Method for preparing platelets

Abstract

Provided is a method of inducing the proliferation and / or differentiation of induced pluripotent cells, embryonic stem cells, hematopoietic stem / progenitor cells and megakaryocytes into platelets, comprising culturing cells with a culture medium containing TPO (thrombopoietin), a SCF (stem cell factor), Flt3 (Fms-related tyrosine kinase 3 ligand), IL-3 (interleukin-3), IL-6 (interleukin-6), an AHR antagonist and PVA (polyvinyl alcohol). Also provided are a culture medium for inducing cell proliferation and / or differentiation into platelets, a composition, a culture platform and the use thereof.

Description

A method for preparing platelets Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for preparing 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] Currently, platelets mainly come from donations from donors. Due to the short storage time of donor-derived platelets and the insufficient number of donors, platelets have become a scarce resource. At the same time, there is a risk of bacterial contamination and deterioration in the process of platelet processing, preparation and transfusion. Therefore, it is very necessary to develop alternative strategies.

[0004] Human pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can be propagated and expanded indefinitely in vitro and possess the ability to differentiate and proliferate into one or more blood cell lineages. Therefore, the efficient in vitro differentiation of platelets from ESCs and iPSCs could be an effective approach to alleviate platelet shortages while also reducing the risk of platelet contamination and deterioration.

[0005] Therefore, it is urgent to develop a method for preparing platelets to quickly obtain high-purity and high-yield platelets to achieve the purpose of treating diseases. Summary of the Invention

[0006] The present application provides a method for inducing induced pluripotent cells, embryonic stem cells, hematopoietic stem / progenitor cells, and megakaryocytes to proliferate and / or differentiate into platelets, which comprises culturing cells in a culture medium containing TPO (thrombopoietin), SCF (stem cell factor), Flt3 (Fms-related tyrosine kinase 3 ligand), IL-3 (interleukin-3), IL-6 (interleukin-6), an AHR antagonist, and PVA (polyvinyl alcohol). The present application also provides a culture medium, a culture platform, and its application for inducing cell proliferation and / or differentiation into platelets. The methods, culture media, compositions and culture platforms provided in the present application have one or more of the following advantages: (1) improving the ability to induce pluripotent cells and embryonic stem cells to proliferate / differentiate into hematopoietic stem / progenitor cells, (2) improving the ability to induce pluripotent cells and embryonic stem cells to proliferate / differentiate into megakaryocytes, (3) improving the ability to induce pluripotent cells and embryonic stem cells to proliferate / differentiate into platelets, and (4) improving the ability of megakaryocytes to proliferate / differentiate into platelets; the preparation of platelets by the method described in the present application can shorten the culture time without changing the cell survival rate, thereby generating high-quality platelets.

[0007] On the one hand, the present application provides a method for inducing the proliferation and / or differentiation of induced pluripotent cells (iPSCs) into platelets, which comprises culturing cells in a culture medium containing TPO (thrombopoietin), SCF (stem cell factor), Flt3 (Fms-related tyrosine kinase 3 ligand), IL-3 (interleukin-3), IL-6 (interleukin-6), an AHR antagonist and PVA (polyvinyl alcohol).

[0008] In one aspect, the present application provides a method for inducing embryonic stem cells (ESCs) to proliferate and / or differentiate into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

[0009] In one aspect, the present application provides a method for inducing hematopoietic stem / progenitor cells (HSCs) to proliferate and / or differentiate into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

[0010] In one aspect, the present application provides a method for inducing megakaryocytes (MKPs) to proliferate and / or differentiate into platelets, comprising culturing cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist, and PVA.

[0011] In certain embodiments, the AHR antagonist in the method is one or more antagonists selected from the group consisting of SR1, GNF351, CH223191, IK-175, AHR antagonist 2, Bay-218, PDM2, PD98059, and BAY-2416964.

[0012] In certain embodiments, the AHR antagonist in the methods is SR1.

[0013] In certain embodiments, the AHR antagonist in the method is GNF351.

[0014] In certain embodiments, the AHR antagonist in the method is CH223191.

[0015] In certain embodiments, the culture medium in the method may further comprise one or more additives selected from the following group: BMP4 (bone morphogenetic protein), VEGF (vascular endothelial growth factor), Activin A (activin A), bFGF (basic fibroblast growth factor), PS (penicillin and streptomycin), GSK inhibitor, KMAD17 inhibitor, ROCK inhibitor.

[0016] In certain embodiments, the GSK inhibitor in the method is CHIR-99021.

[0017] In certain embodiments, the KMAD17 inhibitor in the method is KP457.

[0018] In certain embodiments, the ROCK inhibitor in the method is Y-39983.

[0019] In certain embodiments, the method comprises culturing the cells in a medium comprising TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, and PS.

[0020] In certain embodiments, the method comprises culturing cells in a medium comprising TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS, and PFHM-II.

[0021] In certain embodiments, the method further comprises culturing the cells in a medium containing TPO, SCF, KP457, Y-39983, and PS.

[0022] In certain embodiments, the method further comprises culturing the cells in a medium containing TPO, SCF, KP457, Y-39983, PS, and PFHM-II.

[0023] In certain embodiments, the method further comprises culturing the cells in a culture medium containing BMP4, VEGF, Activin A, CHIR-99021, and PS.

[0024] In certain embodiments, the method further comprises culturing the cells in a medium containing BMP4, VEGF, bFGF, and PS.

