MKP proliferation and differentiation method and use thereof

Abstract

The present application relates to a method for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKPs), comprising adding a human platelet lysate (hPL) and an HDAC inhibitor to an MKP differentiation medium. The present application also provides a culture medium used in the method, and a composition comprising the culture medium.

Description

MKP proliferation and differentiation methods and their applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP). Background Technology

[0002] Megakaryotic progenitor cells (MKPs) differentiate from hematopoietic stem / progenitor cells (HSCs) in the bone marrow and can further differentiate into megakaryocytes, which in turn generate platelets (PLTs). Platelets are crucial for restoring and maintaining normal hemostasis and coagulation functions in the human body. Currently, the main source of platelets in China is voluntary donation, but the amount donated is far less than the clinical demand. Platelets are terminal cells and cannot be expanded through in vitro culture. Therefore, in vitro differentiation and expansion of MKPs to obtain platelets is an effective method to solve the platelet shortage.

[0003] Although MKPs can differentiate from hematopoietic stem cells, they are few in number among hematopoietic cells in the bone marrow, accounting for only 0.05% of the total number of nucleated cells in the bone marrow. Therefore, there is an urgent need for an effective method to induce pluripotent cells to proliferate / differentiate into MKPs, improve the purity of differentiation and expansion, and obtain a large number of MKPs. Summary of the Invention

[0004] This application provides a method for inducing the proliferation and / or differentiation of pluripotent cells into megakaryocyte progenitor (MKP) cells, comprising adding human platelet lysate (hPL) and an HDAC inhibitor to the MKP differentiation culture medium. The method provided in this application can effectively increase the expression of the MKP cell marker CD41 by culturing pluripotent cells using hPL and the HDAC inhibitor, thereby promoting the generation of more MKP cells from pluripotent cells.

[0005] On the one hand, this application provides a method for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP), which includes adding human platelet lysate (hPL) and HDAC inhibitor to the MKP differentiation medium.

[0006] In some embodiments, the pluripotent cells in the method include induced pluripotent stem cells (iPSCs). In some embodiments, the pluripotent cells in the method include hematopoietic stem / progenitor cells (HSCs). In some embodiments, the hematopoietic stem / progenitor cells in the method are CD34+ hematopoietic stem / progenitor cells. In some embodiments, the pluripotent cells in the method include human embryonic stem cells (ESCs).

[0007] In some embodiments, the concentration of hPL in the method can be 0.5-10%. In some embodiments, the concentration of hPL in the method can be 2-8%. In some embodiments, the concentration of hPL in the method can be 5-8%.

[0008] In some embodiments, the HDAC inhibitor in the method may be selected from one or more of hydroxylamine HDAC inhibitors, short-chain fatty acid HDAC inhibitors, benzamide HDAC inhibitors, pyrrolidone HDAC inhibitors, and cyclic peptide HDAC inhibitors. In some embodiments, the HDAC inhibitor in the method may be a non-selective inhibitor or a selective inhibitor. In some embodiments, the HDAC inhibitor in the method may be selected from one or more inhibitors from the group consisting of: butyrate, phenyl butyrate, valproic acid, SA (Trichostatin A), FK-228 (Romidepsin), Chidamide, Tucidinostat, Belinostat, Apidin, Valproic acid (VPA), Trichostatin A (TSA), JNJ-26481585 (Quisinostat), MGCD0103 (Mocetinostat), SAHA (Vorinostat), 4SC-202 (Domatinostat), MS-275 (Entinostat), CAY10603, and Panobinostat (LBH589). In some embodiments, the concentration of the HDAC inhibitor in the method may be 10-1000 nM.

[0009] In some embodiments, the MKP differentiation medium in the method further comprises a basal medium. In some embodiments, the basal medium in the method may be selected from one or more of the following media: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan. TM SFEMII, DMEM, RPMI1640 X-VIVO 10, HPGM, and SCGM. In some embodiments, the basal medium in the method may be a serum-free medium. In some embodiments, the basal medium in the method is SCGM.

[0010] In some embodiments, the MKP differentiation medium in the method further comprises nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

[0011] In some embodiments, the MKP differentiation medium in the method further comprises one or more of the following: M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), Glutamax (glutamine), TGF (transforming growth factor), EGF (epidermal growth factor), TNF (tumor necrosis factor), Ascorbic acid, Transferrin, bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), TPO (thrombopoietin), IGF-I (insulin-like growth factor-I), SCF (stem cell factor), IL-3, IL-6, IL-7, IL-11, ROCK inhibitor, BMP4 (recombinant human bone morphogenetic protein 4), FLt3L (Fms-associated tyrosine kinase 3 ligand), Low-density lipoprotein, 2-mercaptoethanol, and NEAA (non-essential amino acids).

[0012] In some embodiments, the MKP differentiation medium in the method may further contain IL-3, IL-6, TPO, SCF, and Flt3L.

[0013] In some embodiments, the MKP differentiation medium in the method may further contain IL-3, IL-6, TPO, SCF, Flt3L, Glutamax, and NEAA.

[0014] In some embodiments, the concentration of IL-3 in the method is approximately 5-50 ng / mL. In some embodiments, the concentration of IL-6 in the method is approximately 5-50 ng / mL. In some embodiments, the concentration of TPO in the method is approximately 5-50 ng / mL. In some embodiments, the concentration of SCF in the method is approximately 5-50 ng / mL. In some embodiments, the concentration of Flt3L in the method is approximately 5-50 ng / mL.

[0015] In some embodiments, the method involves culturing cells at 35-39°C. In some embodiments, the method involves culturing cells in a CO2 atmosphere of 3-7%. In some embodiments, the method involves culturing cells in a serum-free environment. In some embodiments, the culture time is approximately 12 days.

[0016] In some embodiments, the MKP culture medium in the method comprises SCGM, hPL, HDAC inhibitor, IL-3, IL-6, TPO, SCF, and Flt3L.

[0017] On the other hand, this application provides a method for inducing pluripotent stem cells to differentiate into platelets, which includes obtaining MKPs using the method provided in this application, culturing MKPs, and proliferating and / or differentiating them into platelets.

