for example, T cells, B cells, NK cells).

[0097] In this application, the term "platelet" generally refers to anucleated cytoplasmic body. Platelets are formed from small pieces of cytoplasm that cleave from the cytoplasm of megakaryocyte progenitor cells and play an important role in physiological hemostasis. Platelets can be activated by thrombin, rapidly adhere to wounds, and aggregate into relatively soft hemostatic plugs, which then promote blood coagulation and form a solid hemostatic plug.

[0098] In this application, the term "composition" generally refers to a product comprising a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of the specified amounts of the specified ingredients. In this application, the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0099] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0100] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0101] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0102] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0103] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation 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.

[0104] Detailed Description of the Invention

[0105] method

[0106] In one aspect, the present application provides a method for inducing proliferation and / or differentiation of pluripotent cells into platelets, comprising using a platelet differentiation medium containing N2 additive and B27 additive components.

[0107] On the other hand, the present application provides a method for inducing proliferation and / or differentiation of hematopoietic stem / progenitor cells into platelets, which comprises using a platelet differentiation medium containing N2 additive and B27 additive components.

[0108] On the other hand, the present application provides a method for inducing megakaryocyte progenitor cells to proliferate and / or differentiate into platelets, which comprises using a platelet differentiation medium containing N2 additive and B27 additive components.

[0109] In the present application, the method may also include the process of culturing, proliferating and / or differentiating pluripotent cells; the method may also include the process of culturing and / or differentiating embryoid bodies; the method may also include the process of culturing, proliferating and / or differentiating hematopoietic stem / progenitor cells; the method may also include the process of culturing, proliferating and / or differentiating megakaryocyte progenitor cells; the method may also include the process of culturing platelets; the method may also include the process of proliferating and / or differentiating pluripotent cells into embryoid bodies; the method may also include the process of proliferating and / or differentiating pluripotent cells into hematopoietic stem / progenitor cells; the method may also include the process of proliferating and / or differentiating pluripotent cells into megakaryocyte progenitor cells; the method may also include the process of differentiating embryoid bodies into hematopoietic stem / progenitor cells; the method may also include the process of differentiating embryoid bodies into megakaryocyte progenitor cells; the method may also include the process of differentiating embryoid bodies into platelets; the method may also include the process of proliferating and / or differentiating hematopoietic stem / progenitor cells into megakaryocyte progenitor cells.

[0110] In the present application, the culture method may include the following steps: (1) inoculating pluripotent cells in platelet differentiation medium I to obtain embryoid bodies; (2) inoculating the embryoid bodies in platelet differentiation medium II to obtain hematopoietic stem / progenitor cells; (3) inoculating the hematopoietic stem / progenitor cells in platelet differentiation medium III to obtain megakaryocyte progenitor cells; (4) inoculating the megakaryocyte progenitor cells in platelet differentiation medium IV to obtain platelets.

[0111] In the present application, the culture method may include the following steps: (1) inoculating pluripotent cells in platelet differentiation medium I and culturing for about 5-8 days to obtain embryoid bodies; (2) inoculating the embryoid bodies in platelet differentiation medium II and culturing for about 7 days to obtain hematopoietic stem / progenitor cells; (3) inoculating the hematopoietic stem / progenitor cells in platelet differentiation medium III and culturing for about 7 days to obtain megakaryocyte progenitor cells; (4) inoculating the megakaryocyte progenitor cells in platelet differentiation medium IV and culturing for about 5-10 days to obtain platelets.

[0112] In the present application, the culture method may include the following steps: (1) inoculating hematopoietic stem / progenitor cells into platelet differentiation medium III to obtain megakaryocyte progenitor cells; (2) inoculating megakaryocyte progenitor cells into platelet differentiation medium IV to obtain platelets.

[0113] In the present application, the culture method may include the following steps: (1) inoculating hematopoietic stem / progenitor cells in platelet differentiation medium III and culturing for about 7 days to obtain megakaryocyte progenitor cells; (2) inoculating megakaryocyte progenitor cells in platelet differentiation medium IV and culturing for about 5-10 days to obtain platelets.

[0114] In the present application, the culture method may comprise culturing cells on a matrix-coated surface.

[0115] In certain embodiments, the culture method may comprise laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel. TM In certain embodiments, the matrix in the culture method may be vitronectin or Matrigel. TM .

[0116] In the present application, the culture method may include regularly or irregularly supplementing and / or replacing the culture medium.

[0117] In the present application, the culture method may comprise digesting the cells into single cells after the cells have proliferated to a sufficient number.

[0118] In certain embodiments, the digestion in the culture method uses Accutase digestion solution. In certain embodiments, the digestion in the culture method uses Trypsin and EDTA.

[0119] In the present application, the culturing method may comprise culturing cells at about 37-39°C, for example, about 36.5°C, about 37°C, about 37.5°C, about 38°C, about 38.5°C, about 39°C, about 39.5°C.

[0120] In the present application, the culturing method may include culturing cells under conditions of about 3-7% CO2, for example, about 3% CO2, about 3.5% CO2, about 4% CO2, about 4.5% CO2, about 5% CO2, about 5.5% CO2, about 6% CO2, about 6.5% CO2, about 7% CO, about 7.5% CO2.

[0121] In the present application, the culture method can be performed under serum-containing culture conditions.

[0122] In the present application, the culture method can be performed under serum-free culture conditions.

[0123] In the present application, the culture method can be performed under feeder-free culture conditions.

