Mkp proliferation and differentiation method and use thereof

Abstract

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

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202580002470.8 (22) Application Date 2025.10.27 (66) Domestic Priority Data 202411514593.3 2024.10.28 CN (85) PCT International Application Entering National Phase Date 2025.11.18 (86) PCT International Application Application Data PCT / CN2025 / 130033 2025.10.27 (71) Applicant Suzhou Xueji Biotechnology Co., Ltd. Address 215000 No. 21, Dongyanli Road, Suzhou Industrial Park, Suzhou City, Jiangsu Province, Building 5, Block B, Phase V of Biomedical Industrial Park (72) Inventors Zhu Fangfang Wu Chen (74) Patent Agency Shanghai Dianshi Intellectual Property Agency (General Partnership) 31309 Patent Attorneys Zhang Zheng and Jiang Fangwei (51) Int.Cl. C12N 5 / 0789 (2010.01) C12N 5 / 0735 (2010.01) C12N 5 / 074 (2010.01) C12N 5 / 078 (2010.01) (54) Invention Title: MKP Proliferation and Differentiation Method and Its Application (57) Abstract: This application relates to a method for inducing pluripotent cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP), which includes adding human platelet lysate (hPL) and an HDAC inhibitor to an MKP differentiation medium. This application also provides the medium used in this method, and a composition containing the medium. Claims (3 pages), Description (16 pages), Drawings (4 pages), CN 121712885 A, 2026.03.20, CN 1 21 71 28 85 A 1. A method for inducing the proliferation and / or differentiation of pluripotent cells into megakaryocytic progenitor cells (MKP), comprising adding human platelet lysate (hPL) and an HDAC inhibitor to the MKP differentiation culture medium. 2. The method of claim 1, wherein the pluripotent cells comprise induced pluripotent stem cells (iPSCs). 3. The method of claim 1, wherein the pluripotent cells comprise hematopoietic stem / progenitor cells (HSCs). 4. The method of 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 of any one of claims 1-5, wherein the concentration of hPL is 0.5-10%. 7. The method of 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 of claim 14, wherein the basal culture medium is selected from one or more of the following: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan™ SFEMII, DMEM, RPMI1640X-VIVO 10, HPGM, and SCGM. 16. The method of any one of claims 14-15, wherein the basal culture medium is a serum-free culture medium. 17. The method of any one of claims 14-16, wherein the basal culture medium is SCGM. 18. The method of 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 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). Claims 1 / 3 Page 2 CN 121712885 A 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 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 is approximately 12 days. 30. The method according to any one of claims 1-29, wherein the MKP medium comprises SCGM, hPL, an 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 MKPs is achieved by culturing pluripotent cells using a culture medium containing human platelet lysate (hPL) and an HDAC inhibitor. 33. A culture medium containing human platelet lysate (hPL) and an HDAC inhibitor for inducing pluripotent cells to proliferate and / or differentiate into megakaryocytic progenitor cells (MKPs), which can improve proliferation and / or differentiation efficiency. 34. The culture medium of claim 33, wherein the concentration of hPL is 0.5-10%. 35. The culture medium of any one of claims 33-34, wherein the concentration of hPL is 2-8%. 36. The culture medium of 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 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). 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 of claim 42, wherein the basal culture medium is selected from one or more of the following: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan™ SFEMII, DMEM, RPMI1640X-VIVO 10, HPGM, and SCGM. 44. The culture medium of any one of claims 42-43, wherein the basal culture medium is a serum-free culture medium. 45. The culture medium of any one of claims 42-44, wherein the basal culture medium is SCGM. 46. The culture medium of 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 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, an 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 according to claim 55, wherein the pluripotent cells comprise induced pluripotent stem cells (iPSCs). 57. The composition according to 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 according to 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 cells are modified pluripotent cells. Claims 3 / 3 Page 4 CN 121712885 A MKP Proliferation and Differentiation Method and Its Application 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 Art

[0002] Megakaryocyte progenitor cells (MKP) are differentiated from hematopoietic stem / progenitor cells (HSC) in the bone marrow, and can continue to differentiate into megakaryocytes, and then generate platelets (PLT). Platelets are extremely important for restoring and maintaining the normal hemostasis and coagulation function of the human body. At present, 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 by in vitro culture. Therefore, in vitro differentiation and expansion of MKP to obtain platelets is an effective method to solve the problem of insufficient platelet supply.

