Method for preparing megakaryocytes by differentiation without feeder cells and use thereof

Abstract

The present invention relates to a method for preparing megakaryocytes by differentiation without feeder cells and the use thereof. Specifically, the present invention provides a method for differentiating human stem cells into hematopoietic stem cells and further differentiating into an immortalized megakaryocyte cell line in vitro. By means of adjusting the composition of a medium, culture conditions and the expression of related factors, the immortalized megakaryocytes can be effectively obtained without the need of feeder cells. The immortalized megakaryocytes obtained by the method of the present invention have long-term proliferation capability, and can be used for in-vitro production of platelets, meeting the requirements of large-scale and stable in-vitro preparation of platelets.

Description

A method for differentiating megakaryocytes from trophoblast cells and its application Technical Field

[0001] This invention relates to the field of cell culture, and more specifically to a method and application for differentiating megakaryocytes from trophoblast-free cells. Background Technology

[0002] Platelets play a crucial role in hemostasis and coagulation. Patients with thrombocytopenia caused by chemotherapy, radiotherapy, or bone marrow hematopoietic dysfunction, as well as those who need platelet replacement due to significant blood loss during surgery, have a high demand for platelets. In real-world blood transfusion scenarios, each person requires approximately 2 × 10⁶ platelets per transfusion. 11 Each blood cell contains a small number of platelets; however, because platelets have a short lifespan in the body, typically only lasting a few days, usually 3-7 days, repeated blood transfusions have become commonplace. The platelets donated by volunteers are increasingly insufficient to meet the growing demand for platelets in society.

[0003] In vivo, hematopoietic stem cells differentiate into megakaryocytes through multiple stages, and the cytoplasm of megakaryocytes lyses to produce platelets. By differentiating stem cells in vitro to obtain infinitely proliferating megakaryocytes, platelets can be produced in large quantities in the laboratory as needed, without being limited by the number of blood donors or time constraints, effectively solving the problem of platelet supply shortages.

[0004] Currently known processes for the in vitro differentiation of stem cells or hematopoietic stem cells into megakaryocytes largely rely on co-culture of stem cells or hematopoietic stem cells with trophoblast cells (such as mesenchymal stem cells or stromal cells). Trophoblast cells can effectively mimic the in vivo microenvironment, providing robust physical support for the differentiation process of stem cells or hematopoietic stem cells, and also providing specific cytokines and inhibiting apoptosis. However, trophoblast cells are exogenous proliferative cells, and their addition during culture carries numerous risks, including immunogenicity, tumorigenicity, and viral transmission, leading to difficulties in quality control.

[0005] Therefore, there is an urgent need in this field to develop a method for producing platelets by obtaining megakaryocytes through in vitro differentiation without the involvement of trophoblast cells. Summary of the Invention

[0006] The purpose of this invention is to provide a method for culturing immortalized megakaryocytes with platelet production function, which does not require the participation of trophoblast cells.

[0007] In a first aspect of the invention, a method for producing immortalized megakaryocytes is provided, the method comprising the steps of:

[0008] (a) Providing hematopoietic stem cells;

[0009] (b) The expression cassette of an exogenous immortalization factor is transferred into the hematopoietic stem cells to obtain transduced hematopoietic stem cells; and under the condition of no trophoblast cells and the transduced hematopoietic stem cells expressing the exogenous immortalization factor, the transduced hematopoietic stem cells are differentiated and cultured to obtain immortalized megakaryocytes.

[0010] In another preferred embodiment, in step (a), the hematopoietic stem cells are isolated from umbilical cord blood, peripheral blood, or bone marrow.

[0011] In another preferred embodiment, in step (a), the hematopoietic stem cells are differentiated from pluripotent stem cells.

[0012] In another preferred embodiment, the pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs); preferably iPSCs.

[0013] In another preferred embodiment, the iPSC includes: a universal iPSC and iPSCs derived from various blood types.

[0014] In another preferred embodiment, step (a) includes:

[0015] (a1) Provides iPSC cells;

[0016] (a2) The iPSC cells are cultured in a hematopoietic stem cell induction medium or under certain conditions to obtain hematopoietic stem cells.

[0017] In another preferred embodiment, in step (a2), the differentiation culture time T a2 The time is 7 to 13 days, preferably 8 to 12 days, and more preferably 8 to 10 days.

[0018] In another preferred embodiment, the induction medium includes: a basal medium and a key factor for inducing differentiation.

[0019] In another preferred embodiment, the basal culture medium is selected from the group consisting of APEL2, mTeSR plus, RPMI, and StemPro-34.

[0020] In another preferred embodiment, the key factors for inducing differentiation include: vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), activin A, fibroblast growth factor (FGF), stem cell factor (SCF), thrombopoietin (TPO), cytokines, GSK-3 inhibitors, and TGF-β pathway inhibitors.

[0021] In another preferred embodiment, the GSK-3 inhibitor is CHIR99021.

[0022] In another preferred embodiment, the TGF-β pathway inhibitor is SB431542.

[0023] In another preferred embodiment, the key factors may be introduced simultaneously or in stages.

[0024] In another preferred embodiment, BMP4, ACTIVIN A and CHIR99021 factors are introduced in the first stage of induction culture; the first stage lasts for 1-3 days, for example, about 2 days.

[0025] In another preferred embodiment, VEGF, bFGF, SCF and SB431542 factors are introduced in the second stage of induction culture; the second stage lasts for 2-4 days, for example about 3 days.

[0026] In another preferred embodiment, VEGF, bFGF, SCF, IL-6, TPO and IL-3 factors are introduced in the third stage of induction culture; the third stage lasts for 3-5 days, for example about 4 days; the medium is changed every 2 days.

[0027] In another preferred embodiment, ACTIVIN A and CHIR99021 were added on day 0 of the culture, and SB431542 was added on day 2 of the culture.

[0028] In another preferred embodiment, the concentration of ACTIVIN A is 10-100 ng / mL, preferably 20-50 ng / mL, and more preferably 25-35 ng / mL.

[0029] In another preferred embodiment, the concentration of CHIR99021 is 1-10 μM, preferably 2-8 μM, and more preferably 4-6 μM.

[0030] In another preferred embodiment, the concentration of SB431542 is 2-20 μM, preferably 5-15 μM, and more preferably 8-12 μM.

[0031] In another preferred embodiment, step (b) includes:

[0032] (b1) The expression cassette of the exogenous first immortalization factor is transferred into the hematopoietic stem cells to obtain transduced hematopoietic stem cells; and under the condition of no trophoblast cells and the transduced hematopoietic stem cells expressing the exogenous first immortalization factor, the transduced hematopoietic stem cells are differentiated and cultured to obtain megakaryocytes.

[0033] (b2) The expression cassette of the exogenous second immortalization factor is transferred into the megakaryocytes to obtain transduced megakaryocytes; and the transduced megakaryocytes are immortalized under the conditions of no feeder cells and the transduced megakaryocytes expressing the exogenous first and second immortalization factors to obtain immortalized megakaryocytes.

[0034] In another preferred embodiment, step (b) further includes:

[0035] (b3) Select immortalized megakaryocyte cell lines for inducing the production of functional platelets.

[0036] In another preferred embodiment, the expression cassette is located on an expression vector, which includes a viral vector and a non-viral vector.

[0037] In another preferred embodiment, the viral vector includes: lentiviral vector, adenovirus vector, adeno-associated virus vector, etc.

[0038] In another preferred embodiment, the expression vector has an inducible promoter.

[0039] In another preferred embodiment, the transfer is performed via a gene editing system, including transposons, CRISPR / Cas9, ZFNs, and TALENs.

[0040] In another preferred embodiment, the immortalization factor includes: oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes.

[0041] In another preferred embodiment, the oncogene is selected from the group consisting of the MYC family, the Src family, the Ras / Raf family, and the protein kinase family.

[0042] In another preferred embodiment, the aging-inhibiting gene is selected from the group consisting of BMI1, CDK4, hTERT, B-myb, and hnRNPU.

[0043] In another preferred embodiment, the apoptosis-inhibiting gene is selected from the group consisting of the BCL-2 family, the IAP family, Suvivin, and the MCL-1 gene.

[0044] In another preferred embodiment, the immortalization factors are: c-Myc, BMI1, and BCL-XL.

[0045] In another preferred embodiment, the first immortalization factor is c-Myc or BMI1.

[0046] In another preferred embodiment, the second immortalization factor is BCL-XL.

[0047] In another preferred embodiment, in step (b1), the differentiation culture time T b1 The treatment time is 7-15 days, preferably 8-12 days.

[0048] In another preferred embodiment, in step (b2), the immortalization culture time T b2 The time is 10-18 days, preferably 12-15 days, and more preferably 13-15 days.

[0049] In another preferred embodiment, the differentiation culture is carried out in a differentiation medium or under differentiation conditions.

[0050] In another preferred embodiment, the differentiation medium includes: basal medium and growth factors.

[0051] In another preferred embodiment, the basal culture medium is selected from the group consisting of IMDM, Stemspan SFEMⅡ, Stempro-34 or APEL2 medium.

[0052] In another preferred embodiment, the basal culture medium is Stemspan SFEMⅡ medium.

[0053] In another preferred embodiment, the growth factor is selected from the group consisting of: SCF, Flt3-L, TPO, IL-3, IL-6, IL-11, GM-CSF, SR1, CAY10433, KO-SR, sodium heparin, valproic acid (VPA), or combinations thereof.

[0054] In another preferred embodiment, the combination of growth factors is: SCF, TPO, Flt3L, IL-6, SR1 and valproic acid.