[0124] In the present application, the culture method can be carried out under culture conditions with feeding.

[0125] In the present application, the source of the pluripotent cells in the culture method is not limited and can be of mammalian or non-mammalian origin.

[0126] In some embodiments, the pluripotent cells are derived from human embryonic stem cells. In some embodiments, the pluripotent cells are derived from human induced pluripotent stem cells.

[0127] In the present application, the source of hematopoietic stem / progenitor cells in the culture method is not limited.

[0128] In some embodiments, the hematopoietic stem / progenitor cells are derived from human embryonic stem cells. In some embodiments, the hematopoietic stem / progenitor cells are derived from human induced pluripotent stem cells. In some embodiments, the hematopoietic stem / progenitor cells are derived from ex vivo human blood. In some embodiments, the hematopoietic stem / progenitor cells are derived from umbilical cord blood. In some embodiments, the hematopoietic stem / progenitor cells are derived from bone marrow. In some embodiments, the hematopoietic stem / progenitor cells are CD34 + Hematopoietic stem / progenitor cells.

[0129] In the present application, the source of megakaryocyte progenitor cells in the culture method is not limited.

[0130] In certain embodiments, the megakaryocytic progenitor cells are derived from human embryonic stem cells. In certain embodiments, the megakaryocytic progenitor cells are derived from human induced pluripotent stem cells. In certain embodiments, the megakaryocytic progenitor cells are derived from ex vivo human blood. In certain embodiments, the megakaryocytic progenitor cells are derived from umbilical cord blood. In certain embodiments, the megakaryocytic progenitor cells are derived from bone marrow. In certain embodiments, the megakaryocytic progenitor cells are derived from CD34 + Hematopoietic stem / progenitor cells.

[0131] In the present application, the culture medium in the culture method may be supplemented with one or more substances, including but not limited to: nutrients / extracts, growth factors, hormones, cytokines and culture medium additives.

[0132] In certain embodiments, the culture medium in the culture method may include a basal culture medium for culturing cells. The basal culture medium may be a single component or a combination of multiple culture media. The basal culture medium includes but is not limited to IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM, RPMI1640.

[0133] In certain embodiments, the culture medium in the culture method may be supplemented with one or more of the following substances, including but not limited to serum replacement, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, insulin, selenium, various interleukins (e.g., IL-1 to IL-18), various colony stimulating factors (e.g., granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 supplement, B27 supplement, Fms-related tyrosine kinase 3 ligand (FLt3). The additives are not limited to their sources and can be commercially available, natural, or recombinant.

[0134] In certain embodiments, inhibitors may be added to the culture medium in the culture method. The inhibitors may include but are not limited to GSK-3 inhibitors, MEK inhibitors, ROCK inhibitors, metalloproteinase inhibitors, and the like.

[0135] In certain embodiments, the ROCK inhibitor includes but is not limited to polynucleotides, polypeptides and small molecules that can reduce ROCK expression and / or ROCK activity. In certain embodiments, the ROCK inhibitor can be Y27632. In certain embodiments, the metalloproteinase inhibitor can be GM6001.

[0136] culture medium

[0137] On the other hand, the present application provides a platelet differentiation culture medium that can be used for cell proliferation and / or differentiation, and can be used for platelet differentiation.

[0138] In certain embodiments, the culture medium can be single component, comprising N2 supplement and B27 supplement.

[0139] In certain embodiments, the culture medium may be a combination culture medium, one or more of which may be used to perform the relevant operations, and one or more of the culture media in the combination culture medium may contain an N2 additive and a B27 additive.

[0140] In certain embodiments, the culture medium may be supplemented with one or more substances including, but not limited to, nutrients / extracts, growth factors, hormones, cytokines, and culture medium additives.

[0141] In some embodiments, the culture medium may include a basal culture medium for culturing cells. The basal culture medium may be a single component or a combination of multiple culture media. The basal culture medium includes but is not limited to IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM II, DMEM, RPMI1640.

[0142] In certain embodiments, the culture medium may be supplemented with one or more of the following substances, including but not limited to serum replacement, glutamine, NEAA (non-essential amino acids), ascorbic acid, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF), transferrin, insulin, selenium, various interleukins (e.g., IL-1 to IL-18), various colony stimulating factors (e.g., granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 supplement, B27 supplement, Fms-related tyrosine kinase 3 ligand (FLt3). The additives are not limited to their sources and can be commercially available, natural, or recombinant.

[0143] In certain embodiments, the culture medium may be supplemented with inhibitors, which may include but are not limited to GSK-3 inhibitors, MEK inhibitors, ROCK inhibitors, metalloproteinase inhibitors, and the like.

[0144] In certain embodiments, the ROCK inhibitor includes, but is not limited to, polynucleotides, polypeptides, and small molecules that can reduce ROCK expression and / or ROCK activity. In certain embodiments, the ROCK inhibitor can be Y27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, WAY-624704, H-1152, Azaindole 1 (TC-S 7001), Hydroxyfasudil (HA-1100), Y-39983, Netarsudil (AR-13324), GSK269962A, Ripasudil (K-115) hydrochloride dihydrate, Belumosudil (KD025), AT13148, Emetine hydrochloride, ZINC00881524, or a ROCK pathway-targeted antibody.