[0003] Although MKPs can differentiate from hematopoietic stem cells, their number in bone marrow hematopoietic cells is very small, accounting for only 0.05% of the total number of nucleated cells in 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 pluripotent cells to proliferate and / or differentiate into megakaryocytic progenitor cells (MKPs), which includes 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 an HDAC inhibitor, thereby promoting the generation of more MKPs by pluripotent cells.

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

[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 can 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 can 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 (see specification page 1 / 16, CN 121712885 A (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 culture medium in the method may be selected from one or more of the following culture media: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan™ SFEMII, DMEM, RPMI1640X-VIVO 10, HPGM, and SCGM. In some embodiments, the basal culture medium in the method may be a serum-free culture medium.In some embodiments, the basal culture 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 comprise IL-3, IL-6, TPO, SCF, and Flt3L.

[0013] In some embodiments, the MKP differentiation medium in the method may further comprise 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 containing 3-7%. In some embodiments, the method involves culturing cells under serum-free conditions. 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, comprising obtaining MKP using the method provided in this application, culturing MKP, and proliferating and / or differentiating it into platelets.

[0018] In some embodiments, the process of obtaining MKP in the method is achieved by culturing pluripotent cells using 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 can be optionally 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 can 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 selected from one or more of the following media: IMDM, MEM, Ham's F12, mTeSR1, APEL, StemSpan™SFEMII, DMEM, RPMI1640X-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 further comprise IL-3, IL-6, TPO, SCF, and Flt3L.

[0026] In some embodiments, the culture medium may further comprise 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 comprises 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 (ES). In some embodiments, the pluripotent cells in the composition comprise modified pluripotent cells. Specification 3 / 16 pages 7 CN 121712885 A

[0031] Other aspects and advantages of this application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of this application are shown and described in the following detailed description. As those skilled in the art will recognize, 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 involved in this application. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. Brief Description of the Drawings

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

[0033] Figure 1 shows the effect 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 effect of different cytokines on the proliferation and / or differentiation of MKP.

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

[0037] Figure 5 shows the effect 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 Embodiments

[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 Definitions

[0041] In this application, the term "pluripotent cell" generally refers to a cell with proliferative and differentiation potential.Pluripotent cells can be functionally defined as cells that: (1) have the ability to differentiate into different cells, and in some cases, generate only one specialized cell type; (2) can undergo long-term self-renewal to generate 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 can 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" is also called "megakaryocyte," which usually refers to a cell that can produce platelets. Megakaryocyte progenitor cells are characterized by polyploid nuclei, large cell volume, and abundant cytoplasm, which allows 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 the body.

[0043] In this application, the term "induced pluripotent stem cells" can generally be abbreviated as iPS cells or iPSCs, and usually refers to a type of pluripotent stem cells prepared artificially from non-pluripotent cells. For example, the artificial method can be the introduction of reprogramming factors. For example, the non-pluripotent cells can 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 cells" can generally be abbreviated as HSCs, and usually refers to cells with long-term self-renewal capacity and the potential to differentiate into various 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" is also called "embryonic stem cell" and can be abbreviated as "ES". It generally refers to cells with unlimited proliferation, self-renewal, and multi-lineage differentiation characteristics. Embryonic stem cells are stem cells obtained from the undifferentiated internal cell mass of the blastocyst (early embryonic stage). Their source and preparation method are not limited. Whether in vitro or in vivo, embryonic stem cells can be induced to differentiate into almost all cell types in the body. For example, the cell types can be hematopoietic stem cells, nerve cells, cardiomyocytes, etc.

[0046] In this application, the term “platelet” generally refers to anucleate cytoplasm.Platelets are formed from small fragments of cytoplasm detached from the cytoplasm of megakaryotic progenitor cells. They 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 coagulation and form firm hemostatic plugs.