[0018] In some embodiments, the process of obtaining MKP in the method is achieved by culturing pluripotent cells in a culture medium containing human platelet lysate (hPL) and an HDAC inhibitor.

[0019] On the other hand, this application provides a culture medium containing human platelet lysate (hPL) and an HDAC inhibitor for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP), which can improve proliferation and / or differentiation efficiency.

[0020] In some embodiments, the concentration of hPL in the culture medium can be 0.5-10%. In some embodiments, the concentration of hPL in the culture medium can be 2-8%. In some embodiments, the concentration of hPL in the culture medium can be 5-8%.

[0021] In some embodiments, the HDAC inhibitor in the culture medium may be selected from one or more of hydroxylamine HDAC inhibitors, short-chain fatty acid HDAC inhibitors, benzamide HDAC inhibitors, pyrrolidone HDAC inhibitors, and cyclic peptide HDAC inhibitors. In some embodiments, the HDAC inhibitor in the culture medium may be a non-selective inhibitor or a selective inhibitor. In some embodiments, the HDAC inhibitor in the culture medium may be selected from one or more inhibitors from the group consisting of: butyrate, phenyl butyrate, valproic acid, SA (Trichostatin A), FK-228 (Romidepsin), Chidamide, Tucidinostat, Belinostat, Apidin, Valproic acid (VPA), Trichostatin A (TSA), JNJ-26481585 (Quisinostat), MGCD0103 (Mocetinostat), SAHA (Vorinostat), 4SC-202 (Domatinostat), MS-275 (Entinostat), CAY10603, and Panobinostat (LBH589). In some embodiments, the concentration of the HDAC inhibitor in the culture medium may be 10-1000 nM.

[0022] In some embodiments, the MKP differentiation medium in the culture medium further comprises a basal medium. In some embodiments, the basal medium in the culture medium may be optionally selected from one or more of the following media: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan. TM SFEMII, DMEM, RPMI1640 X-VIVO 10, HPGM, and SCGM. In some embodiments, the basal medium in the culture medium may be a serum-free medium. In some embodiments, the basal medium in the method is SCGM.

[0023] In some embodiments, the culture medium further comprises nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

[0024] In some embodiments, the culture medium further comprises one or more of the following: M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), Glutamax (glutamine), TGF (transforming growth factor), EGF (epidermal growth factor), TNF (tumor necrosis factor), Ascorbic acid, Transferrin, bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), TPO (thrombopoietin), IGF-I (insulin-like growth factor-I), SCF (stem cell factor), IL-3, IL-6, IL-7, IL-11, ROCK inhibitor, BMP4 (recombinant human bone morphogenetic protein 4), FLt3L (Fms-associated tyrosine kinase 3 ligand), Low-density lipoprotein, 2-mercaptoethanol, and NEAA (non-essential amino acids).

[0025] In some embodiments, the culture medium may also contain IL-3, IL-6, TPO, SCF, and Flt3L.

[0026] In some embodiments, the culture medium may also contain IL-3, IL-6, TPO, SCF, Flt3L, Glutamax, and NEAA.

[0027] In some embodiments, the concentration of IL-3 in the culture medium is approximately 5-50 ng / mL. In some embodiments, the concentration of IL-6 in the culture medium is approximately 5-50 ng / mL. In some embodiments, the concentration of TPO in the culture medium is approximately 5-50 ng / mL. In some embodiments, the concentration of SCF in the culture medium is approximately 5-50 ng / mL. In some embodiments, the concentration of Flt3L in the culture medium is approximately 5-50 ng / mL.

[0028] In some embodiments, the culture medium contains SCGM, hPL, HDAC inhibitor, IL-3, IL-6, TPO, SCF, and Flt3L.

[0029] On the other hand, this application also provides a composition comprising pluripotent cells and the culture medium described in this application.

[0030] In some embodiments, the pluripotent cells in the composition comprise induced pluripotent stem cells (iPSCs). In some embodiments, the pluripotent cells in the composition comprise hematopoietic stem / progenitor cells (HSCs). In some embodiments, the hematopoietic stem / progenitor cells in the composition are CD34+ hematopoietic stem / progenitor cells. In some embodiments, the pluripotent cells in the composition comprise human embryonic stem cells (ESCs). In some embodiments, the pluripotent cells in the composition comprise modified pluripotent cells.

[0031] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0032] The features and advantages of the invention related to this application can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0033] Figure 1 shows the effects of different concentrations of HPL on the proliferation and / or differentiation of MKP.

[0034] Figure 2 shows the results of flow cytometry detection of the expression of MKP cell markers CD34 and CD41.

[0035] Figure 3 shows the effects of different cytokines on the proliferation and / or differentiation of MKP.

[0036] Figure 4 shows the effects of different concentrations of SM05 on the proliferation and / or differentiation of MKP.

[0037] Figure 5 shows the effects of different types of HDAC inhibitors on the proliferation and / or differentiation of MKP when hPL is added.

[0038] Figure 6 shows the effects of different types of HDAC inhibitors on the proliferation and / or differentiation of MKP when hPL is added. Detailed Implementation

[0039] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0040] Terminology Definition

[0041] In this application, the term "pluripotent cell" generally refers to a cell with the potential for proliferation and differentiation. A pluripotent cell can be functionally defined as a cell that: (1) has the ability to differentiate into different cell types, and in some cases, generates only one specialized cell type; and (2) is capable of long-term self-renewal, producing one or more cells that are the same as 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 may include induced pluripotent stem cells, hematopoietic stem / progenitor cells, CD34+ hematopoietic stem / progenitor cells, embryonic stem cells, etc.

[0042] In this application, the term "megakaryocyte progenitor cell," also known as "megakaryocyte," generally refers to a cell capable of producing platelets. Megakaryocyte progenitor cells are characterized by polyploid nuclei, large cell volume, and abundant cytoplasm, enabling each cell to produce thousands of platelets. The source and preparation method of megakaryocyte progenitor cells are not limited; for example, the megakaryocyte progenitor cells can be differentiated from pluripotent cells or isolated from vivo.