[0145] In certain embodiments, the metalloproteinase inhibitor can be GM6001, CL-82198, SM-7368, Dendrobii caulis Extract, T-26c, AUDA (compound 43), MMP-9-IN-1 (OUN87710), Isoliquiritin apioside (ISLA, ILA), Nobiletin (NSC 76751, Hexamethoxyflavone), Solasodine, Triolein, Cordycepin (3'-Deoxyadenosine), Doxycycline Hyclate (WC2031), Trans-Zeatina ((E)-Zeatin), Lactobionic acid (Galactosylgluconic acid), Doxycycline, Morroniside, 1,10-Phenanthroline, JNJ0966, Abametapir (HA-44, BRN 0123183), o-Phenanthroline, Batimastat (BB-94), Marimastat (BB-2516, TA2516), SB-3CT, TAPI-1, NSC 405020, T-5224, Auraptene (7-geranyloxycoumarin), Polygalacic acid, ARP 100, Absinthin (Absynthine), Ginkgolide C (BN-52022).

[0146] In the present application, the culture medium in the method and the platelet differentiation medium can be a combination of one or more of the following culture media: platelet differentiation medium I, platelet differentiation medium II, platelet differentiation medium III, platelet differentiation medium IV.

[0147] In certain embodiments, the platelet differentiation medium I may comprise a basal medium, for example, mTeSR.

[0148] In certain embodiments, the platelet differentiation medium I may comprise a basal medium and other substances, for example, a ROCK inhibitor. In certain embodiments, the ROCK inhibitor may be Y27632.

[0149] In certain embodiments, the platelet differentiation medium II may comprise a basal medium, for example, APEL.

[0150] In certain embodiments, the platelet differentiation medium II may comprise a basal medium and other substances. For example, the other substances may comprise one or more selected from the following group: BMP4, VEGF, and SCF.

[0151] In certain embodiments, the platelet differentiation medium III may comprise a basal medium, such as StemSpan TM SFEM II.

[0152] In certain embodiments, the platelet differentiation medium III may comprise a basal medium and other substances. For example, the other substances may comprise one or more selected from the following group: IL3, IL6, SCF, TPO, and FLt3.

[0153] In certain embodiments, the platelet differentiation medium IV may comprise a basal medium, for example, IMDM.

[0154] In certain embodiments, the platelet differentiation medium IV may comprise a basal medium and other substances, for example, the other substances may comprise one or more selected from the following group: N2 additive, B27 additive, NEAA, glutamine, ITS, ascorbic acid, IL6, SCF, TPO.

[0155] In the present application, the concentration of the N2 additive in the culture medium and the platelet differentiation medium in the method can be about 0.5%. For example, the concentration of the N2 additive is about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, about 0.4%, about 0.41%, about 0.42%, about 0.43%, about 0.44%, about 0.45%, about 0.46%, about 0.47%, about 0.48%, about 0.50%, about 0.51%, about 0.52%, about 0.53%, about 0.54%, about 0.55%, about 0.56%, about 0.57%, about 0.58%, about 0.59%, about 0.60%, about 0.61%, about 0.62%, about 0.63%, about 0.64%, about 0.65%, about 0.66%, about 0.67%, about 0.68%, about 0.69%, about 0.70%, about 0.71%, about 0.72%, about 0.73%, about 0.74%, about 0.75%, about 0.76%, about 0.77%, about 0.78%, about 0.79%, about 0.80%, about 0.81%, about 0.82%, about 0.83%, about 0.84%, about 0.85%, about 0.86%, about 0.87%, about 0.88%, about 0.89%, about 0.90%, about 0.91%, about 0.92%, 47%, about 0.48%, about 0.49%, about 0.5%, about 0.51%, about 0.52%, about 0.53%, about 0.54%, about 0.55%, about 0.56%, about 0.57%, about 0.58%, about 0.59%, about 0.6%, about 0.7%, about 0.71%, about 0.72%, about 0.73%, about 0.74%, about 0.75%, about 0.76%, about 0.77%, about 0.78%, about 0.79%, about 0.8%.

[0156] In the present application, the B27 additive concentration in the culture medium and the platelet differentiation medium in the method can be about 1%. For example, the B27 additive concentration is about 0.25%, about 0.3%, about 0.35%, about 0.40%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, about 1.75%.

[0157] In the present application, the Y27632 concentration in the culture medium and the platelet differentiation medium in the method can be about 1-50 μM. For example, the Y27632 concentration is about 1 μM, about 3 μM, about 5 μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM.

[0158] In the present application, in the culture medium and the platelet differentiation culture medium in the method, the BMP4 concentration may be about 5-100 ng / mL. For example, the BMP4 concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0159] In the present application, the VEGF concentration in the culture medium and the platelet differentiation medium in the method can be about 5-100 ng / mL. For example, the VEGF concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0160] In the present application, the SCF concentration in the culture medium and the platelet differentiation medium in the method may be about 5-100 ng / mL. For example, the SCF concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0161] In the present application, the IL3 concentration in the culture medium and the platelet differentiation medium in the method can be about 5-100 ng / mL. For example, the IL3 concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0162] In the present application, the IL6 concentration in the culture medium and the platelet differentiation medium in the method can be about 5-100 ng / mL. For example, the IL6 concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0163] In the present application, the TPO concentration in the culture medium and the platelet differentiation medium in the method can be about 5-100 ng / mL. For example, the TPO concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0164] In the present application, the FLt3 concentration in the culture medium and the platelet differentiation culture medium in the method can be about 5-100 ng / mL. For example, the FLt3 concentration is about 5 ng / mL, 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, or about 100 ng / mL.