[0047] In this application, the term "human platelet lysate" can be abbreviated as "hPL" and is generally derived from human platelets, which contain a variety of 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 donation. 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 differentiations, such as platelets obtained through differentiation of hematopoietic stem cells, platelets obtained through differentiation of induced pluripotent stem cells (iPSCs), and platelets obtained through differentiation of megakaryotic 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 genome, such alteration being the insertion, deletion, substitution, or modification of genes. For example, the modification can include causing the cells to express a specific protein or fragment thereof. For example, the modification can include causing the cells 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 dividing cell. The multiple cell individuals can be cells of the same type or cells of 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 generate growth and differentiation from a cell starting population.

[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-inducible cells are cells that occupy a more specific position 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 removing or releasing a portion 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 to include, encompass, contain, or contain. In some cases, it also means "to be" or "composed of".

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

[0057] Detailed Description of the Invention

[0058] Method

[0059] In one aspect, this application provides 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 culture medium.

[0060] In another aspect, this application provides a method for inducing the differentiation of pluripotent stem cells into platelets, comprising obtaining MKP using the method described in this application, culturing the MKP, and proliferating and / or differentiating it into platelets.

[0061] In this application, the pluripotent cells differentiated into MPK can be any cells 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 of the pluripotent cells can be adjusted. For example, the pluripotent cells can be selected from one or more of the following groups: human induced pluripotent stem cells, human embryonic stem cells, hematopoietic stem / progenitor cells, CD34+ hematopoietic stem / progenitor cells.

[0062] In this application, the source of the pluripotent cells, human induced pluripotent stem cells, human embryonic stem cells, hematopoietic stem / progenitor cells, and CD34+ hematopoietic stem / progenitor cells is not limited. They can be of mammalian origin or non-mammal origin, and can be differentiated from other cells. 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 be derived from bone marrow or blood.

[0063] In this application, the culture can be carried out at a temperature 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 performed 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, about 7.5% CO2.

[0065] In this application, the method can be performed under culture conditions with serum. In this application, the method can be performed under culture conditions without serum.

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

[0067] In this application, the MKP differentiation medium in the culture method can be supplemented with 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 can be a method for non-disease diagnosis and treatment purposes.

[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 megakaryocytic progenitor cells (MKP), which can improve the proliferation and / or differentiation efficiency.

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

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

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

[0076] For example, the hPL may be derived from human platelets collected from blood donation.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 obtained through hematopoietic stem cell differentiation, platelets obtained through induced pluripotent stem cell (iPSC) differentiation, or platelets obtained through megakaryocyte progenitor cell (MKP) differentiation. For example, commercially available hPL products can be purchased directly.

[0077] For example, in the T cell differentiation culture medium, the concentration of hPL is 0.5-10%. For example, about 0.5%, 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%.

[0078] In this application, the types of HDAC inhibitors are not limited, as long as they can inhibit histone deacetylase activity, they 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 usually located in the cell nucleus and are associated with a variety of inhibitory complexes; Class II, further divided into IIa (HDAC4, 5, 7, 9) and IIB (HDAC6 and 10), which shuttle between the cell nucleus and cytoplasm and exhibit tissue specificity; Class IV, currently with only one member, HDAC11; Class III, also known as sirtuins (SIRT1-7), has a different catalytic mechanism than other HDACs and depends 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), which can inhibit multiple HDAC subtypes; selective inhibitors: some inhibitors have a higher affinity for specific HDAC subtypes, for example, some inhibitors may specifically target HDAC6 or HDAC8.

[0081] HDAC inhibitors can be classified according to their chemical structures: hydroxylamines: such as SAHA and TSA, which inhibit enzyme activity by binding to zinc ions in 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 usually contain the chemical structure of pyrrolidone, 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-lymphocytoma.

[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- (Quisinostat), MGCD0103 (Mocetinostat), SAHA (Vorinostat), 4SC-202 (Domatinostat), MS-275 (Entinostat), CAY10603, and Panobinostat (LBH589).

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

[0084] Since the optimal working concentration of different types of inhibitors is different, the concentration of the inhibitor can be adjusted according to the type of inhibitor used, the 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 may 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, StemSpan™ SFEMII, DMEM, RPMI 1640X-VIVO 10, HPGM, and SCGM. For example, the basal culture medium may be a single component of SCGM, and SCGM may be mixed with other culture 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), 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)), and various interferons (e.g., IFN-γ). The additives include stem cell factor (SCF), thrombopoietin (TPO), erythropoietin (EPO), N2 additive, B27 additive, and Fms-associated tyrosine kinase 3 ligand (FLt3). The additives are not limited to any particular source and can 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. For example, 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 inhibitor, 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, 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 (page 8 / 16 of the specification, CN 121712885 A).