[0055] In another preferred embodiment, the differentiation medium further includes other components that are beneficial to cell culture, including: insulin-transferrin-selenium complex (ITS), L-glutamine (e.g., GlutaMAX), 1-thioglycerol, and ascorbic acid.

[0056] In another preferred embodiment, the culture is either static or dynamic.

[0057] In another preferred embodiment, the culture is a suspension culture.

[0058] In another preferred embodiment, the culture temperature is 37℃±5℃; the CO2 content is 5%±1%.

[0059] In another preferred embodiment, in step (b1), the inoculation density is 0.5-3 × 10⁻⁶. 5 / ml, preferably 1-2×10 5 / ml.

[0060] In another preferred embodiment, in step (b2), the cell density is maintained at 1-4 × 10⁻⁴. 5 / ml, preferably 1.5-2.5×10 5 / ml, more preferably 2×10 5 / ml.

[0061] In another preferred embodiment, the immortalization factor combination is expressed under the control of megakaryocyte or platelet-specific promoters, including CD41, CD42a, CD42b, CD42c, CD42d, CD61, PF4, or vWF promoters.

[0062] In another preferred embodiment, step (b) specifically includes:

[0063] (b1) The expression cassette of an exogenous first immortalization factor is transferred into the hematopoietic stem cells to obtain transduced hematopoietic stem cells; and under the conditions of no trophoblast cells and the transduced hematopoietic stem cells expressing the exogenous first immortalization factor, the transduced hematopoietic stem cells are cultured in a first stage to obtain cell population 1, which contains megakaryocytes or is composed primarily of megakaryocytes; and the cell population 1 is cultured in a second stage to obtain cell population 2.

[0064] (b2) The expression cassette of the exogenous second immortalization factor is transferred into the cell population 2 to obtain the transduced cell population 2; and the transduced cell population 2 is cultured in a third stage under the conditions of no feeder cells and the transduced cell population 2 expressing the exogenous first and second immortalization factors to obtain cell population 3, which contains immortalized megakaryocytes or is composed primarily of immortalized megakaryocytes.

[0065] In another preferred embodiment, the number of transduced hematopoietic stem cells before the first stage of culture is N0, and the number of cells in cell population 1 is N1; N1 / N0 ≥ 4, preferably N1 / N0 ≥ 5, and more preferably N1 / N0 ≥ 6.

[0066] In another preferred embodiment, the number of cells in the cell group 2 is N2; N2 / N1≥8, preferably N2 / N1≥10, and more preferably N2 / N1≥12.

[0067] In another preferred embodiment, the number of cells in the cell group 3 is N3; N3 / N2 ≥ 15, preferably N3 / N2 ≥ 20, and more preferably N3 / N2 ≥ 25.

[0068] In another preferred embodiment, N3 / N0 ≥ 500, more preferably N3 / N0 ≥ 800, more preferably N3 / N0 ≥ 1000, and even more preferably N3 / N0 ≥ 1500.

[0069] In another preferred embodiment, the cell viability of cell population 1 and cell population 2 is each independently ≥80%.

[0070] In another preferred embodiment, the cell viability of the cell population 3 is ≥90%, preferably ≥95%.

[0071] In another preferred embodiment, the proportion of CD41+ cells in the cell population 3 is ≥80%, preferably ≥85%, and more preferably ≥90%.

[0072] In another preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the cell population 3 is ≥70%, preferably ≥75%, and more preferably ≥80%.

[0073] In another preferred embodiment, the expression cassette of the exogenous first immortalization factor is a lentiviral vector containing the c-Myc and BMI1 genes.

[0074] In another preferred embodiment, the expression cassette of the exogenous second immortalization factor is a lentiviral vector containing the BCL-XL gene.

[0075] In another preferred embodiment, the immortalization factor combination is expressed under the control of megakaryocyte or platelet-specific promoters, including CD41, CD42a, CD42b, CD42c, CD42d, CD61, PF4, or vWF promoters.

[0076] In another preferred embodiment, the first stage of cultivation lasts for 3-7 days, preferably 4-6 days, and more preferably 5 days.

[0077] In another preferred embodiment, the second stage of cultivation lasts for 7-11 days, preferably 8-10 days, and more preferably 11 days.

[0078] In another preferred embodiment, the third stage of cultivation takes 5-9 days, preferably 6-8 days, and more preferably 7 days.

[0079] In another preferred embodiment, the first, second, and third stages of culture are carried out in the same culture medium or in different culture media; preferably, they are carried out in the same culture medium.

[0080] In another preferred embodiment, the culture medium includes: basal culture medium and growth factors.

[0081] In another preferred embodiment, the basal culture medium is selected from the group consisting of Stemspan SFEMⅡ, IMDM, Stempro-34 or APEL2 medium.

[0082] In another preferred embodiment, the basal culture medium is Stemspan SFEMⅡ medium.

[0083] In another preferred embodiment, the growth factor is selected from the group consisting of: SCF, TPO, Flt3-L, IL-3, IL-6, SR1, heparin or sodium heparin, valproic acid (VPA), UM171, celecoxib, or combinations thereof.

[0084] In another preferred embodiment, the growth factors comprise: SCF, TPO, Flt3-L, IL-6; and the growth factors optionally include SR1, UM171, IL-3, heparin or heparin sodium, valproic acid and / or Celecoxib.

[0085] In another preferred embodiment, the culture medium does not contain IL-3.

[0086] In another preferred embodiment, the concentration of SCF in the culture medium is 20-200 ng / mL; preferably 50-150 ng / mL; preferably 80-120 ng / mL; more preferably 100 ng / mL.

[0087] In another preferred embodiment, the concentration of TPO in the culture medium is 10-100 ng / mL; preferably 20-80 ng / mL; more preferably 40-60 ng / mL; and even more preferably 50 ng / mL.

[0088] In another preferred embodiment, the concentration of Flt3-L in the culture medium is 20-200 ng / mL; preferably 50-150 ng / mL; preferably 80-120 ng / mL; more preferably 100 ng / mL.

[0089] In another preferred embodiment, the concentration of IL-6 in the culture medium is 5-30 ng / mL; preferably 10-25 ng / mL; more preferably 12-20 ng / mL; and even more preferably 15 ng / mL.

[0090] In another preferred embodiment, the concentration of SR1 in the culture medium is 0.5-2 μM; preferably 0.7-1.5 μM; preferably 0.8-1.2 μM; more preferably 1 μM.

[0091] In another preferred embodiment, the concentration of UM171 in the culture medium is 0.5-2 μM; preferably 0.7-1.5 μM; preferably 0.8-1.2 μM; more preferably 1 μM.

[0092] In another preferred embodiment, the concentration of valproic acid in the culture medium is 0.1-0.5 mM; preferably 0.15-0.25 mM; more preferably 0.2 mM.

[0093] In another preferred embodiment, the concentration of Celecoxib in the culture medium is 0.1-1 μM; preferably 0.2-0.8 μM; preferably 0.4-0.6 μM; more preferably 0.5 μM.

[0094] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0095] (i) SCF, TPO, Flt3L, IL-6 and SR1;

[0096] (ii) SCF, TPO, Flt3L, IL-6, SR1 and valproic acid;

[0097] (iii) SCF, TPO, Flt3L, IL-6 and UM171;

[0098] (iv) SCF, TPO, Flt3L, IL-6, SR1, valproic acid and Celecoxib.

[0099] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0100] (i) 20-200ng / mL SCF, 10-100ng / mL TPO, 20-200ng / mL Flt3L, 5-30ng / mL IL-6 and 0.5-2μM SR1;

[0101] (ii) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6, 0.5-2 μM SR1 and 0.1-0.5 mM valproic acid;

[0102] (iii) 20-200ng / mL SCF, 10-100ng / mL TPO, 20-200ng / mL Flt3L, 5-30ng / mL IL-6 and 0.5-2μM UM171;

[0103] (iv) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6, 0.5-2 μM SR1, 0.1-0.5 mM valproic acid and 0.1-1 μM Celecoxib.

[0104] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0105] (i) 80-120ng / mL SCF, 40-60ng / mL TPO, 80-120ng / mL Flt3L, 12-20ng / mL IL-6 and 0.8-1.2μM SR1;

[0106] (ii) 80-120 ng / mL SCF, 40-60 ng / mL TPO, 80-120 ng / mL Flt3L, 12-20 ng / mL IL-6, 0.8-1.2 μM SR1 and 0.15-0.25 mM valproic acid;

[0107] (iii) 80-120ng / mL SCF, 40-60ng / mL TPO, 80-120ng / mL Flt3L, 12-20ng / mL IL-6 and 0.8-1.2μM UM171;

[0108] (iv) 80-120 ng / mL SCF, 40-60 ng / mL TPO, 80-120 ng / mL Flt3L, 12-20 ng / mL IL-6, 0.8-1.2 μM SR1, 0.15-0.25 mM valproic acid and 0.4-0.6 μM Celecoxib.

[0109] In another preferred embodiment, the culture medium further contains doxycycline; preferably, the concentration of doxycycline is 0.5-2 μM; preferably 0.7-1.5 μM; preferably 0.8-1.2 μM; more preferably 1 μM.

[0110] In another preferred embodiment, the culture is either static or dynamic.

[0111] In another preferred embodiment, the culture is a suspension culture.

[0112] In another preferred embodiment, the culture temperature is 37℃±5℃; the CO2 content is 5%±1%.

[0113] In another preferred embodiment, during the first stage of culture, the seeding density of transduced hematopoietic stem cells was 0.5-3 × 10⁻⁶. 5 / ml, preferably 1-2×10 5 / ml.