[0043] In this application, the term "induced pluripotent stem cell" is generally abbreviated as iPS cells or iPSCs, and typically refers to a type of pluripotent stem cell prepared artificially from non-pluripotent cells. For example, the artificial method may be the introduction of reprogramming factors. For example, the non-pluripotent cells may be adult somatic cells or terminally differentiated cells, such as fibroblasts, hematopoietic cells, myocytes, neurons, epidermal cells, etc.

[0044] In this application, the term "hematopoietic stem / progenitor cell" is generally abbreviated as HSC and typically refers to cells with long-term self-renewal capacity and the potential to differentiate into various types of mature blood cells. The source and preparation method of hematopoietic stem / progenitor cells are not limited; for example, the hematopoietic stem / progenitor cells can be differentiated from pluripotent cells and can be isolated from bone marrow or blood. Hematopoietic stem / progenitor cells can differentiate into various cell types, such as bone marrow lineage cells (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage cells (e.g., T cells, B cells, NK cells). For example, hematopoietic stem / progenitor cells can differentiate into megakaryocyte progenitor cells.

[0045] In this application, the term "embryonic stem cell," also known as "embryonic stem cell" or abbreviated as "ES," generally refers to cells with the characteristics of unlimited proliferation, self-renewal, and multi-lineage differentiation. Embryonic stem cells are stem cells obtained from the undifferentiated inner cell mass of the blastocyst (early embryonic stage). Their source and preparation methods are unrestricted. Whether in vitro or in vivo, embryonic stem cells can be induced to differentiate into almost all cell types in the body, such as hematopoietic stem cells, nerve cells, cardiomyocytes, etc.

[0046] In this application, the term "platelet" generally refers to anucleate cytoplasm. Platelets can be formed from small pieces of cytoplasm detached from the cytoplasm of megakaryocyte progenitor cells and play an important role in physiological hemostasis. They can be activated by thrombin, rapidly adhere to the wound site, and aggregate into clusters to form relatively soft hemostatic plugs, which then promote blood clotting and form firm hemostatic plugs.

[0047] In this application, the term "human platelet lysate" can be abbreviated as "hPL," which is generally derived from human platelets and contains various cell growth factors. In this application, the human platelet lysate can contain platelets from various sources; for example, the human platelet lysate can be derived from human platelets collected from blood donations. For example, the human platelet lysate can be derived from platelets isolated and purified from blood samples. For example, the human platelet lysate can be derived from platelets obtained through various cell differentiation processes, such as platelets differentiated from hematopoietic stem cells, platelets differentiated from induced pluripotent stem cells (iPSCs), or platelets differentiated from megakaryocyte progenitor cells (MKPs). For example, commercially available hPL products can be purchased directly.

[0048] In this application, the term "modified" generally refers to alterations or modifications made to cells. For example, the modification can be a genetic operation to change the cell's genome; this change can be the insertion, deletion, substitution, or modification of genes. For example, the modification can include enabling the cell to express a specific protein or fragment thereof. For example, the modification can include enabling the cell to contain a vector capable of expressing the specific protein or fragment thereof.

[0049] In this application, the term "proliferation" generally refers to the generation of multiple cell individuals by a starting cell through division. These multiple cell individuals can be cells of the same type or different types. The starting cell used for proliferation does not need to be the same as the cells generated by proliferation. For example, proliferating cells can arise from the growth and differentiation of a population of starting cells.

[0050] In this application, the term "differentiation" generally refers to the process by which non-specific or less specific cells acquire specific cellular characteristics. Differentiated or differentiation-induced cells are cells that occupy more specific positions in a cell lineage.

[0051] In this application, "composition" generally refers to a product comprising a specified amount of a specified ingredient, and any product produced directly or indirectly from a combination of the specified amounts of the specified ingredients. In this application, the composition may also contain other inactive ingredients, such as carriers, excipients, adjuvants, stabilizers, etc.

[0052] In this application, the term "ex vivo" generally refers to manipulation of cells, tissues, and / or organs that have been removed from a living organism. In some embodiments, the cells, tissues, and / or organs may be returned to the living organism or introduced into another organism by certain methods.

[0053] In this application, the term "in vitro" generally refers to the removal or release of a part of an organism from the organism.

[0054] In this application, the term "and / or" should be understood to mean any one, two, or more of the alternatives or any combination thereof.

[0055] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0056] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as 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 a specified value.

[0057] Invention Details

[0058] method

[0059] On the one hand, this application provides a method for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP), which includes adding human platelet lysate (hPL) and HDAC inhibitor to the MKP differentiation medium.

[0060] On the other hand, this application provides a method for inducing pluripotent stem cells to differentiate into platelets, which includes obtaining MKPs using the method described in this application, culturing MKPs, and proliferating and / or differentiating them into platelets.

[0061] In this application, the pluripotent cells differentiated into MPK can be any cell with MKP differentiation potential, and can be of natural origin or modified. For example, the pluripotent cells can be artificially modified by physical, chemical and / or biological methods. For example, the expression of certain genes in the pluripotent cells can be adjusted. For example, the pluripotent cells can be selected from one or more of the following: human induced pluripotent stem cells, human embryonic stem cells, hematopoietic stem / progenitor cells, and CD34+ hematopoietic stem / progenitor cells.

[0062] In this application, the sources of the pluripotent cells, human induced pluripotent stem cells, human embryonic stem cells, hematopoietic stem / progenitor cells, and CD34+ hematopoietic stem / progenitor cells are not limited; they can be of mammalian origin or non-mammal origin, and can be differentiated from other cell types. For example, hematopoietic stem / progenitor cells and CD34+ hematopoietic stem / progenitor cells can be differentiated from pluripotent cells, human induced pluripotent stem cells, or human embryonic stem cells. For example, hematopoietic stem / progenitor cells and CD34+ hematopoietic stem / progenitor cells can originate from bone marrow or from blood.

[0063] In this application, the culture can be carried out in conditions of about 35-39°C. For example, about 34.5°C, about 35°C, about 35.5°C, about 36°C, about 36.5°C, about 37°C, about 37.5°C, about 38°C, about 38.5°C, about 39°C, and about 39.5°C.

[0064] In this application, the culture can be carried out 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% CO2, and about 7.5% CO2.

[0065] In this application, the method can be performed under culture conditions with serum present. In this application, the method can also be performed under serum-free culture conditions.