[0165] In the present application, the glutamine concentration in the culture medium and the platelet differentiation medium in the method can be about 1%. For example, the glutamine concentration is about 0.25%, about 0.3%, about 0.35%, about 0.40%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, about 1.75%.

[0166] In the present application, the ITS concentration in the culture medium and the platelet differentiation medium in the method may be about 1%. For example, the ITS concentration is about 0.25%, about 0.3%, about 0.35%, about 0.40%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, or about 1.75%.

[0167] In the present application, in the culture medium in the method and the platelet differentiation medium, the ascorbic acid concentration may be about 50-200 μM. For example, the ascorbic acid concentration is about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 105 μM, about 110 μM, about 115 μM, about 120 μM, about 125 μM, about 130 μM, about 135 μM, about 140 μM, about 145 μM, about 150 μM, about 155 μM, about 160 μM, about 165 μM, about 170 μM, about 175 μM, about 180 μM, about 185 μM, about 190 μM, about 195 μM, or about 200 μM.

[0168] Composition and use

[0169] In another aspect, the present application provides a composition comprising pluripotent cells and the culture medium.

[0170] On the other hand, the present application provides a composition comprising hematopoietic stem / progenitor cells and the culture medium.

[0171] In another aspect, the present application provides a composition comprising megakaryocyte progenitor cells and the culture medium.

[0172] In certain embodiments, the composition and content of the composition can be varied. In certain embodiments, the composition and content of the composition can be specified. In certain embodiments, the composition can include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.

[0173] In another aspect, the present application provides use of the method in differentiating platelets, which comprises the culture medium.

[0174] On the other hand, the present application provides use of the culture medium in inducing proliferation of pluripotent cells and / or differentiation of platelets.

[0175] On the other hand, the present application provides use of the culture medium in the proliferation of hematopoietic stem / progenitor cells and / or differentiation of platelets.

[0176] On the other hand, the present application provides use of the culture medium in inducing megakaryocyte progenitor cell proliferation and / or platelet differentiation.

[0177] On the other hand, the present application provides a culture platform for obtaining platelets, which comprises the method and the culture medium.

[0178] On the other hand, the present application provides a use of the culture platform, which comprises the method and the culture medium.

[0179] On the other hand, the present application provides use of the method, the culture medium, the composition, and the culture platform for preparing platelets.

[0180] In another aspect, the present application provides a method for preventing and / or treating a disease, comprising using the method, the culture medium, and the composition.

[0181] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0182] Example

[0183] Example 1 Platelet differentiation culture

[0184] Induce cell differentiation into platelets using the following methods and culture media:

[0185] (1) ESC / iPSC were seeded in Matrigel or vitronectin pre-coated culture dishes or cell culture six-well plates (1×10 cells per 10 cm culture dish). 6 cells or 2 × 10 cells per well of a 6-well plate 5 cells) were cultured in mTeSR1 medium at 37°C, 5% CO2 until the cell saturation was about 70-80%, which usually took about 4-7 days;

[0186] (2) Aspirate the culture supernatant and wash once with DPBS buffer preheated to 37°C; digest with Accutase digestion solution at 37°C for about 5 minutes, and resuspend the cells in preheated DPBS;

[0187] (3) On Day 0, the cell suspension was centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in platelet differentiation medium I and inoculated into 6-well suspension culture plates (3 × 10 cells per well of the 6-well plate).5 cells) and cultured at 37°C with 5% CO2;

[0188] (4) On Day 1, the cell suspension was collected and centrifuged at 300 g for 5 min. After removing the supernatant, the cells were cultured with Platelet Differentiation Medium II at 37°C and 5% CO2.

[0189] (5) From Day 1 to Day 6, cells were cultured continuously and fresh culture medium was replaced regularly;

[0190] (6) On Day 7, the EB suspension was collected and centrifuged at 400 g for 5 min. The supernatant was discarded and digested with 1 ml of preheated 0.05% Trypsin + EDTA in a 37°C water bath for 5 min. 1 ml of 10% FBS was added to terminate the digestion and the cells were centrifuged at 400 g for 5 min.

[0191] (7) Remove the supernatant of the cell suspension and resuspend it in differentiation medium III to obtain CD34 + Hematopoietic stem / progenitor cells were obtained by flow cytometry analysis, and the purity of CD34 cells was greater than 90%. + cell;

[0192] (8) From Day 7 to Day 14, CD34 + Hematopoietic stem / progenitor cell suspension was inoculated into 6-well suspension culture plates (8×10 4 cells) were cultured at 37°C, 5% CO2 for 7 days, with half of the medium changed regularly by adding fresh platelet differentiation medium III;

[0193] (9) On Day 14, megakaryocyte progenitor cells (MKP) were obtained. MKP were collected and centrifuged at 400 g for 10 min. After discarding the supernatant, the cells were resuspended in platelet differentiation medium IV and inoculated into 6-well suspension culture plates (8 × 10 cells per well). 5 cells) and cultured at 37°C with 5% CO2;

[0194] (10) From Day 14 to Day 19, continue culturing cells and regularly replenish with fresh Platelet Differentiation Medium IV;

[0195] (11) On Day 19, fresh platelet differentiation medium IV was added, and the cells were transferred to 39°C, 5% CO2 and cultured, and fresh platelet differentiation medium IV was added regularly;

[0196] (12) On Day 22, GM6001 was added to each well and cultured at 39°C, 5% CO2 for 2 days;

[0197] (13) On Day 24, after the cells were collected, they were centrifuged at 1000 g for 10 min to obtain a high-purity platelet suspension;