[0114] In another preferred embodiment, during the second and / or third stage of culture, the cell density is maintained at 1-4 × 10⁶ cells / year. 5 / ml, preferably 1.5-2.5×10 5 / ml, more preferably 2×10 5 / ml.

[0115] In another preferred embodiment, step (b3) includes: performing monoclonal screening on the functional immortalized megakaryocytes to obtain a functional immortalized megakaryocyte line.

[0116] In another preferred embodiment, the monoclonal screening is performed by methods such as flow cytometry.

[0117] In another preferred embodiment, the number of positive, stably proliferating monoclonal cells accounts for ≥6% of the total number of cells, preferably ≥8%, more preferably ≥10%, more preferably ≥12%, and even more preferably ≥14%.

[0118] In another preferred embodiment, the immortalized megakaryocyte line has a CD41+ / CD42b+ double-positive cell ratio of ≥90%, preferably ≥95%, and more preferably ≥99%.

[0119] In another preferred embodiment, the immortalized megakaryocyte line is continuously expanded for more than 120 days.

[0120] In another preferred embodiment, the doubling time of the immortalized megakaryocyte line is ≤2 days.

[0121] In another preferred embodiment, the method further includes:

[0122] (c) Passage and expand the immortalized megakaryocyte line to obtain immortalized megakaryocytes after passage and expansion;

[0123] (d) The immortalized megakaryocytes that have been passaged and expanded are cultured to mature and form platelets.

[0124] In another preferred embodiment, in step (d), the immortalized megakaryocytes are cultured in platelet differentiation medium under the condition that the megakaryocytes do not express or cease to express exogenous immortalizing factors.

[0125] In a second aspect of the invention, an immortalized megakaryocyte is provided, said megakaryocyte being obtained by the method described in the first aspect of the invention.

[0126] In another preferred embodiment, the megakaryocyte contains oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes.

[0127] In another preferred embodiment, the megakaryocytes are characterized by being CD34 negative or CD34 positive, CD41 positive, and CD42b positive.

[0128] In another preferred embodiment, the immortalized megakaryocytes can be stably expanded in vitro for a long period of time.

[0129] In another preferred embodiment, the immortalized megakaryocytes are stably expanded in vitro for 3-12 months, preferably 3-8 months, more preferably 3-6 months, and most preferably 3-4 months.

[0130] In another preferred embodiment, the megakaryocytes have the ability to differentiate into functional platelets.

[0131] In a third aspect of the invention, the use of immortalized megakaryocytes as described in the second aspect of the invention is provided for the production of functionally active platelets.

[0132] In another preferred embodiment, the immortalized megakaryocytes cease the forced expression of some or all oncogenes, proto-oncogenes, and apoptosis-inhibiting genes.

[0133] In another preferred embodiment, the megakaryocytes are differentiated into platelets in a maturation culture medium.

[0134] In another preferred embodiment, the maturation culture medium comprises: a basal culture medium supplemented with serum, L-glutamine, insulin-transferrin-selenium, ascorbic acid, 1-thioglycerol, SR1, TPO, SCF, heparin sodium, KP457, and Y27632.

[0135] In another preferred embodiment, no trophoblast cells are added to the maturation culture medium.

[0136] In another preferred embodiment, the platelets do not contain heterologous cells.

[0137] In a fourth aspect of the invention, a culture medium for inducing hematopoietic stem cells to differentiate into megakaryocytes is provided, the culture medium comprising: basal culture medium and growth factors.

[0138] In another preferred embodiment, the basal culture medium is selected from the group consisting of IMDM, Stemspan SFEM II, Stempro-34, or APEL2.

[0139] In another preferred embodiment, the basal culture medium is Stemspan SFEM II medium.

[0140] In another preferred embodiment, the growth factor is selected from the group consisting of: SCF, Flt3-L, TPO, IL-3, IL-6, IL-11, GM-CSF, SR1, CAY10433, KO-SR, sodium heparin, valproic acid (VPA), or combinations thereof.

[0141] In another preferred embodiment, the combination of growth factors is: SCF, TPO, Flt3L, IL-6, SR1 and valproic acid.

[0142] In another preferred embodiment, the culture medium further includes other components that are beneficial to cell culture, including: insulin-transferrin-selenium complex (ITS), L-glutamine (e.g., GlutaMAX), 1-thioglycerol, and ascorbic acid.

[0143] In another preferred embodiment, the growth factors comprise: SCF, TPO, Flt3-L, IL-6; and the growth factors optionally include SR1, UM171, IL-3, heparin or heparin sodium, and / or valproic acid.

[0144] In another preferred embodiment, the concentration of SCF in the culture medium is 20-200 ng / mL; preferably 50-150 ng / mL; preferably 80-120 ng / mL; more preferably 100 ng / mL.

[0145] In another preferred embodiment, the concentration of TPO in the culture medium is 10-100 ng / mL; preferably 20-80 ng / mL; more preferably 25-75 ng / mL; and more preferably 50 ng / mL.

[0146] In another preferred embodiment, the concentration of Flt3-L in the culture medium is 20-200 ng / mL; preferably 50-150 ng / mL; preferably 80-120 ng / mL; more preferably 100 ng / mL.

[0147] In another preferred embodiment, the concentration of IL-6 in the culture medium is 5-30 ng / mL; preferably 10-20 ng / mL; more preferably 12-18 ng / mL; and even more preferably 15 ng / mL.

[0148] In another preferred embodiment, the concentration of SR1 in the culture medium is 0.1-5 μM; preferably 0.5-2 μM; preferably 0.8-1.2 μM; more preferably 1 μM.

[0149] In another preferred embodiment, the concentration of UM171 in the culture medium is 0.5-2 μM; preferably 0.7-1.5 μM; preferably 0.8-1.2 μM; more preferably 1 μM.

[0150] In another preferred embodiment, the concentration of valproic acid in the culture medium is 0.1-1 mM; preferably 0.1-0.5 mM; preferably 0.15-0.25 mM; more preferably 0.2 mM.

[0151] In another preferred embodiment, the concentration of Celecoxib in the culture medium is 0.1-1 μM; preferably 0.2-0.8 μM; preferably 0.4-0.6 μM; more preferably 0.5 μM.

[0152] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0153] (i) SCF, TPO, Flt3L, IL-6 and SR1;

[0154] (ii) SCF, TPO, Flt3L, IL-6, SR1 and valproic acid;

[0155] (iii) SCF, TPO, Flt3L, IL-6 and UM171;

[0156] (iv) SCF, TPO, Flt3L, IL-6, SR1, valproic acid and Celecoxib.

[0157] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0158] (i) 20-200ng / mL SCF, 10-100ng / mL TPO, 20-200ng / mL Flt3L, 5-30ng / mL IL-6 and 0.1-5μM SR1;

[0159] (ii) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6, 0.1-5 μM SR1 and 0.1-1 mM valproic acid;

[0160] (iii) 20-200ng / mL SCF, 10-100ng / mL TPO, 20-200ng / mL Flt3L, 5-30ng / mL IL-6 and 0.5-2μM UM171;

[0161] (iv) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6, 0.1-5 μM SR1, 0.1-1 mM valproic acid and 0.1-1 μM Celecoxib.

[0162] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0163] (i) 50-150ng / mL SCF, 25-75ng / mL TPO, 50-150ng / mL Flt3L, 10-20ng / mL IL-6 and 0.5-2μM SR1;

[0164] (ii) 50-150 ng / mL SCF, 25-75 ng / mL TPO, 50-150 ng / mL Flt3L, 10-20 ng / mL IL-6, 0.5-2 μM SR1 and 0.1-0.5 mM valproic acid;

[0165] (iii) 50-150ng / mL SCF, 25-75ng / mL TPO, 50-150ng / mL Flt3L, 10-20ng / mL IL-6 and 0.5-2μM UM171;

[0166] (iv) 50-150 ng / mL SCF, 25-75 ng / mL TPO, 50-150 ng / mL Flt3L, 10-20 ng / mL IL-6, 0.5-2 μM SR1, 0.1-0.5 mM valproic acid and 0.1-1 μM Celecoxib.

[0167] In another preferred embodiment, the combination of growth factors is selected from the group consisting of:

[0168] (i) 80-120ng / mL SCF, 40-60ng / mL TPO, 80-120ng / mL Flt3L, 12-18ng / mL IL-6 and 0.8-1.2μM SR1;

[0169] (ii) 80-120 ng / mL SCF, 40-60 ng / mL TPO, 80-120 ng / mL Flt3L, 12-18 ng / mL IL-6, 0.8-1.2 μM SR1 and 0.15-0.25 mM valproic acid;

[0170] (iii) 80-120ng / mL SCF, 40-60ng / mL TPO, 80-120ng / mL Flt3L, 12-18ng / mL IL-6 and 0.8-1.2μM UM171;

[0171] (iv) 80-120 ng / mL SCF, 40-60 ng / mL TPO, 80-120 ng / mL Flt3L, 12-18 ng / mL IL-6, 0.8-1.2 μM SR1, 0.15-0.25 mM valproic acid and 0.4-0.6 μM Celecoxib.

[0172] In another preferred embodiment, the culture medium further contains doxycycline; preferably, the concentration of doxycycline is 0.5-2 μM; preferably 0.7-1.5 μM; preferably 0.8-1.2 μM; more preferably 1 μM.

[0173] In a fifth aspect of the invention, a method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells is provided, the method comprising:

[0174] (a1) Provides iPSC cells;

[0175] (a2) The iPSC cells are cultured in hematopoietic stem cell induction medium to obtain hematopoietic stem cells.

[0176] In another preferred embodiment, in step (a2), the differentiation culture time T a2 The treatment time is 8 to 10 days; preferably 9 days.