[0025] In certain embodiments, in the method, the culture medium comprises a basal culture medium, and the basal culture medium can be selected from one or more culture media in the following group: STEMdiff TM APEL TM 2 Medium, StemSpan TM -ACF Erythroid Expansion Medium, IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM I, DMEM, RPMI 1640 and PFHM-II.

[0026] In certain embodiments, the method comprises using a STEMdiff TM APEL TM 2. Cells were cultured in culture medium containing TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS, and PFHM-II.

[0027] In certain embodiments, the method further comprises using a STEMdiff TM APEL TM 2. Cells were cultured in medium containing TPO, SCF, KP457, Y-39983, PS, and PFHM-II.

[0028] In certain embodiments, the method further comprises using a STEMdiff TM APEL TM 2. Cells were cultured in culture medium containing BMP4, VEGF, Activin A, CHIR-99021, and PS.

[0029] In certain embodiments, the method further comprises using a StemSpan TM -Cells were cultured in ACF Erythroid Expansion Medium, BMP4, VEGF, bFGF, and PS.

[0030] In certain embodiments, the method comprises the following steps: (1) using a STEMdiff TM APEL TM 2 Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II; and (2) using a STEMdiff TM APEL TM 2. Cells were cultured in medium containing TPO, SCF, KP457, Y-39983, PS, and PFHM-II.

[0031] In certain embodiments, the method comprises the following steps: (1) using a STEMdiff TM APEL TM 2) Culture cells in a medium containing BMP4, VEGF, Activin A, CHIR-99021, and PS; (2) Culture cells in a medium containing StemSpan TM -ACF Erythroid Expansion Medium, BMP4, VEGF, bFGF and PS culture medium; (3) using STEMdiff TM APEL TM 2 Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II culture medium; and (4) using a STEMdiff TM APEL TM 2. Cells were cultured in medium containing TPO, SCF, KP457, Y-39983, PS, and PFHM-II.

[0032] In certain embodiments, the methods involve culturing the cells at about 35-39°C.

[0033] In certain embodiments, the methods involve culturing cells under conditions of about 3-7% CO2.

[0034] In certain embodiments, the methods involve culturing cells under serum-free culture conditions.

[0035] In certain embodiments, the methods involve culturing cells under culture conditions that are free of feeder cells.

[0036] In certain embodiments, in the method, the concentration of BMP4 is 5-100 ng / mL; the concentration of VEGF is 5-100 ng / mL; the concentration of Activin is The concentration of A is 5-100 ng / mL; the concentration of CHIR99021 is 0.5-10uM, the concentration of bFGF is 5-100 ng / mL, the concentration of IL-3 is 5-100 ng / mL; the concentration of IL-6 is 5-100 ng / mL; the concentration of SCF is 5-100 ng / mL; the concentration of TPO is 5-100 ng / mL; the concentration of Flt3 is 5-100 ng / mL; the concentration of PFHM-II is 1%-10%; the concentration of SR1 is 0.5-5μM; the concentration of PVA is 50-200ug / ml, the concentration of Y-39983 is 1-10mM; and the concentration of KP457 is 5-30mM.

[0037] In another aspect, the present application provides a culture medium comprising TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

[0038] In certain embodiments, in the culture medium, the AHR antagonist is selected from one or more antagonists in the group consisting of SR1, GNF351, CH223191, IK-175, AHR antagonist 2, Bay-218, PDM2, PD98059 and BAY-2416964.

[0039] In certain embodiments, the AHR antagonist in the culture medium is SR1.

[0040] In certain embodiments, the AHR antagonist in the culture medium is GNF351.

[0041] In certain embodiments, the AHR antagonist in the culture medium is CH223191.

[0042] In certain embodiments, the culture medium comprises STEMdiff TM APEL TM 2 Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II.

[0043] On the other hand, the present application provides a culture medium comprising TPO, SCF, KP457 and Y-39983.

[0044] In certain embodiments, the culture medium comprises STEMdiff TM APELTM 2 Medium, TPO, SCF, KP457, Y-39983, PS and PFHM-II.

[0045] On the other hand, the present application provides a combination culture medium, which comprises differentiation medium I and differentiation medium II, wherein the differentiation medium I comprises TPO, SCF, Flt3, IL-3, IL-6, SR1 and PVA; and the differentiation medium II comprises TPO, SCF, KP457 and Y-3998.

[0046] In certain embodiments, the culture medium, the differentiation medium I comprises STEMdiff TM APEL TM 2 Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II.

[0047] In certain embodiments, the differentiation medium II in the culture medium comprises STEMdiff TM APEL TM 2 Medium, TPO, SCF, KP457, Y-39983, PS and PFHM-II.

[0048] In certain embodiments, the culture medium further comprises differentiation medium III, wherein the differentiation medium III comprises BMP4, VEGF, Activin A, and CHIR-99021.

[0049] In certain embodiments, the culture medium, the differentiation medium III comprises STEMdiff TM APEL TM 2 Medium, BMP4, VEGF, Activin A, CHIR-99021 and PS.

[0050] In certain embodiments, the culture medium further comprises differentiation medium IV, wherein the differentiation medium IV comprises BMP4, VEGF and bFGF.

[0051] In certain embodiments, the culture medium, the differentiation medium IV comprises StemSpan TM -ACF Erythroid Expansion Medium, BMP4, VEGF, bFGF and PS.

[0052] In another aspect, a composition is provided, comprising induced pluripotent stem cells and the culture medium described herein.