[0094] For example, in the culture medium, the concentration of TPO is about 5-50 ng / mL. For example, 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.

[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 the 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] Cells

[0099] On the other hand, this application also provides an MKP, which 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, which 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 the state of the cells can be determined by cell markers. For example, the cell type and / or the state of the cells can be determined by the marker phenotype.

[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 therein.

[0105] In this application, the pluripotent cells can 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 described therein and the culture medium described therein.

[0107] On the other hand, this application also provides a culture platform for obtaining platelets, comprising 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, wherein the pluripotent cells, MKP, or platelets are obtained by using the method, the culture medium, the composition, and the culture platform.

[0109] For example, the administered pluripotent cells, MKP, or platelets can be pharmaceutically formulated according to any conventional method. For example, a carrier, excipient, or diluent can be used to mix or dilute the active ingredient.Examples of suitable carriers, excipients, or diluents are lactose, dextrose, 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 include, for example, fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc. The compositions of the present invention are formulated using any method known in the art to provide a rapid, sustained, or delayed release of the active ingredient upon administration to a patient.

[0110] The administration of cells in this application may be by injection (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous) or by other methods, such as infusion, to ensure that they enter the bloodstream in an effective form. The cells can also be administered via intratumoral, peritumoral, intralesional, or perilesional routes 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 also be a single dose or multiple doses. For example, the actual dosage of the cells can be determined based on various relevant factors, such as the type of disease; route of administration; patient's age, sex, and / or weight; and severity of patient symptoms.

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

[0113] Examples

[0114] Example 1 Cell Culture

[0115] 1.1. Isolate CD34+ hematopoietic stem / progenitor cells from umbilical cord blood or apheresis blood, and culture the cells using culture medium (SCGM+HDAC inhibitor+HPL+IL-3+IL-6+TPO+SCF+Flt3L). First, the CD34+ hematopoietic stem / progenitor cells are seeded into cell differentiation bags at a 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 are counted every 2 days, and the cell density is maintained at 2-5×10^5 / mL by supplementing the culture medium;

[0116] 1.2. After completing step 1.1, the cells in the cell differentiation bags are counted, and the cell density is increased to 3- The cells were cultured at 6×10^5 / mL at 37℃ and 5% CO2 for 4 days. During this period, the cells in the cell differentiation bag were counted every 2 days, and the cell density was maintained at 3-6×10^5 / mL by supplementing the culture medium.

[0117] 1.3. After completing step 1.2, the cells were collected by centrifugation at 400g for 5min. 1-5×10^5 cells were taken for flow cytometry analysis to detect the expression of the MKP cell marker CD41.

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

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

[0120] Experimental steps

[0121] Resuscitate CD34+ hematopoietic stem / progenitor cells and culture them 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 is used to replenish the medium every 3-4 days, and cell counting and flow cytometry are performed every 7 days.

[0122] Specific steps:

[0123] Resuscitate CD34+ hematopoietic stem / progenitor cells and culture them in basal MKP medium (SCGM + HDAC inhibitor + IL-3 + IL-6 + TPO) +SCF+Flt3L) was seeded into 96-well plates with a U-shaped bottom, with 4 groups × 2 replicates per well, seeding density of 1.0 × 10^5 / mL, and volume of 100 μL / well; among them, 0% HPL was added to one control group, and 2%, 5%, and 8% HPL were added to the three experimental groups, respectively. The cells were cultured at 37℃ and 5% CO2 for 7 days; during this period, 100 μl of culture medium + HPL was added to each well on day 3;

[0124] After completing step 1.1, one well of cells from each group was transferred to a 1.5 mL EP tube, centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting; then flow cytometry analysis was performed to detect the expression of MKP cell markers CD34 and CD41.

[0125] Cells from another well in each group were transferred to a 24-well plate, and 800 μL of culture medium + HPL was added to each well. The cells were cultured at 37°C and 5% CO2 for 7 days. During this period, 500 μL of culture medium + HPL was added to each well on day 4.