[0177] In another preferred embodiment, the induction medium includes: a basal medium and a key factor for inducing differentiation.

[0178] In another preferred embodiment, the basal culture medium is selected from the group consisting of APEL2, mTeSR plus, RPMI, and StemPro-34; preferably APEL2.

[0179] In another preferred embodiment, the key factors for inducing differentiation include: vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), activin A, fibroblast growth factor (FGF), stem cell factor (SCF), thrombopoietin (TPO), cytokines, GSK-3 inhibitors, and TGF-β pathway inhibitors.

[0180] In another preferred embodiment, the GSK-3 inhibitor is CHIR99021.

[0181] In another preferred embodiment, the TGF-β pathway inhibitor is SB431542.

[0182] In another preferred embodiment, the key factors may be introduced simultaneously or in stages.

[0183] In another preferred embodiment, BMP4, ACTIVIN A and CHIR99021 factors are introduced in the first stage of induction culture; the first stage lasts for 1-3 days, for example, about 2 days.

[0184] In another preferred embodiment, VEGF, bFGF, SCF and SB431542 factors are introduced in the second stage of induction culture; the second stage lasts for 2-4 days, for example about 3 days.

[0185] In another preferred embodiment, VEGF, bFGF, SCF, IL-6, TPO and IL-3 factors are introduced in the third stage of induction culture; the third stage lasts for 3-5 days, for example about 4 days; the medium is changed every 2 days.

[0186] In another preferred embodiment, ACTIVIN A and CHIR99021 were added on day 0 of the culture, and SB431542 was added on day 2 of the culture.

[0187] In another preferred embodiment, the concentration of ACTIVIN A is 10-100 ng / mL, preferably 20-50 ng / mL, and more preferably 25-35 ng / mL.

[0188] In another preferred embodiment, the concentration of CHIR99021 is 1-10 μM, preferably 2-8 μM, and more preferably 4-6 μM.

[0189] In another preferred embodiment, the concentration of SB431542 is 2-20 μM, preferably 5-15 μM, and more preferably 8-12 μM.

[0190] In another preferred embodiment, 1-10 ng / ml BMP4, 10-50 ng / mL ACTIVIN A and 1-10 μM CHIR99021 are introduced during the first stage of induction culture.

[0191] In another preferred embodiment, 5 ng / ml BMP4, 30 ng / mL ACTIVIN A and 5 μM CHIR99021 were introduced during the first stage of induction culture.

[0192] In another preferred embodiment, 40-60 ng / ml VEGF, 10-30 ng / ml bFGF, 50-150 ng / ml SCF and 5-20 μM SB431542 are introduced during the second stage of induction culture.

[0193] In another preferred embodiment, 50 ng / ml VEGF, 20 ng / ml bFGF, 100 ng / ml SCF and 10 μM SB431542 were introduced during the second stage of induction culture.

[0194] In another preferred embodiment, 40-60 ng / ml VEGF, 10-30 ng / ml bFGF, 50-150 ng / ml SCF, 5-20 ng / ml IL-6, 40-60 ng / ml TPO and 5-20 ng / ml IL-3 are introduced during the third stage of induction culture.

[0195] In another preferred embodiment, 50 ng / ml VEGF, 20 ng / ml bFGF, 100 ng / ml SCF, 10 ng / ml IL-6, 50 ng / ml TPO and 10 ng / ml IL-3 are introduced during the third stage of induction culture.

[0196] In another preferred embodiment, the method further includes:

[0197] (a3) Collect the cell population obtained from step (a2) and optionally perform detection.

[0198] In another preferred embodiment, the cell viability of the cell population collected in step (a3) ​​is ≥90%, preferably ≥95%, and more preferably ≥98%.

[0199] In another preferred embodiment, the proportion of CD41+ / CD42b+ double-positive cells in the cell population collected in step (a3) ​​is ≥75%, preferably ≥78%, and more preferably ≥80%.

[0200] In another preferred embodiment, the proportion of CD45+ / CD34+ double-positive cells in the cell population collected in step (a3) ​​is ≥60%, preferably ≥63%, and more preferably ≥65%.

[0201] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0202] The beneficial effects of this invention include:

[0203] 1. This invention provides a method for differentiating megakaryocytes from trophoblast cells, which reduces the risk of introducing exogenous heterogeneous cells, thereby avoiding tumorigenicity, immune rejection, and ectopic tissue formation caused by heterogeneous cells.

[0204] 2. This invention induces differentiation by editing the genes of relevant factors and changing the composition of the culture medium, thereby efficiently obtaining immortalized megakaryocyte lines under conditions without feeder cells.

[0205] 3. The immortalized megakaryocytes of the present invention have long-term proliferation function and platelet production activity; the platelets produced by the immortalized megakaryocytes of the present invention do not contain heterologous cells and have very high safety.

[0206] 4. Using the method of the present invention to culture hematopoietic stem cells, immortalized megakaryocytes with a cell count of more than 500 times can be obtained, and the megakaryocytes cultured by the present invention have a CD41+ / CD42b+ double positive cell ratio of more than 80%. Attached Figure Description

[0207] Figure 1 shows a schematic diagram (A) of the platelet production process of the present invention and a schematic diagram (B) of the lentiviral vector structure.

[0208] Figure 2 shows the experimental results of inducing hiPSC differentiation into hematopoietic stem cells using three different methods. A. Schematic diagram showing the process of iPSC induction into hematopoietic stem cells; B. Viability of hematopoietic stem cells harvested on days 9, 11, 13, and 15; C. Flow cytometry results of hematopoietic stem cells harvested on day 9 after induction using the three methods.

[0209] Figure 3 shows the experimental results of culturing megakaryocytes derived from pluripotent stem cells using four different methods. A. Schematic diagram showing the morphology of immortalized megakaryocytes induced by four different culture media; B. Flow cytometry results showing the proportion of CD41+ / CD42b+ cell populations in immortalized megakaryocytes induced by four different culture media.

[0210] Figure 4 shows the experimental results of culturing megakaryocytes derived from umbilical cord blood using four different methods. A. Schematic diagram showing the morphology of immortalized megakaryocytes induced by five different culture media. B. Flow cytometry results of the proportion of CD41+ / CD42b+ cell populations in immortalized megakaryocytes induced by four different culture media.

[0211] Figure 5 shows the results of the optimization experiment on the culture medium for immortalized megakaryocytes. A. Showing the effect of different culture media on the proportion of CD41+ / CD42b+ positive cells; B. Showing the statistical results of the doubling time of monoclonal megakaryocyte lines over 120 days under different culture media conditions; C. Showing the proportion of CD41+ / CD42b+ cell populations of monoclonal megakaryocyte lines over 120 days under different culture media conditions; D. Statistical results of the effect of different culture media on the immortalization efficiency of megakaryocytes.

[0212] Figure 6 shows the experimental results of the maturation and differentiation of immortalized megakaryocytes into platelets. A. Schematic diagram of megakaryocyte differentiation, showing the processes of mature megakaryocytes and the proplatelets at the ends of the processes; B. Calcein-AM staining, showing the active platelets (green) formed by megakaryocyte differentiation; C. Platelet viability (Calcein-AM+) detected by flow cytometry; D. Flow cytometry showing the ratio of CD41+ / CD42b+ expressed on the platelet surface; E. Flow cytometry showing the ratio of CD62p and PAC-1 expressed on platelets before and after activation with ADP (100 μM) and TRAP6 (40 μM); F. Platelet aggregation analysis showing the platelet aggregation curve after activation with ADP (50 μM); G. Platelet contraction response after treatment with 10 U / ml thrombin. Detailed Implementation

[0213] Through extensive and in-depth research, the inventors have constructed a method for stably expanding an immortalized megakaryocyte cell line (i.e., platelet-derived cells) from human stem cells in vitro. Under specific conditions where trophoblast cells are discarded, the culture medium composition is precisely adjusted to stimulate the differentiation potential of megakaryocytes. Simultaneously, during the megakaryocyte differentiation process without trophoblast cell involvement, relevant factors such as oncogenes and apoptosis-inhibiting genes are cleverly introduced into hematopoietic stem cells derived from human stem cells. By rigorously controlling the expression levels of these factors and simultaneously optimizing the culture medium composition, an immortalized megakaryocyte cell line was successfully obtained. When platelet differentiation needs to be induced, simply stopping the expression of these factors and selectively altering the culture medium composition again achieves the desired result, opening up a new avenue for the large-scale and stable preparation of platelets. This invention is based on this foundation.

[0214] definition

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0216] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0217] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0218] As used in this article, the term "pluripotent stem cell (PSC)" refers to a class of undifferentiated cells that have the potential to differentiate into all cell types in the body, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).

[0219] As used herein, the term "human induced pluripotent stem cell" (hiPCS) refers to a type of stem cell with self-renewal and multi-lineage differentiation potential obtained by reprogramming human somatic cells. In this application, the term hiPSC encompasses hiPSCs obtained by reprogramming somatic cells from blood types A, B, AB, and O, as well as hiPSCs from universal cell sources.

[0220] As used in this article, the term "hematopoietic stem cells (HSCs)" refers to a type of adult stem cell capable of differentiating into all blood cell lineages, including erythrocytes, leukocytes, and platelets. Typical cell surface markers include CD34. +Hematopoietic stem cells are found in bone marrow, umbilical cord blood, and peripheral blood, and can be directly isolated from them. Hematopoietic stem cells can also be obtained by inducing differentiation of pluripotent stem cells in vitro. In a preferred embodiment, the hematopoietic stem cells of the present invention are obtained by differentiating and culturing hiPSCs in a culture medium.