[0066] In this application, the method can be performed under culture conditions without feeder cells. In this application, the method can also be performed under culture conditions with feeder cells.

[0067] In this application, the MKP differentiation medium in the culture method may contain one or more substances, including but not limited to: nutrients / extracts, growth factors, hormones, cytokines and / or culture medium additives.

[0068] In this application, the cell culture time in the method can be adaptively adjusted according to the temperature, cell type, and cell density. For example, the culture time can be approximately 7 days, approximately 7.5 days, approximately 8 days, approximately 8.5 days, approximately 9 days, approximately 9.5 days, approximately 10 days, approximately 10.5 days, approximately 11 days, approximately 11.5 days, approximately 12 days, approximately 12.5 days, approximately 13 days, approximately 13.5 days, approximately 14 days, approximately 14.5 days, approximately 15 days, approximately 15.5 days, approximately 16 days, approximately 16.5 days, approximately 17 days, approximately 17.5 days, approximately 18 days, or approximately 18.5 days.

[0069] In this application, the method can be an in vitro method. In this application, the method can be an ex vivo method.

[0070] In this application, the method may be a method for the purpose of non-disease diagnosis and treatment.

[0071] culture medium

[0072] On the other hand, this application provides a culture medium containing human platelet lysate (hPL) and an HDAC inhibitor for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP), which can improve proliferation and / or differentiation efficiency.

[0073] In this application, the culture medium can be used as the MKP differentiation medium and applied to the method described in this application to induce pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP) to obtain MPK.

[0074] In this application, the culture medium can be used as the MKP differentiation medium and applied to the method described in this application to induce pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP) to obtain MPK cells, and then the obtained MKP cells can be proliferated and / or differentiated into platelets.

[0075] In this application, the source of hPL is not restricted, and an appropriate hPL concentration can be selected according to the actual situation.

[0076] For example, the hPL can be derived from human platelets collected from blood donations. For example, the human platelet lysate can be derived from platelets isolated and purified from blood samples. For example, the human platelet lysate can be derived from platelets obtained through various cell differentiation processes, such as platelets differentiated from hematopoietic stem cells, platelets differentiated from induced pluripotent stem cells (iPSCs), or platelets differentiated from megakaryocyte progenitor cells (MKPs). For example, commercially available hPL products can be purchased directly.

[0077] For example, in the T cell differentiation medium, the concentration of hPL is 0.5-10%. For example, approximately 0.5%, approximately 1%, approximately 1.5%, approximately 2%, approximately 2.5%, approximately 3%, approximately 3.5%, approximately 4%, approximately 4.5%, approximately 5%, approximately 5.5%, approximately 6%, approximately 6.5%, approximately 7%, approximately 7.5%, approximately 8%, approximately 8.5%, approximately 9%, approximately 9.5%, and approximately 10%.

[0078] In this application, the type of HDAC inhibitor is not limited, as long as it can inhibit histone deacetylase activity, it can be used in the culture medium described in this application.

[0079] HDAC inhibitors can be classified according to the class of HDACs they act on. HDACs found in humans are divided into four classes: Class I, including HDAC1, 2, 3, and 8. These are typically located in the cell nucleus and are associated with multiple inhibitory complexes; Class II, further divided into IIa (HDAC4, 5, 7, 9) and IIB (HDAC6 and 10), which move between the nucleus and cytoplasm and exhibit tissue specificity; Class IV, currently containing only one member, HDAC11; and Class III, also known as sirtuins (SIRT1-7), whose catalytic mechanism differs from other HDACs, relying on NAD+ as a cofactor. HDAC inhibitors can inhibit the activity of different HDAC subtypes, including class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8); class II HDACs (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10); class III HDACs (also known as sirtuins, including SIRT1-7); and class IV HDACs (HDAC11).

[0080] HDAC inhibitors can be classified according to their inhibitory selectivity against different HDAC subtypes: non-selective inhibitors, such as SAHA (Vorinostat) and TSA (Trichostatin A), can inhibit multiple HDAC subtypes; selective inhibitors, some of which have a higher affinity for specific HDAC subtypes, such as some inhibitors that may specifically target HDAC6 or HDAC8.

[0081] HDAC inhibitors can be classified according to their chemical structure: Hydroxylamines: such as SAHA and TSA, which inhibit enzyme activity by binding to zinc ions at the active site of HDAC; Short-chain fatty acids: such as butyrate and valproic acid, which exert their effects by binding to zinc ions; Benzamides: such as MS-275 (Entinostat), which interact with the active site of HDAC by mimicking the N-terminal tail of histones; Pyrrolidones: these inhibitors typically contain the chemical structure of pyrrolidones, such as Apidin, a natural pyrrolidone HDAC inhibitor with anti-proliferative and apoptosis-inducing effects; Cyclic peptides: these drugs are usually composed of cyclic peptide compounds, such as Romidepsin, which is the only marketed cyclic peptide HDAC inhibitor with a broad spectrum of inhibition and is used as a single-agent injection for the treatment of T-cell lymphoma.

[0082] In some embodiments, the HDAC inhibitor may be selected from one or more inhibitors from the group consisting of: butyrate, phenyl butyrate, valproic acid, SA (Trichostatin A), FK-228 (Romidepsin), Chidamide, Tucidinostat, Belinostat, Apidin, Valproic acid (VPA), Trichostatin A (TSA), JNJ-26481585 (Quisinostat), MGCD0103 (Mocetinostat), SAHA (Vorinostat), 4SC-202 (Domatinostat), MS-275 (Entinostat), CAY10603, and Panobinostat (LBH589).

[0083] For example, the HDAC inhibitors may be SM05 (Quisinostat), SM133 (Mocetinostat, MGCD0103), SM134 (Vorinostat, SAHA), SM135 (Domatinostat, 4SC-202), SM136 (Entinostat, MS-275), SM137 (CAY10603), and SM138 (Panobinostat, LBH589).

[0084] Since different types of inhibitors have different optimal working concentrations, the concentration of the inhibitor can be adjusted according to the type of inhibitor used, culture conditions, and the state of the cultured cells.

[0085] In this application, the culture medium may include a basal culture for cell culture.