[0053] In another aspect, a composition is provided, comprising embryonic stem cells and the culture medium described herein.

[0054] In another aspect, a composition is provided, comprising hematopoietic stem / progenitor cells and a culture medium as described herein.

[0055] In another aspect, a composition is provided, comprising megakaryocytes and the culture medium described herein.

[0056] On the other hand, a culture platform for obtaining platelets is provided, which comprises the method and / or culture medium described in the present application.

[0057] On the other hand, a method for preventing and / or treating a disease is provided, which comprises administering the method, culture medium, composition and / or culture platform described in the present application.

[0058] 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

[0059] 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:

[0060] FIG1 shows the staining results of mesoderm cells described in the present application.

[0061] FIG2 shows the staining results of hematopoietic stem / progenitor cells described in the present application.

[0062] FIG3 shows the megakaryocyte staining results described in the present application.

[0063] FIG4 shows the platelet detection results described in this application.

[0064] FIG5 shows the test results of cells cultured using different differentiation medium III as described in the present application.

[0065] FIG6 shows the results of cell culture assays using different AHR inhibitors as described in the present application.

[0066] FIG. 7 shows the test results of cells cultured using different differentiation medium IV as described in the present application.

[0067] FIG8 shows the test results of cells cultured using different differentiation medium IV as described in the present application. DETAILED DESCRIPTION

[0068] 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.

[0069] Definition of terms

[0070] 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.

[0071] 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.

[0072] In this application, the term "megakaryocyte," also known as "megakaryocyte progenitor cell," 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.

[0073] 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.

[0074] 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.

[0075] In the present application, the term "hematopoietic stem / progenitor cell" is also referred to as "hematopoietic stem cell", which can be used interchangeably in the present application and 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 be differentiated 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).

[0076] 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.

[0077] 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.

[0078] 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.

[0079] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0080] 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..."

[0081] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0082] 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.

[0083] Detailed Description of the Invention

[0084] method

[0085] On the one hand, the present application provides a method for inducing the proliferation and / or differentiation of induced pluripotent cells (iPSCs) into platelets, which comprises culturing cells in a culture medium containing TPO (thrombopoietin), SCF (stem cell factor), Flt3 (Fms-related tyrosine kinase 3 ligand), IL-3 (interleukin-3), IL-6 (interleukin-6), an AHR antagonist and PVA (polyvinyl alcohol).

[0086] In one aspect, the present application provides a method for inducing embryonic stem cells (ESCs) to proliferate and / or differentiate into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

[0087] In one aspect, the present application provides a method for inducing hematopoietic stem / progenitor cells (HSCs) to proliferate and / or differentiate into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

[0088] In one aspect, the present application provides a method for inducing megakaryocytes (MKPs) to proliferate and / or differentiate into platelets, comprising culturing cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist, and PVA.

[0089] In the present application, the method may further include the process of culturing, proliferating and / or differentiating induced pluripotent cells; the method may further include the process of culturing, proliferating and / or differentiating embryonic stem cells; the method may further include the process of culturing and / or differentiating embryoid bodies; the method may further include the process of culturing, proliferating and / or differentiating hematopoietic stem / progenitor cells; the method may further include the process of culturing, proliferating and / or differentiating megakaryocytes; the method may further include the process of culturing platelets; the method may further include the process of proliferating and / or differentiating induced pluripotent cells into embryoid bodies; the method may further include the process of proliferating and / or differentiating induced pluripotent cells into hematopoietic stem / progenitor cells; the method may further include the process of proliferating and / or differentiating induced pluripotent cells into megakaryocytes; the method may further include the process of differentiating embryoid bodies into hematopoietic stem / progenitor cells; the method may further include the process of differentiating embryoid bodies into megakaryocytes; the method may further include the process of differentiating embryoid bodies into platelets; the method may further include the process of proliferating and / or differentiating hematopoietic stem / progenitor cells into megakaryocytes.

[0090] In the present application, the method may include the following steps: (1) inoculating induced pluripotent cells or embryonic stem cells in differentiation medium I for mesodermal differentiation; (2) inoculating the cells in differentiation medium II to obtain hematopoietic stem / progenitor cells; (3) inoculating the hematopoietic stem / progenitor cells in differentiation medium III to obtain megakaryocytes; (4) inoculating megakaryocytes in differentiation medium IV to obtain platelets.

[0091] In the present application, the method may include the following steps: (1) inoculating induced pluripotent cells or embryonic stem cells in differentiation medium I for about 2 days for mesodermal differentiation; (2) inoculating the cells in differentiation medium II for about 5 days to obtain hematopoietic stem / progenitor cells; (3) inoculating the hematopoietic stem / progenitor cells in differentiation medium III for about 7 days to obtain megakaryocytes; (4) inoculating megakaryocytes in differentiation medium IV for about 4 days to obtain platelets.

[0092] In the present application, the method may comprise the following steps: (1) inoculating hematopoietic stem / progenitor cells into platelet differentiation medium III to obtain megakaryocytes; (2) inoculating megakaryocytes into platelet differentiation medium IV to obtain platelets.

[0093] In the present application, the method may include the following steps: (1) inoculating hematopoietic stem / progenitor cells in platelet differentiation medium III and culturing them for about 7 days to obtain megakaryocyte progenitor cells; (2) inoculating megakaryocyte progenitor cells in platelet differentiation medium IV and culturing them for about 4-8 days to obtain platelets.