[0221] As used herein, the term "megakaryocyte (MK)" refers to a type of large, polyploid cell found in the bone marrow, derived from hematopoietic stem cells, and is the source cell of platelets. Its development involves differentiation from hematopoietic stem cells into megakaryotic progenitor cells (MkPs), followed by intranuclear replication to form polyploid cells, and finally, cytoplasmic extension to form proplatelets. Megakaryocytes express specific surface markers CD41 and CD42b. Immature megakaryocytes are mononuclear (2N) with a diameter of 10-15 μm, while mature megakaryocytes are multinucleated (8-64N) with a diameter >30 μm. Mature megakaryocytes rapidly produce platelets.

[0222] As used herein, the term "immortalized megakaryocyte (imMK)" refers to a megakaryocyte line that can proliferate indefinitely, obtained through gene editing technologies (such as lentiviral transduction of oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes). It retains the basic characteristics of natural megakaryocytes, including polyploid nucleation, proplatelet release, and platelet production. imMK exhibits stable proliferation in culture systems, with a double positivity rate of >80% for the surface markers CD41 and CD42b. An immortalized megakaryocyte line (imMKCL) can be established through monoclonal selection.

[0223] As used in this article, the term "platelet" refers to anucleate cells formed by the fragmentation of megakaryocyte cytoplasm. Platelets play key roles in blood circulation, such as hemostasis, thrombosis, and immune regulation. Platelet transfusion is crucial for patients with thrombocytopenia caused by surgical blood loss, chemotherapy, radiotherapy, or bone marrow hematopoietic dysfunction.

[0224] In vitro platelet production system without trophoblast cells

[0225] In this invention, a culture system without trophoblast cells is provided, which does not rely on trophoblast cells in the in vitro culture of multiple stages of platelet generation, including: differentiation of pluripotent stem cells into hematopoietic stem cells, differentiation and culture of hematopoietic stem cells into megakaryocytes, and further differentiation of megakaryocytes into platelets.

[0226] As used in this article, the term "feeder cell" also refers to a type of auxiliary cell used in the in vitro culture of supporting cells. Common sources include mesenchymal stem cells and fibroblasts. Feeder cells provide a suitable microenvironment for cells by secreting extracellular matrix and growth factors.

[0227] Trophoblast cells play an important role in cell culture in vitro; however, the introduction of trophoblast cells brings risks.

[0228] First, heterologous trophoblast cells pose an immunogenicity risk. Currently used trophoblast cells, specifically OP9 and C3H10T1 / 2, are both derived from mice and differ significantly from human cells. When used in the production of human cell therapy products, their cellular components, such as proteins and glycoproteins, could potentially act as foreign antigens, inducing an immune response in the human body. Even with rigorous purification procedures throughout the production process, it is difficult to completely eliminate all residues derived from mouse cells. This could lead to immune rejection in patients receiving cell therapy, affecting treatment efficacy and posing potential health risks.

[0229] Secondly, trophoblast cells pose a certain risk of tumorigenesis. OP9 and C3H10T1 / 2, as mouse-derived trophoblast cells, possess certain proliferative characteristics. However, due to the highly complex cell culture environment and the inherent limitations of current monitoring techniques, there remains a small probability that these mouse cells can escape control, proliferate abnormally, and ultimately form tumors. If such an extreme situation occurs in a patient receiving cell therapy, the consequences will be extremely serious.

[0230] Furthermore, trophoblast cells pose a risk of viral transmission. Mouse cells carry the potential risk of specific murine viruses, such as mouse parvovirus and lactate dehydrogenase-enhancing virus. These viruses may not be pathogenic in mice themselves, but once they are involved in the production of human cell therapy products, they immediately pose a risk of infecting human cells, thus seriously threatening the lives and health of patients receiving cell therapy. Even with rigorous testing procedures to carefully screen for viruses, the potential risk of viral transmission cannot be completely eliminated.

[0231] Given that OP9 or C3H / 10T1 / 2 trophoblast cells are not derived from humans, their application in the production of human cell therapy products is likely to encounter more complex regulatory challenges. Regulatory authorities typically set stricter and more detailed standards for cell therapy products involving allogeneic cells, particularly in key areas such as safety assessments and residue testing. This inevitably leads to lengthy and complex product development and approval processes, significantly increasing both research and development costs and time.

[0232] To overcome the above-mentioned defects, in this invention, through precise adjustment of the culture medium composition and screening of culture conditions, a method and culture system for in vitro platelet production from feeder cells were finally obtained.

[0233] In vitro induced pluripotent stem cells to generate hematopoietic stem cells

[0234] In this invention, a method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells in vitro is provided. By adding specific factors, pluripotent stem cells are induced to differentiate into hematopoietic lineages, and this method does not require the addition of trophoblast cells.

[0235] In this invention, pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In a preferred embodiment, the pluripotent stem cells are iPSCs, preferably human iPSCs (hiPSCs). The source and type of the hiPSCs are not limited, including but not limited to universal iPSCs and iPSCs from various blood types. The methods for culturing and dissociating iPSCs described in this invention can employ any method known in the art, which can be routinely mastered by those skilled in the art. For example, before differentiating and culturing iPSCs, iPSC cells can be cultured in a stem cell-suitable medium such as mTeSR plus medium. One day before differentiation culture, the iPSCs are treated into cell clumps using a mild dissociation agent, such as Accutase, EDTA, TrypLE SELECT Enzyme, and ReLeSR.

[0236] In this invention, a culture medium for inducing pluripotent stem cell differentiation into hematopoietic stem cells in vitro is provided, referred to as an "induction medium". The induction medium includes: a basal culture medium suitable for stem cells / hematopoietic stem cells, and differentiation-inducing factors and growth factors added to the basal culture medium.

[0237] In this invention, the type and source of the basal culture medium suitable for stem cells / hematopoietic stem cells are not limited, such as APEL2 medium, DMEM / F12, mTeSR series medium, Essential 8 series medium, StemFlex series medium, TeSR-E8 series medium, Repro series medium, NutriStem hPSC series medium, STEMdiff series medium, and STEMFit series medium, as long as these media can provide a suitable nutritional environment for the growth of iPSC cells and promote their differentiation into hematopoietic stem cells. Preferably, the basal culture medium is APEL2 medium.

[0238] Factors added to the basal culture medium include, but are not limited to: vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), activin A, fibroblast growth factor (FGF), stem cell factor (SCF), thrombopoietin (TPO), cytokines, GSK-3 inhibitors (e.g., CHIR99021), and TGF-β pathway inhibitors (e.g., SB431542), which can be introduced into the culture process simultaneously or stepwise. Preferably, the key factors are introduced into the culture process stepwise and sequentially.

[0239] In a preferred embodiment, the above-mentioned additive factors are introduced in stages. In the first stage, BMP4, ACTIVIN A, and CHIR99021 factors are introduced into the basal medium and cultured for about 2 days. In the second stage, the basal medium supplemented with VEGF, bFGF, SCF, and SB431542 factors is replaced and cultured for about 3 days. In the third stage, the basal medium supplemented with VEGF, bFGF, SCF, IL-6, TPO, and IL-3 factors is replaced and cultured for about 4 days, with the medium being replaced with a medium of the same composition every 2 days.

[0240] In a preferred embodiment, the induction culture time is 7–12 days, preferably 8–10 days. In another preferred embodiment, the suspension cells are collected on approximately day 9 of culture to obtain hematopoietic stem cells. Compared with methods with an induction time longer than 12 days, the hematopoietic stem cells obtained by the method of the present invention have excellent differentiation into megakaryocytes.

[0241] Hematopoietic stem cells differentiated by the method of this invention possess molecular surface markers such as CD34, CD45, CD41, CD43, and CD42b. Compared with hematopoietic stem cells obtained by other methods, the hematopoietic stem cells obtained by the method of this invention are particularly suitable for further differentiation and culture to obtain immortalized megakaryocytes.

[0242] Inducing hematopoietic stem cells to produce megakaryocytes

[0243] In this invention, a method for inducing hematopoietic stem cells to generate immortalized megakaryocytes in vitro is provided, and the method does not involve the addition of trophoblast cells.

[0244] The method of the present invention is applicable to the induction and differentiation of hematopoietic stem cells from various sources, including (i) hematopoietic stem cells isolated from umbilical cord blood, peripheral blood or bone marrow; and (ii) hematopoietic stem cells induced and differentiated in vitro from iPSC cells or embryonic stem cells, such as hematopoietic stem cells differentiated by the method of the present invention.

[0245] For example, CD34 derived from umbilical cord blood +Hematopoietic stem cells can be commercially available. The collection and separation of cells are based on the "Regulations on the Management of Human Genetic Resources" and the "Good Clinical Practice for Drug Clinical Trials," and are conducted with valid donor informed consent and cell source certificates to ensure compliance, quality, and safety.

[0246] In this invention, a culture medium for inducing the differentiation of hematopoietic stem cells into megakaryocytes in vitro is provided, referred to as a "differentiation culture medium". The differentiation culture medium includes: a basal culture medium, and growth factors added to the basal culture medium.

[0247] The basal culture medium includes, but is not limited to, IMDM, SFEM II, Stempro-34, or APEL2 medium; the added growth factors include, but are not limited to, SCF, Flt3-L, TPO, IL-3, IL-6, IL-11, GM-CSF, SR1, CAY10433, KO-SR, Sodium Heparin, and VPA. To ensure cell growth and nutrition, other additives, including ITS, GlutaMAX, 1-Thioglycerol, and Ascorbic acid, may optionally be added to the differentiation medium.