[0198] (14) During the culture of MKP, the culture medium components were optimized and improved, and platelet differentiation medium IV with different components was used, the components of which are shown in Table 1 ;

[0199] (15) Platelet differentiation medium I: can contain mTeSR basal medium and ROCK inhibitor Y27632, with the inhibitor concentration being approximately 1-50 μM;

[0200] (16) Platelet differentiation medium II: may include APEL basal medium, BMP4 concentration of about 5-100 ng / mL, VEGF concentration of about 5-100 ng / mL, and SCF concentration of about 5-100 ng / mL;

[0201] (17) Platelet Differentiation Medium III: Can contain StemSpan TM SFEM II medium, IL3 concentration is about 5-100 ng / mL, IL6 concentration is about 5-100 ng / mL, SCF concentration is about 5-100 ng / mL, TPO concentration is about 5-100 ng / mL, and FLt concentration is about 5-100 ng / mL;

[0202] (18) Platelet differentiation medium IV: may include IMDM basal medium, N2 additive concentration of about 0.5%, B27 additive concentration of about 1%, glutamine concentration of about 1%, ITS concentration of about 1%, ascorbic acid concentration of about 50-200 μM, IL6 concentration of about 5-100 ng / mL, SCF concentration of about 5-100 ng / mL, TPO concentration of about 5-100 ng / mL, and NEAA.

[0203] Table 1. Platelet differentiation medium IV different differentiation systems

[0204] Group IMDMBSAN2+B27ITSNEAA+Glutamine Ascorbic Acid IL6+SCF+TPOS1√√√√√√√S2√ √√√√√√S3√√ √√√√√S4√√√ √√√S5√√√√ √√S6√√√√√√

[0205] The experimental results are shown in Figures 1 to 6. Cell surface protein staining (①PE-CD41; APC-CD42b; DAPI ②APC-CD61; FITC-CD63; DAPI) was performed, and then flow cytometry analysis was performed. The statistical results are shown in Table 2, indicating that the N2 additive and the B27 additive have a decisive effect on platelet differentiation.

[0206] Table 2. Flow cytometry results statistics

[0207] Group CD41 + CD42b + CD41 + CD42b + CD61 + CD63 + CD61 + CD63 + S159.12%25.50%24.50%60.65%33.72%26.95%S249.52%27.47%27.14%53.46%18.49%15.04%S359.21%12.44%11.09%62.51%37.02%29.79 %S454.59%20.34%19.63%56.57%30.7%22.95%S561.32%26.49%27.17%59.37%33.32%26.57%S656.83%23.58%23.20%57.43%29.26%23.58%

Claims

1. A method for inducing proliferation and / or differentiation of pluripotent cells into platelets, comprising using a platelet differentiation medium containing N2 supplement and B27 supplement components.

2. A method for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into platelets, comprising using a platelet differentiation medium containing N2 additive and B27 additive components.

3. A method for inducing megakaryocyte progenitor cells to proliferate and / or differentiate into platelets, comprising using a platelet differentiation medium containing N2 additive and B27 additive components.

4. The method of any one of claims 1-3, wherein the platelet differentiation medium comprises N2 supplement at a concentration of about 0.5%.

5. The method of any one of claims 1-4, wherein the platelet differentiation medium comprises B27 supplement at a concentration of about 1%.

6. The method according to any one of claims 1 to 5, wherein the platelet differentiation medium further comprises one or more selected from the group consisting of TPO (thrombopoietin), SCF (stem cell factor), IL6, ITS (insulin, transferrin, selenium), glutamine, NEAA (non-essential amino acids), ascorbic acid, BMP4 (bone morphogenetic protein), VEGF (vascular endothelial growth factor), IL3 and FLt3 (Fms-related tyrosine kinase 3 ligand).

7. The method according to any one of claims 1 to 6, wherein the platelet differentiation medium comprises TPO at a concentration of 5-100 ng / mL.

8. The method according to any one of claims 1 to 7, wherein the platelet differentiation medium comprises SCF at a concentration of 5-100 ng / mL.

9. The method according to any one of claims 1 to 8, wherein the platelet differentiation medium comprises IL6 at a concentration of 5-100 ng / mL.

10. The method of any one of claims 1-9, wherein the platelet differentiation medium comprises about 1% ITS.

11. The method of any one of claims 1-10, wherein the platelet differentiation medium comprises about 1% glutamine.

12. The method according to any one of claims 1 to 11, wherein the platelet differentiation medium comprises ascorbic acid at a concentration of 50-200 μM.

13. The method according to any one of claims 1 to 12, wherein the platelet differentiation medium comprises BMP4 at a concentration of 5-100 ng / mL.

14. The method of any one of claims 1-13, wherein the platelet differentiation medium comprises VEGF at a concentration of 5-100 ng / mL.

15. The method of any one of claims 1-14, wherein the platelet differentiation medium comprises IL3 at a concentration of 5-100 ng / mL.

16. The method according to any one of claims 1 to 15, wherein the platelet differentiation medium comprises FLt3 at a concentration of 5-100 ng / mL.

17. The method of any one of claims 1-16, wherein the platelet differentiation medium further comprises a basal medium.

18. The method according to claim 17, wherein the basal medium comprises one or more selected from the group consisting of IMDM, StemSpan TM SFEM II, APEL, and mTeSR.