[0094] As is common knowledge in the art, the culture time of each step can be adjusted during the culture process.

[0095] In the present application, the method may comprise culturing cells on a matrix-coated surface.

[0096] In certain embodiments, the matrix in the method can be laminin, vitronectin, gelatin, polylysine, thrombospondin, or Matrigel. TM In certain embodiments, the matrix in the culture method may be vitronectin, and the matrix in the culture method may be Matrigel (a soluble preparation of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

[0097] In the present application, the method may comprise regularly or irregularly supplementing and / or replacing the culture medium.

[0098] In the present application, the method may comprise digesting the cells into single cells after the cells have proliferated to a sufficient number.

[0099] 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.

[0100] In the present application, the 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.

[0101] In the present application, the culturing method may comprise 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.

[0102] In the present application, the method can be performed under serum-containing culture conditions. In the present application, the method can be performed under serum-free culture conditions.

[0103] In the present application, the method can be performed under feeder-free culture conditions. In the present application, the method can be performed under feeder-containing culture conditions.

[0104] In the present application, the source of the induced pluripotent cells in the method is not limited and can be of mammalian or non-mammalian origin.

[0105] In the present application, the source of human embryonic stem cells in the method is not limited and can be of mammalian or non-mammalian origin.

[0106] In the present application, the source of the hematopoietic stem / progenitor cells in the method is not limited. In certain embodiments, the hematopoietic stem / progenitor cells are derived from human embryonic stem cells. In certain embodiments, the hematopoietic stem / progenitor cells are derived from human induced pluripotent stem cells. In certain embodiments, the hematopoietic stem / progenitor cells are derived from ex vivo human blood. In certain embodiments, the hematopoietic stem / progenitor cells are derived from umbilical cord blood. In certain embodiments, the hematopoietic stem / progenitor cells are derived from bone marrow. In certain embodiments, the hematopoietic stem / progenitor cells are CD34 + Hematopoietic stem / progenitor cells.

[0107] In the present application, the source of megakaryocytes in the method is not limited. In certain embodiments, the megakaryocytes are derived from human embryonic stem cells. In certain embodiments, the megakaryocyte progenitor cells are derived from human induced pluripotent stem cells. In certain embodiments, the megakaryocytes are derived from ex vivo human blood. In certain embodiments, the megakaryocytes are derived from umbilical cord blood. In certain embodiments, the megakaryocytes are derived from bone marrow. In certain embodiments, the megakaryocytes are derived from CD34 + Hematopoietic stem / progenitor cells.

[0108] In the present application, the culture medium in the method may be supplemented with one or more substances, including but not limited to: nutrients / extracts, growth factors, hormones, cytokines and culture medium additives.

[0109] In certain embodiments, the 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 SFEMII, DMEM, RPMI1640.

[0110] In certain embodiments, the culture medium in the 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.

[0111] In the present application, the AHR antagonist in the method is one or more antagonists selected from the group consisting of SR1, GNF351, CH223191, IK-175, AHR antagonist 2, Bay-218, PDM2, PD98059, and BAY-2416964. In certain embodiments, the AHR antagonist may be SR1, the AHR antagonist may be GNF351, and the AHR antagonist may be CH223191.

[0112] In the present application, inhibitors may be added to the culture medium during the method. The inhibitors may include, but are not limited to, GSK inhibitors, KMAD17 inhibitors, ROCK inhibitors, and the like. In certain embodiments, the GSK inhibitor may be CHIR-99021. In certain embodiments, the KMAD17 inhibitor may be KP457. In certain embodiments, the ROCK inhibitor may be Y-39983.

[0113] culture medium

[0114] On the other hand, the present application provides a culture medium that can be used for proliferation and / or differentiation into platelets, which comprises TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

[0115] In certain embodiments, the culture medium may be a combination culture medium, one or more of which may be used for relevant operations, and one or more culture media in the combination culture medium may contain TPO, SCF, Flt3, IL-3, IL-6, AHR antagonist and PVA.

[0116] On the other hand, the present application provides a culture medium that can be used for proliferation and / or differentiation into platelets, which comprises TPO, SCF, KP457 and Y-39983.

[0117] 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 culture media in the combination culture medium may contain TPO, SCF, KP457, and Y-39983.

[0118] 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.

[0119] 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 STEMdiff TM APELTM 2Medium, StemSpan TM -ACF Erythroid Expansion Medium, IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM I, DMEM, RPMI 1640 and PFHM-II.

[0120] 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.

[0121] In the present application, inhibitors may be added to the culture medium in the method, and the inhibitors may include but are not limited to AHR antagonists, GSK inhibitors, KMAD17 inhibitors, ROCK inhibitors, and the like.

[0122] In the present application, the AHR antagonist in the method is one or more antagonists selected from the group consisting of SR1, GNF351, CH223191, IK-175, AHR antagonist 2, Bay-218, PDM2, PD98059, and BAY-2416964. In certain embodiments, the AHR antagonist may be SR1, the AHR antagonist may be GNF351, and the AHR antagonist may be CH223191.

[0123] 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.

[0124] In the present application, the culture medium may be a combination of one or more of the following culture media: differentiation medium I, differentiation medium II, differentiation medium III, and differentiation medium IV.

[0125] In some embodiments, the differentiation medium I may include a basal medium and other substances, for example, the basal medium may be STEMdiff TM APEL TM 2 Medium, the other substances may include one or more selected from the following group: BMP4, VEGF, Activin A, CHIR-99021 and PS.