[0248] In a preferred embodiment, the growth factors in the culture medium of the present invention preferably include: SCF, TPO, Flt3-L, and IL-6; and the growth factors optionally include SR1, UM171, IL-3, heparin or heparin sodium, and / or valproic acid. For example, the preferred combination of growth factors in the culture medium of the present invention is selected from the following group:

[0249] (i) SCF, TPO, Flt3L, IL-6 and SR1;

[0250] (ii) SCF, TPO, Flt3L, IL-6, SR1 and valproic acid;

[0251] (iii) SCF, TPO, Flt3L, IL-6 and UM171;

[0252] (iv) SCF, TPO, Flt3L, IL-6, SR1, valproic acid and Celecoxib.

[0253] In this invention, a method for generating immortalized megakaryocytes in the above-mentioned differentiation culture medium is provided, comprising the steps of: seeding hematopoietic stem cells into the differentiation culture medium at a suitable culture density (e.g., 1-5 × 10⁻⁶). 5 / ml, more preferably 1-2×10 5Cells were seeded ( / ml) into suitable culture dishes, such as 6-well plates. An expression vector containing a combination of immortalizing factors (oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes) was introduced into the culture medium and co-incubated. During culture, the medium was changed to maintain cell density until the cell population differentiated to the surface molecular marker CD41. + CD42b + The proportion of megakaryocytes (i.e., large cells) is >80%.

[0254] As used in this article, the term "immortification factor" refers to exogenous genes introduced through genetic engineering during the immortalization of megakaryocytes, including oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes.

[0255] As used in this article, the term "oncogene" refers to regulatory factors that activate cell proliferation signaling pathways or inhibit the cell cycle, thus endowing cells with the ability to proliferate indefinitely. Commonly used oncogenes include the MYC family, the Src family, the Ras / Raf family, and the protein kinase family.

[0256] As used in this article, the term "senescence-inhibiting gene" refers to factors that inhibit cellular senescence-related signaling pathways or regulate telomerase activity, thereby delaying the cellular senescence process. Common senescence-inhibiting genes include BMI1, CDK4, hTERT, B-myb, and hnRNPU.

[0257] As used in this article, the term "apoptosis inhibitory gene" refers to factors that inhibit apoptosis-related signaling pathways or regulate the activity of apoptosis-executing proteins, thereby improving cell survival. Commonly used apoptosis inhibitory genes include the BCL-2 family, the IAP family, Suvivin, and MCL-1 genes.

[0258] In some embodiments of the present invention, the immortalization factor combination includes two or three of the following: oncogenes, senescence inhibitory genes, and apoptosis inhibitory genes; in some embodiments, the immortalization factor combination includes one oncogene, one senescence inhibitory gene, and one apoptosis inhibitory gene; in some embodiments, the immortalization factor combination includes one senescence inhibitory gene and one apoptosis inhibitory gene.

[0259] As used herein, the genetic engineering methods for introducing the immortalization factor into cells are not limited, and gene editing systems known in the art, including transposons, CRISPR / Cas9, ZFNs, and TALENs, can be used. In a preferred embodiment, the immortalization factor is introduced into cells via a viral vector, such as a lentiviral vector, adenovirus vector, or adeno-associated virus vector, preferably a lentiviral vector.

[0260] In one embodiment, the immortalization factor combination can be introduced into cells simultaneously or stepwise; preferably, the immortalization factor combination is added stepwise, for example, first adding oncogenes and senescence inhibitor genes, and then adding apoptosis inhibitor genes. Exemplarily, oncogenes and senescence inhibitor genes are added on day 0 of differentiation culture, and apoptosis inhibitor genes are introduced after a large number of megakaryocytes are generated, for example, on days 9-18 of differentiation culture, preferably days 9-13.

[0261] In a preferred embodiment, the immortalization factor combination is expressed under the control of megakaryocyte or platelet-specific promoters, including CD41, CD42a, CD42b, CD42c, CD42d, CD61, PF4, or vWF promoters.

[0262] The megakaryocytes produced by the method of the present invention have characteristics such as CD34 negative or CD34 positive, CD41 positive, and CD42b positive.

[0263] After obtaining megakaryocytes, the forced expression of some or all oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes is stopped, and mature megakaryocytes are obtained through further culture. The expression of immortalization factors can be stopped by, for example, removing inducers from the culture medium (such as doxycycline, p-isopropylbenzoic acid, coumarin) or by using other gene-editing techniques to remove the relevant genes.

[0264] After several days of cell passage culture, CD41 with a relatively stable ratio can be obtained. + CD42b + Immortalized megakaryocyte (imMK) population with double positivity. Cells with shorter population doubling time were selected and analyzed by flow cytometry to ensure that the population was more than 80% double positive for the cell surface molecular markers CD41 and CD42b.

[0265] These cells underwent induced differentiation tests, revealing that during maturation, the cells increased in size and exhibited multinucleation, resulting in a megakaryocyte cell line capable of producing functional platelets. The corresponding megakaryocytes were then subjected to monoclonal screening using flow cytometry, ultimately identifying a functional, immortalized megakaryocyte cell line (imMKCL) with long-term proliferative capacity suitable for platelet production.

[0266] The immortalized megakaryocyte line obtained by the method of the present invention has the ability to be stably expanded in vitro for a long period of time, and the stable expansion time in vitro can reach 3-6 months, and in some embodiments it can reach 3-4 months.

[0267] Immortalized megakaryocytes produce platelets

[0268] After obtaining mature megakaryocytes through the above-mentioned mature megakaryocyte production method, platelets can be obtained through further differentiation and culture.

[0269] Mature megakaryocytes can be cultured in suspension or statically. In culture without a feeder layer, the culture temperature is approximately 37°C, containing approximately 5% CO2, and the culture period is approximately 2-6 days. Any known suitable medium or equivalent medium for platelet production can be used. During culture, stirring or shaking can be performed at a speed of approximately 30-120 rpm. The produced platelets can be stored and prepared in suspension in a suitable solution for approximately 3-7 days at a temperature of approximately 20-24°C with shaking. The methods for culturing and storing platelets are routinely chosen by those skilled in the art.

[0270] Platelet counts were performed using flow cytometry, and the expression of cell surface molecular markers such as CD41 and CD42b was also detected using flow cytometry. After platelet activation with the addition of a platelet stimulant, the expression of cell surface molecular markers CD41, CD42b, PAC-1, and CD62P was detected again using flow cytometry.

[0271] The platelets produced by the method of this invention have excellent contractile function and exhibit good aggregation response under the stimulation of different agonists in vitro.

[0272] The platelets produced by the method of this invention do not contain foreign cells, have high safety, and can be used for platelet transfusion in patients with thrombocytopenia caused by chemotherapy, radiotherapy, bone marrow hematopoietic dysfunction, etc., as well as patients who need to replenish platelets after massive blood loss during surgery.

[0273] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0274] Example 1: Screening of hiPSC-induced differentiation into hematopoietic stem cells under trophoblast-free conditions

[0275] In this embodiment, human induced pluripotent stem cells (hiPSCs) are induced to differentiate into hematopoietic stem cells using three different methods. The specific steps are as follows:

[0276] Before incubation, hiPSCs were cultured in mTeSR plus medium until cell confluence reached approximately 70-80%. On day -1, hiPSCs were treated into cell clumps using a mild dissociation agent. The cell clumps were then seeded at an appropriate density into matrix gel-coated 6-well plates and cultured in mTeSR plus medium for 24 hours.

[0277] (I) Method 1:

[0278] On day 0, the medium was replaced with RPMI medium supplemented with 5 ng / ml BMP4, 50 ng / ml VEGF, and 25 ng / ml hWnt3a. On day 2, the medium was changed with RPMI medium supplemented with 5% KO-SR, 50 ng / ml VEGF, 20 ng / ml bFGF, and 5 ng / ml BMP4. On day 3, the medium was changed with StemPro medium supplemented with 5 ng / ml BMP4, 50 ng / ml VEGF, and 5 ng / ml bFGF. On day 4, the medium was changed with StemPro medium supplemented with 50 ng / ml VEGF and 5 ng / ml bFGF. On day 6, the medium was changed with IMDM medium containing 25% Ham's-F12 supplemented with 50 ng / ml VEGF, 100 ng / ml bFGF, 100 ng / ml SCF, and 25 ng / ml Flt3L. On day 7, the medium was changed to IMDM medium containing 25% Ham's-F12 supplemented with 50 ng / ml VEGF, 5 ng / ml bFGF, 100 ng / ml SCF, 25 ng / ml Flt3L, 10 ng / ml IL-6, 50 ng / ml TPO, 0.5 U / ml EPO, and 0.2 μM FICZ. Starting on day 9, suspension cells were collected at days 9, 11, 13, and 15.

[0279] (II) Method 2:

[0280] On day 0, the medium was replaced with RPMI medium supplemented with 5% KO-SR, 0.4 mM MTG, 0.3 mM 2PAA, 25 ng / ml hWnt3a, 5 ng / ml BMP4, and 50 ng / ml VEGF. On day 2, the medium was replaced with RPMI medium supplemented with 5% KO-SR, 0.4 mM MTG, 0.3 mM 2PAA, 25 ng / ml hWnt3a, 5 ng / ml BMP4, 50 ng / ml VEGF, and 5 ng / ml bFGF. On day 4, the medium was replaced with StemPro medium supplemented with 0.3 mM 2PAA, 0.4 mM MTG, 50 ng / ml VEGF, and 20 ng / ml bFGF. On day 6, the medium was changed with IMDM medium containing 25% Ham's-F12, supplemented with 0.5% N-2, 1% B-27, 0.5% BSA, 0.4mM MTG, 0.3mM 2PAA, 50 ng / ml VEGF, 5 ng / ml bFGF, 100 ng / ml SCF, and 25 ng / ml Flt3L. On day 7, the medium was changed with IMDM medium containing 25% Ham's-F12, supplemented with 0.5% N-2, 1% B-27, 0.5% BSA, 0.4mM MTG, 0.3mM 2PAA, 50 ng / ml VEGF, 5 ng / ml bFGF, 50 ng / ml SCF, 25 ng / ml Flt3L, 50 ng / ml TPO, 10 ng / ml IL-6, 0.5 U / ml EPO, and 0.2 μM FICZ. Starting from day 9 of culture, suspension cells were collected on days 9, 11, 13, and 15.