[0086] In some embodiments, the basal culture medium can be a single component or a combination of multiple culture media. In some embodiments, the basal culture medium includes, but is not limited to, IMDM, MEM, Ham's F12, mTeSR1, APEL, and StemSpan. TM SFEM II, DMEM, RPMI 1640 X-VIVO 10, HPGM, and SCGM. For example, the basal medium can be a single-component SCGM, or SCGM can be mixed with other media in a specific ratio.

[0087] In this application, the culture medium may contain one or more of nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

[0088] For example, the culture medium may contain one or more of the following substances, including but not limited to serum substitutes, 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), and vascular endothelial growth factor. The additives include: 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 additive, B27 additive, and Fms-associated tyrosine kinase 3 ligand (FLt3). These additives are not limited to any particular source and may be commercially available, natural, or recombinant.

[0089] For example, the culture medium may contain one or more of the following: M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), Glutamax (glutamine), TGF (transforming growth factor), EGF (epidermal growth factor), TNF (tumor necrosis factor), Ascorbic acid, Transferrin, bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), TPO (thrombopoietin), IGF-I (insulin-like growth factor-I), SCF (stem cell factor), IL-3, IL-6, IL-7, IL-11, ROCK inhibitor, BMP4 (recombinant human bone morphogenetic protein 4), FLt3L (Fms-associated tyrosine kinase 3 ligand), Low-density lipoprotein, 2-mercaptoethanol, and NEAA (non-essential amino acids).

[0090] For example, the culture medium may contain IL-3, IL-6, TPO, SCF, and Flt3L. Alternatively, the culture medium may contain IL-3, IL-6, TPO, SCF, Flt3L, Glutamax, and NEAA.

[0091] For example, the culture medium may contain SCGM, hPL, HDAC inhibitors, IL-3, IL-6, TPO, SCF, and Flt3L.

[0092] For example, in the culture medium, the concentration of IL-3 is approximately 5-50 ng / mL. For example, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL.

[0093] For example, in the culture medium, the concentration of IL-6 is approximately 5-50 ng / mL. For example, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL.

[0094] For example, in the culture medium, the concentration of TPO is approximately 5-50 ng / mL. For example, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL.

[0095] For example, in the culture medium, the concentration of the SCF is approximately 5-50 ng / mL. For example, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL.

[0096] For example, in the culture medium, the concentration of Flt3L is approximately 5-50 ng / mL. For example, approximately 5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL.

[0097] In this application, the culture medium can be used alone or in combination with other culture media. For example, different differentiation media can be used for different differentiation steps.

[0098] cell

[0099] On the other hand, this application also provides an MKP that can be prepared by the method described in this application or cultured using the culture medium described in this application.

[0100] On the other hand, this application also provides a platelet that can be prepared by the method described in this application or cultured using the culture medium described in this application.

[0101] In this application, the cells and / or their state can be determined using cell markers. For example, cell type and / or cell state can be determined using marker phenotypes.

[0102] In some embodiments, the cells described in this application are isolated.

[0103] Composition and Use

[0104] On the other hand, this application also provides a composition comprising pluripotent cells and the culture medium described herein.

[0105] In this application, the pluripotent cells may be one or more of induced pluripotent stem cells, embryonic stem cells, hematopoietic stem / progenitor cells, and CD34+ hematopoietic stem / progenitor cells.

[0106] On the other hand, this application also provides a culture platform for obtaining MKP, which includes the method and the culture medium.

[0107] On the other hand, this application also provides a culture platform for obtaining platelets, which includes the method and the culture medium.

[0108] On the other hand, this application also provides a method for preventing and / or treating a disease, comprising administering the pluripotent cells, MKP, or platelets to a subject in need, the pluripotent cells, MKP, or platelets being obtained by using the method, the culture medium, the composition, and the culture platform.

[0109] For example, the administered pluripotent cells, MKPs, or platelets can be pharmaceutically formulated according to any conventional method. For example, carriers, excipients, or diluents can be used to mix or dilute the active ingredient. Examples of suitable carriers, excipients, or diluents are lactose, dextran, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The formulation may additionally contain, for example, fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc. The compositions of the present invention can be formulated using any method known in the art to provide a rapid, sustained, or delayed release of the active ingredient after administration to a patient.

[0110] The cells in this application can be administered via injection (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous) or other methods such as infusion to ensure they enter the bloodstream in an effective form. The cells can also be administered intratumorally, peritumorally, intralesionally, or peritumorally to exert local and systemic therapeutic effects. For example, they can be administered via local or intravenous injection.

[0111] In this application, the dosage of the cells can be a single dose or multiple doses. For example, the actual dosage of the cells can be determined based on a variety of relevant factors, such as the type of disease; the route of administration; the patient's age, sex, and / or weight; and the severity of the patient's symptoms.

[0112] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0113] Example

[0114] Example 1: Cell Culture

[0115] 1.1. CD34+ hematopoietic stem / progenitor cells were isolated from umbilical cord blood or apheresis blood and cultured in a culture medium (SCGM+HDAC inhibitor+HPL+IL-3+IL-6+TPO+SCF+Flt3L). First, CD34+ hematopoietic stem / progenitor cells were seeded into cell differentiation bags at a cell density of 1-5×10^5 / mL and cultured at 37℃ and 5% CO2 for 8 days. During this period, the cells in the cell differentiation bags were counted every 2 days, and the cell density was maintained at 2-5×10^5 / mL by supplementing the culture medium.

[0116] 1.2. After completing step 1.1, count the cells in the cell differentiation bag and increase the cell density to 3-6×10^5 / mL. Continue culturing at 37℃ and 5% CO2 for 4 days. During this period, count the cells in the cell differentiation bag every 2 days and maintain the cell density at 3-6×10^5 / mL by supplementing the culture medium.

[0117] 1.3. After completing step 1.2, centrifuge at 400g for 5 min to collect cells, and take 1-5×10^5 cells for flow cytometry analysis to detect the expression of MKP cell marker CD41.

[0118] The following exploration and improvement of culture medium components all use the experimental procedures in this example.

[0119] Example 2: Testing the effects of different concentrations of hPL on the proliferation and / or differentiation of MKP.