[0126] In some embodiments, the differentiation medium II may include a basal medium and other substances, for example, the basal medium may be StemSpan TM -ACF Erythroid Expansion Medium, the other substances may include one or more selected from the following group: BMP4, VEGF, bFGF and PS.

[0127] In some embodiments, the differentiation medium III may include a basal medium and other substances, for example, the basal medium may be STEMdiff TM APEL TM 2 Medium, the other substances may include one or more selected from the following group: TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II.

[0128] In some embodiments, the differentiation medium IV may include a basal medium and other substances, for example, the basal medium may be STEMdiff TM APEL TM 2 Medium, the other substances may include one or more selected from the following group: TPO, SCF, KP457, Y-39983, PS and PFHM-II.

[0129] In certain embodiments, in the differentiation medium, the concentration of BMP4 is 5-100 ng / mL; the concentration of VEGF is 5-100 ng / mL; the concentration of Activin The concentration of A is 5-100 ng / mL; the concentration of CHIR99021 is 0.5-10uM, the concentration of bFGF is 5-100 ng / mL, the concentration of IL-3 is 5-100 ng / mL; the concentration of IL-6 is 5-100 ng / mL; the concentration of SCF is 5-100 ng / mL; the concentration of TPO is 5-100 ng / mL; the concentration of Flt3 is 5-100 ng / mL; the concentration of PFHM-II is 1%-10%; the concentration of SR1 is 0.5-5μM; the concentration of PVA is 50-200μg / ml, the concentration of Y-39983 is 1-10mM; and the concentration of KP457 is 5-30mM.

[0130] 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, about 100 ng / mL.

[0131] 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, about 100 ng / mL.

[0132] For example, the Activin A 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, about 100 ng / mL.

[0133] For example, the CHIR99021 concentration is about 0.5 μM, about 1.0 μM, about 1.5 μM, about 2.0 μM, about 2.5 μM, about 3.0 μM, about 3.5 μM, about 4.0 μM, about 4.5 μM, about 5.0 μM, about 5.5 μM, about 6.0 μM, about 6.5 μM, about 7.0 μM, about 7.5 μM, about 8.0 μM, about 8.5 μM, about 9.0 μM, about 9.5 μM, or about 10.0 μM.

[0134] For example, the bFGF 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.

[0135] For example, the IL-3 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, about 100 ng / mL.

[0136] For example, the IL-6 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, about 100 ng / mL.

[0137] 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, about 100 ng / mL.

[0138] 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, about 100 ng / mL.

[0139] 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.

[0140] For example, the PFHM-II additive concentration is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%.

[0141] For example, the SR1 concentration is about 0.5 μM, 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, or about 5.5 μM.

[0142] For example, the PVA concentration is about 5 μg / mL, 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 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, about 95 μg / mL, about 100 μg / mL, 105 μg / mL. / mL, about 110 μg / mL, about 115 μg / mL, about 120 μg / mL, about 125 μg / mL, about 130 μg / mL, about 135 μg / mL, about 140 μg / mL, about 145 μg / mL, about 150 μg / mL, about 155 μg / mL, about 160 μg / mL, about 165 μg / mL, about 170 μg / mL, about 175 μg / mL, about 180 μg / mL, about 185 μg / mL, about 190 μg / mL, about 195 μg / mL, about 200 μg / mL,.

[0143] For example, the Y-39983 concentration is about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM.

[0144] For example, the KP457 concentration is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM.

[0145] Composition and use

[0146] In another aspect, the present application provides a composition comprising induced pluripotent cells and the culture medium.

[0147] On the other hand, the present application provides a composition comprising embryonic stem cells and the culture medium

[0148] On the other hand, the present application provides a composition comprising hematopoietic stem / progenitor cells and the culture medium.

[0149] In another aspect, the present application provides a composition comprising megakaryocytes and the culture medium.

[0150] 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.

[0151] In another aspect, the present application provides use of the method in proliferating and / or differentiating platelets, which comprises the culture medium.

[0152] On the other hand, the present application provides use of the culture medium in inducing proliferation of pluripotent cells and / or differentiation of platelets.

[0153] 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.

[0154] On the other hand, the present application provides use of the culture medium in inducing megakaryocyte proliferation and / or platelet differentiation.

[0155] On the other hand, the present application provides a culture platform for obtaining platelets, which comprises the method and the culture medium.

[0156] On the other hand, the present application provides a use of the culture platform, which comprises the method and the culture medium.

[0157] On the other hand, the present application provides use of the method, the culture medium, the composition, and the culture platform for preparing platelets.

[0158] 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.

[0159] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0160] Example

[0161] Example 1 iPSC cell recovery / maintenance / passaging-seeding and differentiation

[0162] 1. Prepare a Matrigel-coated culture plate (without trophoblast cells) one day in advance and turn on the water bath to control the water temperature to 37°C. Remove the cells from the liquid nitrogen tank, spray the tube wall with 75% alcohol, and quickly transfer them to the cell compartment. Remove the water bath when only a small amount of solid matter remains in the cryopreservation tube. After complete dissolution, add 1mL of mTesR1 and centrifuge at 1000rpm for 3 minutes. Carefully discard the supernatant and resuspend the cells in 1mL of mTesR1. Count the cells and calculate the number of cells to 2x10 5 cells / well were added to a 6-well plate that had been plated in advance, and culture medium was added to 2 mL (containing 10 μM Y27632). After gentle shaking, the plate was quickly placed in the incubator.