19. The method according to any one of claims 17-18, wherein the basal medium is IMDM.

20. The method of claims 1-19, wherein the basal medium is serum-free.

21. The method of any one of claims 1-20, wherein the method comprises culturing the cells at about 37-39°C.

22. The method of any one of claims 1-21, wherein the method comprises culturing cells under conditions of about 5% CO2.

23. The method according to any one of claims 1 to 22, wherein the method comprises supplementing and / or replacing the culture medium.

24. The method of any one of claims 1-23, wherein the method comprises adding a metalloproteinase inhibitor to the platelet differentiation medium.

25. The method of claim 24, wherein the metalloproteinase inhibitor is GM6001.

26. The method of claim 1 and any one of claims 4-25, wherein the method comprises culturing and / or maintaining pluripotent cells in a stem cell culture medium.

27. The method of any one of claims 1 and 4-26, wherein the stem cell culture medium is mTeSR.

28. The method of any one of claims 1 and 4-27, wherein the method comprises culturing cells on a matrix-coated surface.

29. The method of claim 28, wherein the matrix is ​​laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel TM .

30. The method according to any one of claims 28-29, wherein the matrix is ​​vitronectin or Matrigel TM .

31. The method according to any one of claims 1 and claims 4-30, wherein the method comprises digesting the pluripotent cells into single cells after the pluripotent cells have proliferated to a sufficient number.

32. The method of any one of claims 1 and 4-31, wherein the method uses an Accutase digestion solution.

33. The method of any one of claims 1 and 4 to 32, wherein the method comprises culturing cells in Platelet Differentiation Medium I containing a ROCK inhibitor.

34. The method of claim 33, wherein the ROCK inhibitor is Y27632.

35. The method of any one of claims 33-34, wherein the Y27632 concentration is about 1-50 μM.

36. The method according to any one of claims 33-35, wherein the base medium of the platelet differentiation medium I is mTeSR.

37. The method of any one of claims 1 and claims 4-36, wherein the pluripotent cells form embryoid bodies.

38. The method of any one of claims 1 and 4 to 37, wherein the method comprises culturing cells using Platelet Differentiation Medium II containing one or more of BMP4, VEGF, and SCF.

39. The method according to any one of claims 1 and 4 to 38, wherein the platelet differentiation medium II comprises BMP4 at a concentration of 5 to 100 ng / mL.

40. The method of any one of claims 1 and 4-39, wherein the platelet differentiation medium II comprises VEGF at a concentration of 5-100 ng / mL.

41. The method according to any one of claims 1 and 4 to 40, wherein the platelet differentiation medium II comprises SCF at a concentration of 5 to 100 ng / mL.

42. The method according to any one of claims 1 and claims 4-41, wherein the base medium of the platelet differentiation medium II is APEL.

43. The method of any one of claims 1 and 4-42, wherein the method comprises culturing the cells at 37-39°C.

44. The method of any one of claims 1 and 4-43, wherein the method comprises culturing cells under conditions of 5% CO2.

45. The method of any one of claims 1 and 4-44, wherein the method comprises culturing the cells for 6 days.

46. ​​The method according to any one of claims 1 and claims 4-45, wherein the method comprises supplementing and / or replacing Platelet Differentiation Medium II.

47. The method of any one of claims 1 and 4-46, wherein the method comprises harvesting and digesting the cells after culture.

48. The method of any one of claims 1 and 4-47, wherein the method comprises digesting the cultured cells using 0.05% Trypsin and EDTA.

49. The method according to any one of claims 1-2 and claims 4-48, wherein the method comprises culturing cells using platelet differentiation medium III containing one or more of IL3, IL6, SCF, TPO, and FLt3.

50. The method of any one of claims 1-2 and claims 4-49, wherein the platelet differentiation medium III comprises IL3 at a concentration of 5-100 ng / mL.

51. The method of any one of claims 1-2 and claims 4-50, wherein the platelet differentiation medium III comprises IL6 at a concentration of 5-100 ng / mL.

52. The method of any one of claims 1-2 and claims 4-51, wherein the platelet differentiation medium III comprises SCF at a concentration of 5-100 ng / mL.

53. The method of any one of claims 1-2 and claims 4-52, wherein the platelet differentiation medium III comprises TPO at a concentration of 5-100 ng / mL.

54. The method according to any one of claims 1-2 and claims 4-53, wherein the platelet differentiation medium III comprises FLt3 at a concentration of 5-100 ng / mL.

55. The method according to any one of claims 1-2 and claims 4-54, wherein the base medium of the platelet differentiation medium III is StemSpan TM SFEM II.

56. The method of any one of claims 1-2 and claims 4-55, wherein the method comprises culturing the cells at 37°C.

57. The method of any one of claims 1-2 and claims 4-56, wherein the method comprises culturing cells under conditions of 5% CO2.

58. The method of any one of claims 1-2 and claims 4-57, wherein the method comprises culturing the cells for 7 days.

59. The method of any one of claims 1-2 and claims 4-58, wherein the method comprises supplementing and / or replacing Platelet Differentiation Medium III.

60. The method of any one of claims 1-59, wherein the method comprises culturing cells using Platelet Differentiation Medium IV containing one or more of N2 Supplement, B27 Supplement, NEAA, glutamine, ITS, ascorbic acid, IL6, SCF, and TPO.