[0120] Experimental steps

[0121] CD34+ hematopoietic stem / progenitor cells were revived and cultured in basal MKP medium (SCGM + HDAC inhibitor + IL-3 + IL-6 + TPO + SCF + Flt3L with different proportions of human platelet lysis buffer (HPL)) for 14 days. The differentiation medium was replenished every 3-4 days, and cell counting and flow cytometry were performed every 7 days.

[0122] Specific steps:

[0123] CD34+ hematopoietic stem / progenitor cells were resuscitated and seeded into 96-well U-bottom plates using basal MKP medium (SCGM++HDAC inhibitor+IL-3+IL-6+TPO+SCF+Flt3L), with 4 groups × 2 replicates per well. The seeding density was 1.0×10^5 / mL, and the volume was 100 μL / well. One control group received 0% HPL, while the three experimental groups received 2%, 5%, and 8% HPL, respectively. Cells were cultured at 37℃ and 5% CO2 for 7 days. On day 3, 100 μL of medium + HPL was added to each well.

[0124] After completing step 1.1, take one well of cells from each group and put it into a 1.5 mL EP tube. Centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells. Then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0125] The cells from the other well of each group were transferred to a 24-well plate, and 800 μL of medium + HPL was added to each well. The plates were then cultured at 37°C and 5% CO2 for 7 days. On day 4, 500 μL of medium + HPL was added to each well.

[0126] After completing step 1.3, centrifuge at 400g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells; then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0127] The experimental results are shown in the table below:

[0128] Figure 1 shows that different concentrations of hPL can promote the proliferation and / or differentiation of MKP.

[0129] Figure 2 shows the results of flow cytometry analysis of the expression of MKP cell markers CD34 and CD41.

[0130] Example 3: Effects of different cytokines on the differentiation stage of MKP

[0131] Experimental steps

[0132] CD34+ hematopoietic stem / progenitor cells were resuscitated in basal MKP medium (SCGM + HDAC inhibitor + hPL + IL-3 + IL-6 + TPO + SCF + Flt3L), with cytokine concentrations added in different proportions, and cultured for 14 days. The differentiation medium was replenished every 3-4 days, and cell counting and flow cytometry were performed every 7 days.

[0133] Specific steps:

[0134] CD34+ hematopoietic stem / progenitor cells were resuscitated and seeded into 96-well U-bottom plates with basal MKP medium (SCGM + HDAC inhibitor + hPL 5% + IL-3 + IL-6 + TPO + SCF + Flt3L) at a seeding density of 1.0 × 10^5 / mL and a volume of 100 μL / well. The specific concentrations of cytokines added are shown in the table below. Cells were cultured at 37℃ and 5% CO2 for 7 days; during this period, 100 μL of medium was added to each well on day 3.

[0135] (-IL-3 indicates that the culture medium does not contain IL-3; -IL-6 indicates that the culture medium does not contain IL-6; -IL-3-IL-6 indicates that the culture medium does not contain either IL-3 or IL-6; cytokine concentration unit is ng / mL)

[0136] After completing step 1.1, take one well of cells from each group and put it into a 1.5 mL EP tube. Centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells. Then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0137] The cells from another well in each group were transferred to a 24-well plate, and 800 μL of culture medium was added to each well. The plates were then cultured at 37°C and 5% CO2 for 7 days. On day 4, 500 μL of culture medium was added to each well.

[0138] After completing step 1.3, centrifuge at 400g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells; then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0139] The experimental results are shown below:

[0140] Figure 3 shows the test results on day 7. Adding hPL can compensate for the effects of IL-3 and IL-6 deficiency on MKP proliferation and / or differentiation.

[0141] Example 4: HDAC Inhibitor (SM05) Concentration Test

[0142] Experimental steps

[0143] CD34+ hematopoietic stem / progenitor cells were revived and cultured for 14 days in basal MKP medium (SCGM+hPL 5%+IL-3+IL-6+TPO+SCF+Flt3L) with different proportions of HDAC inhibitor (SM05). The cells were replenished with the differentiation medium every 3-4 days, and cell counting and flow cytometry were performed every 7 days.

[0144] Specific steps:

[0145] CD34+ hematopoietic stem / progenitor cells were resuscitated and seeded into 96-well U-bottom plates with basal MKP medium (SCGM + hPL 5% + IL-3 + IL-6 + TPO + SCF + Flt 3L) at a seeding density of 1.0 × 10^5 / mL and a volume of 100 μL / well. A commonly used concentration of 6 nM SM05 was used as a positive control for comparison, while the concentrations for other groups were 1.5 nM, 3 nM, 6 nM, 9 nM, 12 nM, and 18 nM. The cells were cultured at 37℃ and 5% CO2 for 7 days; on day 3, 100 μL of medium + SM05 was added to each well.

[0146] After completing step 1.1, take one well of cells from each group and put it into a 1.5 mL EP tube. Centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells. Then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0147] The cells from another well in each group were transferred to a 24-well plate, and 800 μL of medium + SM05 was added to each well. The plates were then cultured at 37°C and 5% CO2 for 7 days. On day 4, 500 μL of medium + SM05 was added to each well.

[0148] After completing step 1.3, centrifuge at 400g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells; then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0149] Experimental Groups:

[0150] The experimental results are shown in the table below:

[0151] Figure 4 shows the effect of different concentrations of SM05 on the proliferation and / or differentiation of MKP on day 14. The results show that within the appropriate working concentration range, HDAC inhibitors can promote the proliferation and / or differentiation of MKP.

[0152] Example 5: Concentration Test Experiment of Different Types of HDAC Inhibitors (with hPL Added)

[0153] Experimental steps

[0154] CD34+ hematopoietic stem / progenitor cells were revived and cultured in basal MKP medium (SCGM+hPL 5%+IL-3+IL-6+TPO+SCF+Flt3L) with different proportions of HDAC inhibitors (SM133, SM135) for 14 days. The differentiation medium was replenished every 3-4 days, and cell counting and flow cytometry were performed every 7 days.