[0126] After completing step 1.3, the cells were centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Then, flow cytometry analysis was performed to detect the expression of MKP cell markers CD34 and CD41.

[0127] The experimental results are shown in the table below: Instruction manual 10 / 16 pages 14 CN 121712885 A

[0128]

[0129] Figure 1 shows that different concentrations of hPL can promote the proliferation and / or differentiation of MKP.

[0130] Figure 2 shows the results of flow cytometry detection of the expression of MKP cell markers CD34 and CD41.

[0131] Example 3 Effects of different cytokines on the differentiation stage of MKP

[0132] Experimental steps

[0133] Resuscitate CD34+ hematopoietic stem / progenitor cells, and culture them in basal MKP medium (SCGM + HDAC inhibitor + hPL + IL-3 + IL-6 + TPO + SCF + Flt3L), wherein the cytokine concentrations are added in different proportions, and culture them continuously for 14 days. The differentiation medium is used to replenish the medium every 3-4 days, and cell counting and flow cytometry are performed every 7 days.

[0134] Specific steps:

[0135] Resuscitate CD34+ hematopoietic stem / progenitor cells, and seed them in basal MKP medium (SCGM + HDAC inhibitor + hPL 5% + IL-3 + IL-6 + TPO + SCF + Flt3L) into 96-well U-bottom plates. The seeding density is 1.0 × 10^5 / mL, and the volume is 100 μL / well. The specific concentrations of cytokines added are shown in the table below. The cells were cultured at 37℃ and 5% CO2 for 7 days; during this period, 100 μl of culture medium was added to each well on the 3rd day;

[0136]

[0137] (-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 IL-3 and IL-6; cytokine concentration unit ng / mL)

[0138] After completing step 1.1, one well of cells from each group was taken into a 1.5 mL EP tube, centrifuged at 400g for 5 min and the supernatant was discarded. The cells were resuspended in 1 mL and counted; then flow cytometry analysis was performed to detect the expression of MKP cell markers CD34 and CD41.

[0139] 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 cells were cultured at 37°C and 5% CO2 for 7 days. During this period, 500 μL of culture medium was added to each well on day 4.

[0140] After completing step 1.3, the cells were centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Flow cytometry analysis was then performed to detect the expression of MKP cell markers CD34 and CD41.

[0141] The experimental results are shown below:

[0142]

[0143]

[0144] Figure 3 shows the detection results on day 7. The addition of hPL can compensate for the effect of IL-3 and IL-6 deficiency on MKP proliferation and / or differentiation.

[0145] Example 4 HDAC Inhibitor (SM05) Concentration Test

[0146] Experimental Procedure

[0147] CD34+ hematopoietic stem / progenitor cells were resuscitated and cultured for 14 days with different proportions of HDAC inhibitor (SM05) added to basal MKP medium (SCGM+hPL 5%+IL-3+IL-6+TPO+SCF+Flt3L). The differentiation medium was used to replenish the solution every 3-4 days, and cell counting and flow cytometry were performed every 7 days.

[0148] Specific steps:

[0149] Resuscitate CD34+ hematopoietic stem / progenitor cells and seed them 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 100μL / well; among them, the concentration of 6nM SM05 was used as a positive control for comparison, and the concentrations of other groups were 1.5nM, 3nM, 6nM, 9nM, 12nM and 18nM, respectively. The cells were cultured at 37°C and 5% CO2 for 7 days. During this period, 100 μl of culture medium + SM05 was added to each well on day 3.

[0150] After completing step 1.1, one well of cells from each group was transferred to a 1.5 mL EP tube, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Flow cytometry analysis was then performed to detect the expression of MKP cell markers CD34 and CD41.

[0151] The other well of cells from each group was transferred to a 24-well plate, and 800 μL of culture medium + SM05 was added to each well. The cells were cultured at 37°C and 5% CO2 for 7 days. During this period, 500 μl of culture medium + SM05 was added to each well on day 4.

[0152] After completing step 1.3, the cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Flow cytometry analysis was then performed to detect the expression of MKP cell markers CD34 and CD41.

[0153] Experimental grouping:

[0154]

[0155] The experimental results are shown in the table below:

[0156]

[0157] 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 a suitable working concentration range, HDAC inhibitors can promote the proliferation and / or differentiation of MKP.