61. The method of any one of claims 1-60, wherein the platelet differentiation medium IV comprises about 0.5% N2.

62. The method of any one of claims 1-61, wherein the platelet differentiation medium IV comprises about 1% B27.

63. The method of any one of claims 1-62, wherein the platelet differentiation medium IV comprises about 1% glutamine.

64. The method of any one of claims 1-63, wherein the platelet differentiation medium IV comprises about 1% ITS.

65. The method of any one of claims 1-64, wherein the platelet differentiation medium IV comprises ascorbic acid at a concentration of 50-200 μM.

66. The method of any one of claims 1-65, wherein the platelet differentiation medium IV comprises IL6 at a concentration of 5-100 ng / mL.

67. The method of any one of claims 1-66, wherein the platelet differentiation medium IV comprises SCF at a concentration of 5-100 ng / mL.

68. The method of any one of claims 1-67, wherein the platelet differentiation medium IV comprises TPO at a concentration of 5-100 ng / mL.

69. The method according to any one of claims 1 to 68, wherein the base medium of the platelet differentiation medium IV is IMDM.

70. The method of any one of claims 1-69, wherein the method comprises culturing cells at 37-39°C.

71. The method of any one of claims 1-70, wherein the method comprises culturing cells under conditions of 5% CO2.

72. The method of any one of claims 1-71, wherein the method comprises culturing the cells using Platelet Differentiation Medium IV for 5-10 days.

73. The method of any one of claims 1-72, wherein the method comprises supplementing and / or replacing Platelet Differentiation Medium IV.

74. The method of any one of claims 1-73, wherein the method comprises adding a metalloproteinase inhibitor to Platelet Differentiation Medium IV.

75. The method of claim 74, wherein the metalloproteinase inhibitor is GM6001.

76. The method of any one of claims 1-75, wherein the method comprises one or more of the following steps: (1) Inoculating pluripotent cells into platelet differentiation medium I to obtain embryoid bodies; (2) Inoculating the embryoid bodies in platelet differentiation medium II to obtain hematopoietic stem / progenitor cells; (3) inoculating hematopoietic stem / progenitor cells in platelet differentiation medium III to obtain megakaryocyte progenitor cells; (4) Megakaryocyte progenitor cells are inoculated into platelet differentiation medium IV to obtain platelets.

77. The method of any one of claims 1 to 76, wherein the method comprises one or more of the following steps: (1) Inoculating pluripotent cells in platelet differentiation medium I and culturing for approximately 5-8 days to obtain embryoid bodies; (2) The embryoid bodies were inoculated in platelet differentiation medium II and cultured for about 7 days to obtain hematopoietic stem / progenitor cells; (3) inoculating hematopoietic stem / progenitor cells in platelet differentiation medium III and culturing for approximately 7 days to obtain megakaryocyte progenitor cells; (4) The megakaryocyte progenitor cells are inoculated into platelet differentiation medium IV and cultured for about 5-10 days to obtain platelets.

78. The method of any one of claims 2 and 49-75, wherein the method comprises one or more of the following steps: (1) Hematopoietic stem / progenitor cells were inoculated into platelet differentiation medium III to obtain megakaryocyte progenitor cells; (2) Megakaryocyte progenitor cells are inoculated into platelet differentiation medium IV to obtain platelets.

79. The method of any one of claims 2, 49-75, and 78, wherein the method comprises one or more of the following steps: (1) Hematopoietic stem / progenitor cells were inoculated into platelet differentiation medium III and cultured for approximately 7 days to obtain megakaryocyte progenitor cells; (2) Megakaryocyte progenitor cells are inoculated into platelet differentiation medium IV and cultured for about 5-10 days to obtain platelets.

80. The method of any one of claims 1 and claims 4-79, wherein the pluripotent cells are derived from a mammal.

81. The method of any one of claims 1 and claims 4-80, wherein the pluripotent cells are human embryonic stem cells.

82. The method of any one of claims 1 and claims 4-80, wherein the pluripotent cells are human induced pluripotent stem cells.

83. The method of any one of claims 2 and claims 4-79, wherein the hematopoietic stem / progenitor cells are derived from human embryonic stem cells.

84. The method of any one of claims 2 and claims 4-79, wherein the hematopoietic stem / progenitor cells are derived from human induced pluripotent stem cells.

85. The method of any one of claims 2 and claims 4-79, wherein the hematopoietic stem / progenitor cells are derived from ex vivo human blood.

86. The method of any one of claims 2 and claims 4-79, wherein the hematopoietic stem / progenitor cells are derived from umbilical cord blood.

87. The method of any one of claims 2 and claims 4-79, wherein the hematopoietic stem / progenitor cells are derived from bone marrow.

88. The method according to any one of claims 2 and claims 4-79, wherein the hematopoietic stem / progenitor cells are CD34 + Hematopoietic stem / progenitor cells.

89. The method of any one of claims 3-79, wherein the megakaryocyte progenitor cells are derived from human embryonic stem cells.

90. The method of any one of claims 3-79, wherein the megakaryocytic progenitor cells are derived from human induced pluripotent stem cells.

91. The method of any one of claims 3-79, wherein the megakaryocyte progenitor cells are derived from ex vivo human blood.

92. The method of any one of claims 3-79, wherein the megakaryocyte progenitor cells are derived from umbilical cord blood.

93. The method of any one of claims 3-79, wherein the megakaryocytic progenitor cells are derived from bone marrow.

94. The method according to any one of claims 3-79, wherein the megakaryocyte progenitor cells are derived from CD34 + Hematopoietic stem / progenitor cells.