[0155] Specific steps:

[0156] CD34+ hematopoietic stem / progenitor cells were resuscitated and seeded into 96-well U-bottom plates with basal MKP medium (SCGM + hPL 5% + IL-3 + IL-6 + TPO + SCF + Flt3L) at a seeding density of 1.0 × 10^5 / mL and a volume of 100 μL / well. The concentrations of HDAC inhibitors used in each group are shown in the table below. Cells were cultured at 37℃ and 5% CO2 for 7 days; on day 3, 100 μL of medium plus HDAC inhibitor was added to each well.

[0157] After completing step 1.1, take one well of cells from each group and put it into a 1.5 mL EP tube. Centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells. Then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0158] The cells from the other well of each group were transferred to a 24-well plate, and 800 μL of culture medium plus HDAC inhibitor was added to each well. The cells were then cultured for 7 days at 37°C and 5% CO2. On day 4, 500 μL of culture medium plus HDAC inhibitor was added to each well.

[0159] After completing step 1.3, centrifuge at 400g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells; then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0160] The experimental results are shown in the table below:

[0161] Figure 5 shows that different types of HDAC inhibitors, SM133 and SM135, have optimal concentrations that promote MKP numbers within a suitable working concentration range (the dashed line in the figure represents the number of cells in the Control group).

[0162] Example 6: Concentration Test Experiment of Different Types of HDAC Inhibitors (with hPL Added)

[0163] Experimental steps

[0164] CD34+ hematopoietic stem / progenitor cells were revived and cultured in basal MKP medium (SCGM+hPL 5%+IL-3+IL-6+TPO+SCF+Flt3L) with different proportions of HDAC inhibitors (SM136, SM138) for 14 days. The differentiation medium was replenished every 3-4 days, and cell counting and flow cytometry were performed every 7 days.

[0165] Specific steps:

[0166] CD34+ hematopoietic stem / progenitor cells were resuscitated and seeded into 96-well U-bottom plates with basal MKP medium (SCGM + hPL 5% + IL-3 + IL-6 + TPO + SCF + Flt3L) at a seeding density of 1.0 × 10^5 / mL and a volume of 100 μL / well. The concentrations of HDAC inhibitors used in each group are shown in the table below. Cells were cultured at 37℃ and 5% CO2 for 7 days; on day 3, 100 μL of medium plus HDAC inhibitor was added to each well.

[0167] After completing step 1.1, take one well of cells from each group and put it into a 1.5 mL EP tube. Centrifuge at 400 g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells. Then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0168] The cells from the other well of each group were transferred to a 24-well plate, and 800 μL of culture medium plus HDAC inhibitor was added to each well. The cells were then cultured for 7 days at 37°C and 5% CO2. On day 4, 500 μL of culture medium plus HDAC inhibitor was added to each well.

[0169] After completing step 1.3, centrifuge at 400g for 5 min, discard the supernatant, resuspend in 1 mL and count the cells; then perform flow cytometry analysis to detect the expression of MKP cell markers CD34 and CD41.

[0170] The experimental results are shown in the table below:

[0171] Figure 6 shows that different types of HDAC inhibitors, SM136 and SM138, have the optimal concentrations that promote MKP numbers within a suitable working concentration range (the dashed line in the figure represents the number of cells in the Control group).

Claims

1. A method for inducing the proliferation and / or differentiation of pluripotent cells into megakaryocyte progenitor cells (MKP), comprising adding human platelet lysate (hPL) and an HDAC inhibitor to the MKP differentiation medium.

2. The method according to claim 1, wherein the pluripotent cells include induced pluripotent stem cells (iPSCs).

3. The method according to claim 1, wherein the pluripotent cells comprise hematopoietic stem / progenitor cells (HSCs).

4. The method according to claim 3, wherein the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

5. The method of claim 1, wherein the pluripotent cells comprise human embryonic stem cells (ES).

6. The method according to any one of claims 1-5, wherein the concentration of hPL is 0.5-10%.

7. The method according to any one of claims 1-6, wherein the concentration of hPL is 2-8%.

8. The method according to any one of claims 1-7, wherein the concentration of hPL is 5-8%.

9. The method according to any one of claims 1-8, wherein the HDAC inhibitor is selected from one or more of hydroxylamine HDAC inhibitors, short-chain fatty acid HDAC inhibitors, benzamide HDAC inhibitors, pyrrolidone HDAC inhibitors, and cyclic peptide HDAC inhibitors.

10. The method according to any one of claims 1-9, wherein the HDAC inhibitor is a non-selective inhibitor.

11. The method according to any one of claims 1-9, wherein the HDAC inhibitor is a selective inhibitor.

12. The method according to any one of claims 1-11, wherein the HDAC inhibitor is optionally selected from one or more inhibitors from the group consisting of: butyrate, phenyl butyrate, valproic acid, SA (Trichostatin A), FK-228 (Romidepsin), Chidamide, Tucidinostat, Belinostat, Apidin, Valproic acid (VPA), Trichostatin A (TSA), JNJ-26481585 (Quisinostat), MGCD0103 (Mocetinostat), SAHA (Vorinostat), 4SC-202 (Domatinostat), MS-275 (Entinostat), CAY10603, and Panobinostat (LBH589).

13. The method according to any one of claims 1-12, wherein the concentration of the HDAC inhibitor is 10-1000 nM.

14. The method according to any one of claims 1-13, wherein the MKP differentiation medium further comprises a basal medium.

15. The method according to claim 14, wherein the basal culture medium is selected from one or more of the following culture media: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEMII, DMEM, RPMI1640X-VIVO 10, HPGM and SCGM.

16. The method according to any one of claims 14-15, wherein the basal culture medium is a serum-free culture medium.

17. The method according to any one of claims 14-16, wherein the basal culture medium is SCGM.

18. The method according to any one of claims 1-17, wherein the MKP differentiation medium further comprises nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

19. The method according to any one of claims 1-18, wherein the MKP differentiation medium further comprises one or more of M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), Glutamax (glutamine), TGF (transforming growth factor), EGF (epidermal growth factor), TNF (tumor necrosis factor), Ascorbic acid, Transferrin, bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), TPO (thrombopoietin), IGF-I (insulin-like growth factor-I), SCF (stem cell factor), IL-3, IL-6, IL-7, IL-11, ROCK inhibitor, BMP4 (recombinant human bone morphogenetic protein 4), FLt3L (Fms-associated tyrosine kinase 3 ligand), Low-density lipoprotein, 2-mercaptoethanol, and NEAA (non-essential amino acids).