[0281] (III) Method 3:

[0282] On day 0, the medium was replaced with APEL2 medium supplemented with 5 ng / ml BMP4, 30 ng / ml ACTIVIN A, and 5 μM CHIR99021. On day 2, the medium was changed again with APEL2 medium supplemented with 50 ng / ml VEGF, 20 ng / ml bFGF, 100 ng / ml SCF, and 10 μM SB431542. On days 5 and 7, the medium was changed again with APEL2 medium supplemented with 50 ng / ml VEGF, 20 ng / ml bFGF, 100 ng / ml SCF, 10 ng / ml IL-6, 50 ng / ml TPO, and 10 ng / ml IL-3. Starting on day 9, suspension cells were collected at days 9, 11, 13, and 15.

[0283] Cell morphology was observed and recorded on days 9, 11, 13, and 15 of the three methods described above, cell viability was detected, and the positive rates of CD34, CD45, CD41, and CD42b were detected by flow cytometry.

[0284] The results are shown in Figure 2. Figure 2B shows the cell viability of cells cultured using the three methods on days 9, 11, 13, and 15. The results indicate that the viability of the hematopoietic stem cell population harvested on day 9 was significantly higher than that harvested on days 11, 13, and 15. Subsequent experiments used hematopoietic stem cells harvested on day 9 as the material. Furthermore, on day 9, the cell viability obtained by method 3 was close to 100%, significantly higher than the approximately 80% obtained by methods 2 and 3.

[0285] The flow cytometry results are shown in Figure 2C. The results show that among the hematopoietic stem cells induced by the three methods, method 3 can obtain the hematopoietic stem cell population with the highest expression rates of CD34, CD45, CD41, and CD42b.

[0286] Example 2: Immortalized Culture of Megakaryotic Cells Derived from Pluripotent Stem Cells

[0287] The hematopoietic stem cells collected on day 9 in Example 1 were used as day 1 of immortalization culture, at a concentration of 2x10⁹ / L. 5 Cells were seeded at a density of 1 / ml into 6-well plates. Lentiviral vectors containing the target genes c-Myc and BMI1 (MOI = 10, see Figure 1B for a schematic diagram of the lentiviral vector) were added to the culture medium. The virus was removed 24 hours after infection. The medium was changed every 3 days to maintain a cell density of 2 x 10⁻⁶ cells / well. 5 Density per ml.

[0288] On day 14 of immortalized culture, lentivirus with the target gene BCL-XL (MOI=10) was added, and the virus was removed 24 hours after infection. On day 21 of immortalized culture, flow cytometry was used to detect CD41 and CD42b molecular surface markers; CD41 / CD42b double positivity represented megakaryocytes.

[0289] Methods 1 through 4 were performed using the following four immortalized culture media, with all other conditions remaining the same:

[0290] Method 1 culture medium: IMDM medium supplemented with 15% FBS, 2mM L-glutamine, 1x insulin-transferrin-selenium, 50mg / mL ascorbic acid, 450μM 1-thioglycerol, 32.3μg / ml SR1, 50ng / ml TPO, 50ng / ml SCF, 25U / ml heparin sodium and 1μg / ml doxycycline.

[0291] Method 2 culture medium: Stemspan SFEMII medium supplemented with 100 ng / mL SCF, 50 ng / mL TPO, 100 ng / mL Flt3L, 15 ng / mL IL-6, 1 μM SR1, 0.2 mM valproic acid and 1 μg / mL doxycycline.

[0292] Method 3 culture medium: APEL 2 medium supplemented with 25 ng / ml TPO, 25 ng / ml SCF, 25 ng / ml Flt3L, 10 ng / ml IL-3, 10 ng / ml IL-6, 5 U / ml heparin and 1 μg / ml doxycycline.

[0293] Method 4 culture medium: StemPro 34 medium supplemented with 2 mM L-glutamine, 50 ng / mL TPO, 20 ng / mL SCF, 10 ng / mL IL-6, 10 ng / mL IL-9 and 1 μg / mL doxycycline.

[0294] The results are shown in Figure 3. The results indicate that all the above-mentioned different culture conditions can induce hematopoietic stem cells to form immortalized megakaryocytes. Among them, method 2 yielded the highest proportion of CD41 and CD42b double-positive immortalized megakaryocytes, exceeding 80%. Methods 3 and 4 also yielded a high proportion of CD41 and CD42b double-positive cells, while method 1 yielded a lower proportion of CD41 and CD42b double-positive cells, only about 50%.

[0295] Method 2 yielded cells with the optimal expansion rate, cell viability, and CD41 positivity. The concentrations of various factors in the culture medium were varied based on Method 2, and the culture effects were compared, as detailed below:

[0296] The culture medium for Method 2-1 was Stemspan SFEMII medium supplemented with 50 ng / mL SCF, 25 ng / mL TPO, 50 ng / mL Flt3L, 10 ng / mL IL-6, 0.5 μM SR1, 0.1 mM valproic acid and 1 μg / mL doxycycline.

[0297] The culture medium for Method 2-2 was Stemspan SFEMII medium supplemented with 150 ng / mL SCF, 75 ng / mL TPO, 150 ng / mL Flt3L, 20 ng / mL IL-6, 2 μM SR1, 0.5 mM valproic acid and 1 μg / mL doxycycline.

[0298] Table 1 shows the percentage of CD41+ / CD42b+ double-positive cells, cell viability, and cell proliferation fold during the culture processes of Method 2, Method 2-1, and Method 2-2.

[0299] Table 1 Note: Cells were cultured at a density of 2E5 / ml, and the theoretical amplification fold was calculated based on the doubling time of each test.

[0300] The results showed that methods 2, 2-1, and 2-2 could all culture immortalized megakaryocytes efficiently and with high quality. Method 2 showed better proportion of CD41+ / CD42b+ double-positive cells and cell viability at day 21 of culture.

[0301] Example 3: Immortalized Culture of Megakaryocytes Derived from Umbilical Cord Blood

[0302] The CD34+ cells derived from umbilical cord blood were cultured using a method similar to that in Example 2, specifically including: purchasing CD34+ cells derived from umbilical cord blood and culturing them at a density of 2 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / ml into 6-well plates, and c-Myc and BMI1 lentiviruses were added to the culture medium. The medium was changed every 3 days to maintain a density of 2 x 10⁶ cells / ml. 5 The density was set at / ml. BCL-XL virus was added on day 14. On day 21, flow cytometry was used to detect molecular surface markers such as CD34, CD41, and CD42. Monoclonal cell lines with stable doubling time and stable CD41 / CD42b expression levels were selected through monoclonal screening.

[0303] Methods 1 through 4 were performed using the following four immortalized culture media, with all other conditions remaining the same:

[0304] Method 1 culture medium: IMDM medium supplemented with 15% FBS, 2mM L-glutamine, 1x insulin-transferrin-selenium, 50mg / mL ascorbic acid, 450μM 1-thioglycerol, 32.3μg / ml SR1, 50ng / ml TPO, 50ng / ml SCF, 25U / ml heparin sodium and 1μg / ml doxycycline.

[0305] Method 2 culture medium: Stemspan SFEMII medium supplemented with 100 ng / mL SCF, 50 ng / mL TPO, 100 ng / mL Flt3L, 15 ng / mL IL-6, 1 μM SR1, 0.2 mM valproic acid and 1 μg / mL doxycycline.

[0306] Method 3 culture medium: APEL 2 medium supplemented with 25 ng / ml TPO, 25 ng / ml SCF, 25 ng / ml Flt3L, 10 ng / ml IL-3, 10 ng / ml IL-6, 5 U / ml heparin and 1 μg / ml doxycycline.

[0307] Method 4 culture medium: StemPro 34 medium supplemented with 2 mM L-glutamine, 50 ng / mL TPO, 20 ng / mL SCF, 10 ng / mL IL-6, 10 ng / mL IL-9 and 1 μg / mL doxycycline.

[0308] The results are shown in Figure 4. The results show that all of the above methods can induce immortalized megakaryocytes from hematopoietic stem cells derived from umbilical cord blood. Among them, method 2 can obtain immortalized megakaryocytes with a double positivity of CD41 and CD42b of over 80%.

[0309] The results of Examples 2 and 3 together demonstrate that the culture medium of Method 2 is suitable for culturing megakaryocytes from different sources such as pluripotent stem cells or umbilical cord blood, has universality, and has better culture effect than other culture media.

[0310] Example 4: Optimization of Culture Medium for Immortalized Megakaryotic Cells

[0311] The hematopoietic stem cells collected on day 9 in Example 1 were used as day 1 of immortalization culture, at a concentration of 2x10⁹ / L. 5 Cells were seeded at a density of 1 / ml into 6-well plates, and c-Myc and BMI1 lentiviruses were added to the culture medium. The medium was changed every 3 days to maintain a density of 2 x 10⁶ cells / ml. 5 The density was [density] / ml. BCL-XL virus was added on day 14 of immortalized culture. On day 21 of immortalized culture, flow cytometry was used to detect CD41 and CD42 molecular surface markers, following the same steps as in Example 2. Monoclonal cell lines with stable doubling time and stable CD41 / CD42b expression levels were selected through monoclonal screening.