95. The method of any one of claims 1-94, wherein the method is in serum-free conditions.

96. The method of any one of claims 1-95, wherein the method does not require co-cultivation.

97. A culture medium comprising an N2 supplement and a B27 supplement.

98. The culture medium of claim 97, wherein the culture medium comprises an N2 additive at a concentration of about 0.5%.

99. The culture medium of any one of claims 97-98, wherein the culture medium comprises a B27 supplement at a concentration of about 1%.

100. The culture medium according to any one of claims 97-99, wherein the culture medium comprises platelet differentiation medium I, and the platelet differentiation medium I comprises a basal medium.

101. The culture medium of claim 100, wherein the basal medium is mTeSR.

102. The culture medium of any one of claims 97-101, wherein the culture medium further comprises a ROCK inhibitor.

103. The culture medium of claim 102, wherein the ROCK inhibitor is Y27632.

104. The culture medium of any one of claims 102-103, wherein the Y27632 concentration is about 1-50 μM.

105. The culture medium according to any one of claims 97-103, wherein the culture medium comprises platelet differentiation medium II, and the platelet differentiation medium II comprises a basal medium.

106. The culture medium of claim 105, wherein the basal medium is APEL.

107. The culture medium of any one of claims 105-106, wherein the culture medium further comprises one or more of BMP4, VEGF, and SCF.

108. The culture medium of any one of claims 107, wherein the concentration of BMP4 is 5-100 ng / mL.

109. The culture medium of any one of claims 107-108, wherein the concentration of VEGF is 5-100 ng / mL.

110. The culture medium of any one of claims 107-109, wherein the concentration of SCF is 5-100 ng / mL.

111. The culture medium according to any one of claims 97-110, wherein the culture medium comprises platelet differentiation medium III, and the platelet differentiation medium III comprises a basal medium.

112. The culture medium of claim 111, wherein the basal medium is StemSpan TM SFEM II.

113. The culture medium of any one of claims 111-112, wherein the culture medium further comprises one or more of IL3, IL6, SCF, TPO and FLt3.

114. The culture medium according to claim 113, wherein the concentration of IL3 is 5-100 ng / mL.

115. The culture medium of any one of claims 113-114, wherein the concentration of IL6 is 5-100 ng / mL.

116. The culture medium of any one of claims 113-115, wherein the concentration of SCF is 5-100 ng / mL.

117. The culture medium of any one of claims 113-116, wherein the concentration of TPO is 5-100 ng / mL.

118. The culture medium according to any one of claims 113-117, wherein the concentration of FLt3 is 5-100 ng / mL.

119. The culture medium of any one of claims 97-118, wherein the culture medium comprises platelet differentiation medium IV, and the platelet differentiation medium IV comprises basal medium.

120. The culture medium of claim 119, wherein the basal medium is IMDM.

121. The culture medium of any one of claims 119-120, wherein the culture medium further comprises one or more of N2 supplement, B27 supplement, NEAA, glutamine, ITS, ascorbic acid, IL6, SCF, and TPO.

122. The culture medium of claim 121, wherein the concentration of N2 is about 0.5%.

123. The culture medium of any one of claims 121-122, wherein the concentration of B27 is about 1%.

124. The culture medium of any one of claims 121-123, wherein the concentration of glutamine is about 1%.

125. The culture medium of any one of claims 121-124, wherein the concentration of ITS is about 1%.

126. The culture medium of any one of claims 121-125, wherein the concentration of ascorbic acid is 50-200 μM.

127. The culture medium of any one of claims 121-126, wherein the concentration of IL6 is 5-100 ng / mL.

128. The culture medium of any one of claims 121-127, wherein the concentration of SCF is 5-100 ng / mL.

129. The culture medium of any one of claims 121-128, wherein the concentration of TPO is 5-100 ng / mL.

130. Use of the method of any one of claims 1-96 for differentiating platelets.

131. Use of the culture medium according to any one of claims 97 to 129 for inducing proliferation of pluripotent cells and / or differentiation of platelets, comprising the culture medium.

132. Use of the culture medium described in any one of claims 97-129 in inducing proliferation of hematopoietic stem / progenitor cells and / or differentiation of platelets.

133. Use of the culture medium according to any one of claims 97 to 129 for inducing proliferation of megakaryocyte progenitor cells and / or differentiation of platelets.

134. A composition comprising pluripotent cells and the culture medium of any one of claims 97-129.

135. A composition comprising hematopoietic stem / progenitor cells and the culture medium of any one of claims 97-129.

136. A composition comprising megakaryocytic progenitor cells and the culture medium of any one of claims 97-129.

137. A culture platform for obtaining platelets, comprising the method of any one of claims 1-96 and / or any one of the culture media described in claims 97-129.

138. Use of the culture platform according to claim 137, comprising the method according to any one of claims 1 to 96 and / or any one of the culture media according to claims 97 to 129.

139. The method of any one of claims 1-96, the culture medium of any one of claims 97-129, the composition of any one of claims 134-136, and / or the culture platform of claim 137, for preparing platelets.

140. The method of any one of claims 1-96, the culture medium of any one of claims 97-129, the composition of any one of claims 134-136, and / or the culture platform of claim 137, for use in preventing and / or treating a disease.

141. Use of any method of claims 1-96, any culture medium of claims 97-129, any composition of claims 134-136, and / or the culture platform of claim 137 for pharmaceutical production.

142. A method for preventing and / or treating a disease, comprising administering the method of any one of claims 1-96, the culture medium of any one of claims 97-129, the composition of any one of claims 134-136, and / or the culture platform of claim 137.

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