[0163] 2. Replace the iPSC maintenance medium mTesR1 daily and observe the cell status (cell clone size, edge stereochemistry, nuclear-cytoplasmic ratio and distribution). When the cells are in good condition and the confluence is 70-80%, prepare to digest and inoculate the cells for differentiation.

[0164] 3. Aspirate and discard mTesR1, wash the cells once with 1 mL DPBS, add 1 mL Accutase and place in the incubator for 4 minutes, remove the Accutase, gently pipette the bottom of the well plate with 1 mL mTesR1, resuspend the cells, pipette evenly and count them. Add 2 mL mTesR1 (containing 10 uM Y27632) to the 6-well plate, and iPSCs are counted at 1.0x10 5 Cells were seeded per well, shaken and immediately placed in the incubator. After 24 hours, the cells were replaced with mTesR1 without Y27632 and differentiation was initiated after one day of culture.

[0165] Example 2 iPSC Differentiation of Platelets

[0166] Experimental steps:

[0167] Stage 1 (Day 0-Day 2): Before starting differentiation, wash the cells once with DPBS, then add 2 mL of Differentiation Medium I. No medium change is required. On Day 2, the clones are completely dispersed and reach the mesoderm.

[0168] Stage II (Day 2-Day 7): During this stage, 2 mL of differentiation medium II needs to be replaced every day. On Day 7, the cells reach the hemogenic endothelium and hematopoietic stem / progenitor cell stage.

[0169] Stage III (Day 7-Day 14):

[0170] On Day 7-Day 12, cells will be suspended in the supernatant. When changing the medium, centrifuge at 400g / 3min to discard the supernatant, resuspend the pellet in 2mL of differentiation medium III and add it to the original well plate.

[0171] On Day 12, the suspended cells in the supernatant and the lower adherent cells were processed separately. The suspended cells in the supernatant were collected by centrifugation at 400g / 3min into ultra-low adsorption 6-well plates and cultured with 2mL differentiation medium III until Day 14 without changing the medium in the middle. The lower adherent cells were digested with 1mL collagenase IV at 37℃ for 30min. The cells were dissociated by gently blowing the bottom of the well plate. The suspension was collected into a 15mL centrifuge tube, 1mL DMEM (high glucose, pyruvate) was added and mixed, centrifuged at 400g for 3min, the supernatant was discarded, 1mL Trypsin-EDTA was added to the centrifuge tube, and placed in a 37℃ incubator for 3min. Then, 2mL Trypsin-EDTA digestion stop solution (1ml DMEM high glucose + 1mL Fetal Bovine Serum) was added, mixed and centrifuged at 400g for 3min, the supernatant was discarded, and 2mL differentiation medium III was added. The cells were resuspended and inoculated into 6-well plates coated with Matrigel in advance at a 1:1 ratio. On the first day, 10μM For Y-27632, on Day 13, centrifuge at 400g for 3 minutes to discard the supernatant. Resuspend the pellet in 2 mL of Differentiation Medium III and add it to the original wells of the plate.

[0172] Stage IV (Day 14-Day 22) uses Differentiation Medium IV to culture cells. On Day 14, all suspended cells (automatically suspended and suspended cells after digestion) are collected by centrifugation at 400g / 3min, and the cells are counted. 100,000 cells are taken to measure CD34 / CD41. The total amount of suspended cells differentiated from iPSCs in a single well of a 6-well plate is 4-8 million, with a cell viability of more than 80% and a CD34+ / CD41+ ratio of 50%-80%. The cells collected on Day 14 are inoculated into a 6-well ultra-low adsorption microplate at 1.5 million / mL. No fluid change is required during subsequent culture. Only 1.5mL of fluid needs to be replenished on Day 18. Samples are taken on Day 18 to test CD41 / CD42b, and on Day 22 to test CD41 / CD42b and platelet count.

[0173] Differentiation medium used during culture:

[0174] Differentiation medium I: STEMdiff TM APEL TM 2 Medium+BMP4(30ng / mL)+VEGF(50ng / mL)+Activin A(25ng / mL)+CHIR-99021(1.5uM)+1%PS

[0175] Differentiation Medium II: StemSpan TM-ACF Erythroid Expansion Medium+BMP4(30ng / mL)+VEGF(50ng / mL)+bFGF(50ng / mL)+1%PS

[0176] Differentiation medium III: STEMdiff TM APEL TM 2 Medium+TPO(50ng / mL)+SCF(50ng / mL)+Flt3(25ng / mL)+IL-3(10ng / mL)+IL-6(10ng / mL)+PFHM-II(5%)+SR1(1uM)+PVA(100μg / mL)+1%PS

[0177] Differentiation Medium IV: STEMdiff TM APEL TM 2 Medium+TPO(50ng / mL)+SCF(50ng / mL)+KP457(15mM)+Y-39983(5mM)++PFHM-II(5%)+1%PS

[0178] Experimental results:

[0179] As shown in Figure 1, cells differentiated into mesoderm on Day 2. Staining for the mesoderm marker Brachyury revealed that 90% of cells were mesoderm, demonstrating that ESC / iPSCs can be efficiently differentiated into mesoderm cells via Stage I.

[0180] As shown in FIG2 , after the cells continued to differentiate until Day 7, they were stained with CD34 and CD43 antibodies, and it was found that more than 70% of CD34+CD43+ hematopoietic stem / progenitor cells could be obtained.