[0158] Example 5 HDAC Inhibitor Concentration Test Experiment (with hPL)

[0159] Experimental Steps

[0160] Resuscitate CD34+ hematopoietic stem / progenitor cells and culture them for 14 days with different proportions of HDAC inhibitors (SM133, SM135) added to basal MKP medium (SCGM+hPL 5%+IL-3+IL-6+TPO+SCF+Flt3L). The differentiation medium is used to replenish the solution every 3-4 days, and cell counting and flow cytometry are performed every 7 days.

[0161] Specific steps:

[0162] Resuscitate CD34+ hematopoietic stem / progenitor cells and seed them 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 100μL / well; the concentration of HDAC inhibitor used in each group is shown in the table below, which is on pages 13 / 16 of the instruction manual, CN 121712885 A. The cells were cultured at 37°C and 5% CO2 for 7 days. During this period, on day 3, 100 μl of culture medium + HDAC inhibitor was added to each well.

[0163]

[0164] After completing step 1.1, one well of cells from each group was transferred to a 1.5 mL EP tube, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Flow cytometry analysis was then performed to detect the expression of MKP cell markers CD34 and CD41.

[0165] The other well of cells from each group was transferred to a 24-well plate, and 800 μL of culture medium + HDAC inhibitor was added to each well. The cells were cultured at 37°C and 5% CO2 for 7 days. During this period, on day 4, 500 μl of culture medium + HDAC inhibitor was added to each well.

[0166] After completing step 1.3, the cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Flow cytometry analysis was then performed to detect the expression of MKP cell markers CD34 and CD41.

[0167] The experimental results are shown in the table below:

[0168]

[0169] Figure 5 shows that different types of HDAC inhibitors SM133 and SM135, within a suitable working concentration range, have the optimal concentration to promote the number of MKPs (the dashed line in the figure represents the number of cells in the Control group).

[0170] Example 6: HDAC Inhibitor Concentration Test Experiment (with hPL)

[0171] Experimental Steps

[0172] Resuscitate CD34+ hematopoietic stem / progenitor cells and culture them for 14 days with different proportions of HDAC inhibitors (SM136, SM138) added to basal MKP medium (SCGM+hPL 5%+IL-3+IL-6+TPO+SCF+Flt3L). Replenish the cells with the differentiation medium every 3-4 days and perform cell counting and flow cytometry every 7 days.

[0173] Specific Steps:

[0174] Resuscitate CD34+ hematopoietic stem / progenitor cells and seed them 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 100μL / well; the concentrations of HDAC inhibitors used in each group are shown in the table below. The cells were cultured at 37℃ and 5% CO2 for 7 days. During this period, 100 μl of culture medium + HDAC inhibitor was added to each well on the 3rd day.

[0175]

[0176]

[0177] After completing step 1.1, one well of cells from each group was taken into a 1.5 mL EP tube, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL and counted. Then, flow cytometry analysis was performed to detect the expression of MKP cell markers CD34 and CD41.

[0178] Cells from another well in each group were transferred to a 24-well plate, and 800 μL of culture medium + HDAC inhibitor was added to each well. The plates were then cultured at 37°C and 5% CO2 for 7 days. During this period, 500 μL of culture medium + HDAC inhibitor was added to each well on day 4.

[0179] After completing step 1.3, the cells were centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL for cell counting. Flow cytometry analysis was then performed to detect the expression of MKP cell markers CD34 and CD41.

[0180] The experimental results are shown in the table below:

[0181]

[0182]

[0183] Figure 6 shows that different types of HDAC inhibitors SM136 and SM138 have the optimal concentration to promote MKP cell numbers within a suitable working concentration range (the dashed line in the figure represents the number of cells in the Control group).Instruction manual, page 16 / 16, 20 CN 121712885 A, Figure 1; Instruction manual, Figure 1 / 4, page 21 CN 121712885 A, Figure 2; Instruction manual, Figure 2 / 4, page 22 CN 121712885 A, Figure 3; Figure 4; Instruction manual, Figure 3 / 4, page 23 CN 121712885 A, Figure 5; Figure 6; Instruction manual, Figure 4 / 4, page 24 CN 121712885 A.

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-VIVO10, 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-VIVO10, 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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