20. The method according to any one of claims 1-19, wherein the MKP differentiation medium further comprises IL-3, IL-6, TPO, SCF and Flt3L.

21. The method according to any one of claims 19-20, wherein the concentration of said IL-3 is about 5-50 ng / mL.

22. The method according to any one of claims 19-21, wherein the concentration of IL-6 is about 5-50 ng / mL.

23. The method according to any one of claims 19-22, wherein the concentration of TPO is about 5-50 ng / mL.

24. The method according to any one of claims 19-23, wherein the concentration of SCF is about 5-50 ng / mL.

25. The method according to any one of claims 19-24, wherein the concentration of Flt3L is about 5-50 ng / mL.

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

27. The method according to any one of claims 1-26, wherein the method involves culturing cells under conditions containing 3-7% CO2.

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

29. The method according to any one of claims 1-28, wherein the culture time of the method is about 12 days.

30. The method according to any one of claims 1-29, wherein the MKP medium comprises SCGM, hPL, HDAC inhibitor, IL-3, IL-6, TPO, SCF and Flt3L.

31. A method for inducing pluripotent stem cells to differentiate into platelets, comprising obtaining MKPs using the method of any one of claims 1-30, culturing the MKPs, and proliferating and / or differentiating them into platelets.

32. The method of claim 31, wherein the process of obtaining MKP is achieved by culturing pluripotent cells in a culture medium containing human platelet lysate (hPL) and an HDAC inhibitor.

33. A culture medium containing human platelet lysate (hPL) and HDAC inhibitors, used to induce the proliferation and / or differentiation of pluripotent cells into megakaryocyte progenitor cells (MKP), which can improve proliferation and / or differentiation efficiency.

34. The culture medium according to claim 33, wherein the concentration of hPL is 0.5-10%.

35. The culture medium according to any one of claims 33-34, wherein the concentration of hPL is 2-8%.

36. The culture medium according to any one of claims 33-35, wherein the concentration of hPL is 5-8%.

37. The culture medium according to any one of claims 33-36, wherein the HDAC inhibitor is selected from one or more of hydroxylamine HDAC inhibitors, short-chain fatty acid HDAC inhibitors, benzamide HDAC inhibitors, pyrrolidone HDAC inhibitors and cyclic peptide HDAC inhibitors.

38. The culture medium according to any one of claims 33-37, wherein the HDAC inhibitor is a non-selective inhibitor.

39. The culture medium according to any one of claims 33-37, wherein the HDAC inhibitor is a selective inhibitor.

40. The culture medium according to any one of claims 33-39, wherein the HDAC inhibitor is selected from one or more inhibitors from the group consisting of: butyrate, phenyl butyrate, valproic acid, SA (Trichostatin A), FK-228 (Romidepsin), Chidamide, Tucidinostat, Belinostat, Apidin, Valproic acid (VPA), Trichostatin A (TSA), JNJ-26481585 (Quisinostat), MGCD0103 (Mocetinostat), SAHA (Vorinostat), 4SC-202 (Domatinostat), MS-275 (Entinostat), CAY10603, and Panobinostat (LBH589).

41. The culture medium according to any one of claims 33-40, wherein the concentration of the HDAC inhibitor is 10-1000 nM.

42. The culture medium according to any one of claims 33-41, wherein the culture medium further comprises a basal culture medium.

43. The culture medium according to claim 42, wherein the basal culture medium is selected from one or more of the following: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan TM SFEMII, DMEM, RPMI1640X-VIVO 10, HPGM and SCGM.

44. The culture medium according to any one of claims 42-43, wherein the basal culture medium is a serum-free culture medium.

45. The culture medium according to any one of claims 42-44, wherein the basal culture medium is SCGM.

46. ​​The culture medium according to any one of claims 33-45, wherein the culture medium further comprises nutrients, extracts, growth factors, hormones, cytokines, and culture medium additives.

47. The culture medium according to any one of claims 33-46, wherein the culture medium further comprises one or more of M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), Glutamax (glutamine), TGF (transforming growth factor), EGF (epidermal growth factor), TNF (tumor necrosis factor), Ascorbic acid, Transferrin, bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor), TPO (thrombopoietin), IGF-I (insulin-like growth factor-I), SCF (stem cell factor), IL-3, IL-6, IL-7, IL-11, ROCK inhibitor, BMP4 (recombinant human bone morphogenetic protein 4), FLt3L (Fms-associated tyrosine kinase 3 ligand), Low-density lipoprotein, 2-mercaptoethanol, and NEAA (non-essential amino acids).

48. The culture medium according to any one of claims 33-47, wherein the culture medium further comprises IL-3, IL-6, TPO, SCF and Flt3L.

49. The culture medium according to any one of claims 47-48, wherein the concentration of said IL-3 is about 5-50 ng / mL.

50. The culture medium according to any one of claims 47-49, wherein the concentration of IL-6 is about 5-50 ng / mL.

51. The culture medium according to any one of claims 47-50, wherein the concentration of TPO is about 5-50 ng / mL.

52. The culture medium according to any one of claims 47-51, wherein the concentration of SCF is about 5-50 ng / mL.

53. The culture medium according to any one of claims 47-52, wherein the concentration of Flt3L is about 5-50 ng / mL.

54. The culture medium according to any one of claims 33-53, wherein the culture medium comprises SCGM, hPL, HDAC inhibitor, IL-3, IL-6, TPO, SCF and Flt3L.

55. A composition comprising pluripotent cells and the culture medium according to any one of claims 33-54.

56. The composition of claim 55, wherein the pluripotent cells comprise induced pluripotent stem cells (iPSCs).

57. The composition of claim 55, wherein the pluripotent cells comprise hematopoietic stem / progenitor cells (HSCs).

58. The composition according to claim 57, wherein the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

59. The composition of claim 55, wherein the pluripotent cells comprise human embryonic stem cells (ES).

60. The composition according to any one of claims 55-59, wherein the pluripotent cell is a modified pluripotent cell.

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