[0181] As shown in Figure 3, after continued differentiation to Day 14, a large number of blood cells and megakaryocytes floated out. After collection and staining with CD34 and CD41 antibodies, it was found that more than 67% of the megakaryocytes were CD41+, of which CD34+ accounted for more than 95%.

[0182] As shown in Figure 4 , a large number of platelets can be collected after cell differentiation to Day 22, with CD41+CD42b+ accounting for more than 50%.

[0183] Example 3: Culturing cells using different differentiation medium III

[0184] In Stage III, cells were cultured using different differentiation medium III. The experimental results are shown in Figure 5 . When SR1 or PVA was not added to the culture medium, the cell proliferation multiple and platelet differentiation efficiency were lower than those when both SR1 and PVA were added.

[0185] Different AHR inhibitors were used in Stage III. The experimental results are shown in Figure 6. Different AHR inhibitors, specifically SR1, GNF351, and CH223191, can promote platelet proliferation and differentiation, and the effects of different AHR inhibitors are similar.

[0186] Example 4 Using different differentiation medium IV to culture cells

[0187] Different differentiation medium IV was used in Stage IV. The experimental results are shown in Figure 7. Not adding Y-39983 will affect the cell survival rate. Although it will not affect the subsequent differentiation process, the small number of surviving cells will eventually affect the production capacity.

[0188] Different differentiation medium IV was used in Stage IV. The experimental results are shown in Figure 8. Failure to add KP457 will affect platelet differentiation, while the addition of KP457 can effectively maintain the expression of CD42b, making it easier to obtain mature platelets.

Claims

1. A method for inducing the proliferation and / or differentiation of induced pluripotent cells (iPSCs) into platelets, comprising culturing the cells in a culture medium containing TPO (thrombopoietin), SCF (stem cell factor), Flt3 (Fms-related tyrosine kinase 3 ligand), IL-3 (interleukin-3), IL-6 (interleukin-6), an AHR antagonist, and PVA (polyvinyl alcohol).

2. A method for inducing embryonic stem cells (ESCs) to proliferate and / or differentiate into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist, and PVA.

3. A method for inducing hematopoietic stem / progenitor cells (HSCs) to proliferate and / or differentiate into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist, and PVA.

4. A method for inducing megakaryocyte (MKP) proliferation and / or differentiation into platelets, comprising culturing the cells in a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

5. The method according to any one of claims 1 to 4, wherein the AHR antagonist is selected from one or more antagonists in the group consisting of SR1, GNF351, CH223191, IK-175, AHR antagonist 2, Bay-218, PDM2, PD98059 and BAY-2416964.

6. The method of any one of claims 1-5, wherein the AHR antagonist is SR1.

7. The method of any one of claims 1-5, wherein the AHR antagonist is GNF351.

8. The method according to any one of claims 1-5, wherein the AHR antagonist is CH223191.

9. The method according to any one of claims 1 to 8, wherein the culture medium may further comprise one or more additives selected from the group consisting of BMP4 (bone morphogenetic protein), VEGF (vascular endothelial growth factor), Activin A (activin A), bFGF (basic fibroblast growth factor), PS (penicillin and streptomycin), GSK inhibitors, KMAD17 inhibitors, and ROCK inhibitors.

10. The method of claim 9, wherein the GSK inhibitor is CHIR-99021. The method according to claim 9 , wherein the KMAD17 inhibitor is KP457.

12. The method of claim 9, wherein the ROCK inhibitor is Y-39983.

13. The method according to any one of claims 1 to 12, wherein the method comprises culturing cells using a culture medium containing TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA and PS.

14. The method of any one of claims 1 to 13, wherein the method comprises culturing cells using a medium containing TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS, and PFHM-II.

15. The method according to any one of claims 1 to 14, wherein the method further comprises culturing the cells using a culture medium containing TPO, SCF, KP457, Y-39983, and PS.

16. The method according to any one of claims 1 to 15, wherein the method further comprises culturing the cells using a culture medium containing TPO, SCF, KP457, Y-39983, PS, and PFHM-II.

17. The method according to any one of claims 1 to 16, wherein the method further comprises culturing the cells using a culture medium containing BMP4, VEGF, Activin A, CHIR-99021 and PS.

18. The method according to any one of claims 1 to 17, wherein the method further comprises culturing the cells using a culture medium containing BMP4, VEGF, bFGF and PS.

19. The method according to any one of claims 1 to 18, wherein the culture medium comprises a basal culture medium, and the basal culture medium can be selected from one or more culture media in the following group: STEMdiff TM APEL TM 2Medium, StemSpan TM -ACF Erythroid Expansion Medium, IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEM I, DMEM, RPMI 1640 and PFHM-II.

20. The method according to any one of claims 1 to 19, wherein the method comprises using a STEMdiff TM APEL TM The cells were cultured in medium containing 2Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS, and PFHM-II.

21. The method according to any one of claims 1 to 20, wherein the method further comprises using a STEMdiff TM APEL TM Cells were cultured in 2Medium, TPO, SCF, KP457, Y-39983, PS, and PFHM-II media.

22. The method according to any one of claims 1 to 21, wherein the method further comprises using a STEMdiff TM APEL TM The cells were cultured in a medium containing 2Medium, BMP4, VEGF, Activin A, CHIR-99021 and PS.

23. The method of any one of claims 1-22, wherein the method further comprises using a TM -Cells were cultured in ACF Erythroid Expansion Medium, BMP4, VEGF, bFGF, and PS.

24. The method according to any one of claims 1 to 23, wherein the method comprises the following steps: (1) using a TM APEL TM 2Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II culture medium; and (2) using a STEMdiff TM APEL TM Cells were cultured in 2Medium, TPO, SCF, KP457, Y-39983, PS, and PFHM-II media.

25. The method according to any one of claims 1 to 24, wherein the method comprises the following steps: (1) using a TM APEL TM 2) Culture cells in a medium containing BMP4, VEGF, Activin A, CHIR-99021, and PS; (2) Culture cells in a medium containing StemSpan TM -ACF Erythroid Expansion Medium, BMP4, VEGF, bFGF and PS culture medium; (3) using STEMdiff TM APEL TM 2Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II culture medium; and (4) using a STEMdiff TM APEL TM Cells were cultured in 2Medium, TPO, SCF, KP457, Y-39983, PS, and PFHM-II media.

26. The method of any one of claims 1-25, wherein the method comprises culturing cells at about 35-39°C.

27. The method of any one of claims 1-26, wherein the method comprises culturing cells under conditions of about 3-7% CO2.

28. The method of any one of claims 1-27, wherein the method comprises culturing cells under serum-free culture conditions.

29. The method of any one of claims 1-28, wherein the method comprises culturing cells under culture conditions that are free of feeder cells.

30. The method according to any one of claims 1 to 29, wherein the concentration of BMP4 is 5-100 ng / mL; the concentration of VEGF is 5-100 ng / mL; the concentration of Activin The concentration of A is 5-100ng / mL; the concentration of CHIR99021 is 0.5-10uM, the concentration of bFGF is 5-100ng / mL, the concentration of IL-3 is 5-100ng / mL; the concentration of IL-6 is 5-100ng / mL; the concentration of SCF is 5-100ng / mL; the concentration of TPO is 5-100ng / mL; the concentration of Flt3 is 5-100ng / mL; the concentration of PFHM-II is 1%-10%; the concentration of SR1 is 0.5-5uM; the concentration of PVA is 50-200ug / ml, the concentration of Y-39983 is 1-10mM; and the concentration of KP457 is 5-30mM.

31. A culture medium comprising TPO, SCF, Flt3, IL-3, IL-6, an AHR antagonist and PVA.

32. The culture medium of claim 31, wherein the AHR antagonist is one or more antagonists selected from the group consisting of SR1, GNF351, CH223191, IK-175, AHR antagonist 2, Bay-218, PDM2, PD98059, and BAY-2416964.

33. The culture medium of any one of claims 31-32, wherein the AHR antagonist is SR1.

34. The culture medium of any one of claims 31-32, wherein the AHR antagonist is GNF351.

35. The culture medium of any one of claims 31-32, wherein the AHR antagonist is CH223191.

36. The culture medium according to any one of claims 31 to 35, wherein the culture medium comprises STEMdiff TM APEL TM 2Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II.

37. A culture medium comprising TPO, SCF, KP457 and Y-39983.

38. The culture medium of claim 37, wherein the culture medium comprises STEMdiff TM APEL TM 2Medium, TPO, SCF, KP457, Y-39983, PS and PFHM-II.

39. A combination culture medium comprising differentiation medium I and differentiation medium II, wherein the differentiation medium I comprises TPO, SCF, Flt3, IL-3, IL-6, SR1 and PVA; and the differentiation medium II comprises TPO, SCF, KP457 and Y-3998.

40. The combination culture medium according to claim 39, wherein the differentiation medium I comprises STEMdiff TM APEL TM 2Medium, TPO, SCF, Flt3, IL-3, IL-6, SR1, PVA, PS and PFHM-II.

41. The combination culture medium according to any one of claims 39-40, wherein the differentiation medium II comprises STEMdiff TM APEL TM 2Medium, TPO, SCF, KP457, Y-39983, PS and PFHM-II.

42. The combination culture medium according to any one of claims 39 to 41, further comprising differentiation culture medium III, wherein the differentiation culture medium III comprises BMP4, VEGF, Activin A and CHIR-99021.

43. The combination culture medium according to claim 42, wherein the differentiation medium III comprises STEMdiff TM APEL TM 2Medium, BMP4, VEGF, Activin A, CHIR-99021 and PS.

44. The combination culture medium according to any one of claims 39-43, further comprising differentiation culture medium IV, wherein the differentiation culture medium IV comprises BMP4, VEGF and bFGF.

45. The combination culture medium according to claim 44, wherein the differentiation medium IV comprises StemSpan TM -ACF Erythroid Expansion Medium, BMP4, VEGF, bFGF and PS.

46. ​​A composition comprising induced pluripotent stem cells and the culture medium according to any one of claims 31 to 45.

47. A composition comprising embryonic stem cells and the culture medium of any one of claims 31-45.

48. A composition comprising hematopoietic stem / progenitor cells and the culture medium of any one of claims 31-45.

49. A composition comprising megakaryocytes and the culture medium of any one of claims 31-45.

50. A culture platform for obtaining platelets, comprising the method according to any one of claims 1 to 30 and / or the culture medium according to any one of claims 31 to 45.

51. A method for preventing and / or treating a disease, comprising administering the method according to any one of claims 1 to 30, the culture medium according to any one of claims 31 to 45, the composition according to any one of claims 46 to 49, and / or the culture platform according to claim 50.

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