[0312] They were cultured in the following four immortalized culture media respectively:

[0313] (a) Stemspan SFEMII medium was supplemented with 100 ng / mL SCF, 50 ng / mL TPO, 100 ng / mL Flt3L, 15 ng / mL IL-6, 1 μM SR1, 0.2 mM valproic acid and 1 μg / mL doxycycline.

[0314] (ii) Stemspan SFEMII medium was supplemented with 100 ng / mL SCF, 50 ng / mL TPO, 100 ng / mL Flt3L, 15 ng / mL IL-6, 1 μM UM171, 0.2 mM valproic acid and 1 μg / mL doxycycline.

[0315] (III) Stemspan SFEMII medium was supplemented with 100 ng / mL SCF, 50 ng / mL TPO, 100 ng / mL Flt3L, 15 ng / mL IL-6, 1 μM SR1 and 1 μg / mL doxycycline.

[0316] (iv) Add 100 ng / mL SCF, 50 ng / mL TPO, 100 ng / mL Flt3L, 15 ng / mL IL-6, 1 μM SR1, 0.5 μM Celecoxib, 0.2 mM valproic acid and 1 μg / mL doxycycline to Stemspan SFEMII medium.

[0317] The results are shown in Figure 5. The results indicate that the above method can induce immortalized megakaryocytes with >80% being positive for both CD41 and CD42b. The immortalization efficiency of megakaryocytes reaches over 10%, where immortalization efficiency refers to the proportion of monoclonal cell lines that can be rapidly expanded after monoclonal screening.

[0318] After 21 days of immortalized culture, monoclonal selection was performed using flow cytometry to select monoclonal cells expressing CD41+ / CD42b+ and transfer them to 96-well plates. The medium was changed every 3 days. After three weeks, the expression levels of CD41+ / CD42b+ in all rapidly proliferating monoclonal cell lines were detected by flow cytometry, and the cell count was assessed using a cell counter.

[0319] The results are shown in Table 2. The immortalized megakaryocytes cultured by each method maintained a relatively stable doubling time within 120 days, and stable expansion in vitro could last for 3-4 months.

[0320] Table 2

[0321] In summary, compared to the control group, megakaryocytes induced to immortalize in culture medium (III) exhibited a faster doubling rate, more stable CD41+ / CD42b+ expression levels, and higher immortalization efficiency.

[0322] Example 5: Maturation and differentiation of immortalized megakaryocytes into platelets

[0323] The immortalized megakaryocytes prepared by method 3 in Example 4 above were used at a ratio of 1x10⁻⁶.5 The solution was resuspended in maturation medium at a density of / ml and incubated for 6 days either statically or in a shake flask. The maturation medium was prepared by adding 5% human AB serum, 2mM L-glutamine, 1x insulin-transferrin-selenium, 50mg / mL ascorbic acid, 450μM 1-thioglycerol, 32.3μg / ml SR1, 50ng / ml TPO, 50ng / ml SCF, 10U / ml heparin sodium, 15μKP457, and 10μM Y27632 to IMDM medium.

[0324] Platelets were collected and counted using a cell counter to determine the number of platelets produced by each megakaryocyte. The expression of cell surface molecular markers CD41 and CD42b was detected using flow cytometry. After platelet activation with a platelet stimulant, the expression of cell surface molecular markers PAC-1 and CD62P was again detected by flow cytometry, along with aggregation and contraction abilities.

[0325] The results showed that the immortalized megakaryocytes cultured by the method of this invention had the ability to differentiate into platelets in vitro (Figure 6A). Under shake-flask culture conditions, a single megakaryocyte could produce 40 platelets (Figure 6B). Furthermore, the produced platelets had a high viability (Calcein-AM+% reached 69.1%) and a CD41+ / CD42b+ ratio exceeding 80% (Figure 6C&D). In vitro stimulation of platelets with ADP (100 μM) and TRAP6 (40 μM) showed an increase in the levels of CD62p and PAC1 proteins on the platelet membrane surface (Figure 6E); platelets treated with 50 μM ADP showed excellent aggregation; and platelets exhibited good contractile function under in vitro stimulation with 10 U / ml thrombin (Figure 6G).

[0326] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for producing immortalized megakaryocytes, characterized in that, The method includes the following steps: (a) Providing hematopoietic stem cells; (b) Transform the expression cassette of the exogenous immortalization factor into the hematopoietic stem cells to obtain transduced hematopoietic stem cells; and differentiate and culture the transduced hematopoietic stem cells under the conditions of no trophoblast cells and the transduced hematopoietic stem cells expressing the exogenous immortalization factor to obtain immortalized megakaryocytes. Step (b) specifically includes: (b1) The expression cassette of the exogenous first immortalization factor is transferred into the hematopoietic stem cells to obtain transduced hematopoietic stem cells; and under the condition of no trophoblast cells and the transduced hematopoietic stem cells expressing the exogenous first immortalization factor, the transduced hematopoietic stem cells are differentiated and cultured to obtain megakaryocytes. (b2) The expression cassette of the exogenous second immortalization factor is transferred into the megakaryocytes to obtain transduced megakaryocytes; and the transduced megakaryocytes are immortalized under the conditions of no feeder cells and the transduced megakaryocytes expressing the exogenous first and second immortalization factors to obtain immortalized megakaryocytes. The differentiation culture and / or immortalization culture are carried out in a differentiation medium; the differentiation medium includes: basal medium and growth factors; the growth factors include: SCF, Flt3-L, TPO and IL-6.

2. The method as described in claim 1, characterized in that, The growth factors also include SR1, UM171, IL-3, heparin or heparin sodium, valproic acid and / or celecoxib.

3. The method as described in claim 1, characterized in that, The combination of the growth factors is as follows: (i) SCF, TPO, Flt3L, IL-6 and SR1; (ii) SCF, TPO, Flt3L, IL-6, SR1 and valproic acid; (iii) SCF, TPO, Flt3L, IL-6, and UM171; or (iv) SCF, TPO, Flt3L, IL-6, SR1, valproic acid and Celecoxib.

4. The method as described in claim 1, characterized in that, The combination of the growth factors is as follows: (i) 20-200ng / mL SCF, 10-100ng / mL TPO, 20-200ng / mL Flt3L, 5-30ng / mL IL-6 and 0.1-5μM SR1; (ii) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6, 0.1-5 μM SR1 and 0.1-1 mM valproic acid; (iii) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6 and 0.5-2 μM UM171; or (iv) 20-200 ng / mL SCF, 10-100 ng / mL TPO, 20-200 ng / mL Flt3L, 5-30 ng / mL IL-6, 0.1-5 μM SR1, 0.1-1 mM valproic acid and 0.1-1 μM Celecoxib.

5. The method as described in claim 1, characterized in that, The number of transduced hematopoietic stem cells before culture in step (b1) is N0, and the number of immortalized megakaryocytes obtained in step (b2) is N3; N3 / N0 ≥ 500.

6. The method as described in claim 1, characterized in that, In the immortalized megakaryocytes obtained in step (b2), the proportion of CD41+ cells is ≥80%, and / or the proportion of CD41+ / CD42b+ double positive cells is ≥70%.

7. The method as described in claim 1, characterized in that, Step (b) also includes: (b3) Select immortalized megakaryocyte cell lines for inducing the production of functional platelets.

8. The method as described in claim 1, characterized in that, The immortalization factors include: oncogenes, senescence-inhibiting genes, and apoptosis-inhibiting genes.

9. The method according to any one of claims 1-8, characterized in that, The first immortalization factor is c-Myc, BMI1; and / or the second immortalization factor is BCL-XL.

10. The method as described in claim 1, characterized in that, The method further includes: (c) Passage and expand the immortalized megakaryocyte line to obtain immortalized megakaryocytes after passage and expansion; (d) The immortalized megakaryocytes that have been passaged and expanded are cultured to mature and form platelets.

11. An immortalized megakaryocyte, characterized in that, The megakaryocytes are obtained by the method of claim 1.

12. The use of the immortalized megakaryocyte as described in claim 11, characterized in that, Used to produce functional platelets.

13. A culture medium for inducing hematopoietic stem cells to differentiate into megakaryocytes, characterized in that, The culture medium includes: basal culture medium and growth factors.

14. The culture medium as described in claim 13, characterized in that, The growth factors include: SCF, TPO, Flt3-L and IL-6; and optionally, the growth factors also include SR1, UM171, IL-3, heparin or heparin sodium, valproic acid and / or Celecoxib.

15. A method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, the method comprising: (a1) Provides iPSC cells; (a2) The iPSC cells are cultured in hematopoietic stem cell induction medium to obtain hematopoietic stem cells; The induction medium includes: basal medium and key factors for inducing differentiation.

16. The method as described in claim 15, characterized in that, In step (a2), the differentiation culture time T a2 It takes 8 to 10 days.

17. The method as described in claim 15, characterized in that, The key factors for inducing differentiation include: vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), activin A, fibroblast growth factor (FGF), stem cell factor (SCF), thrombopoietin (TPO), cytokines, GSK-3 inhibitors, and TGF-β pathway inhibitors.

18. The method as described in claim 15, characterized in that, BMP4, ACTIVIN A, and CHIR99021 factors were introduced in the first stage of induction culture; the first stage lasted 1-3 days. VEGF, bFGF, SCF, and SB431542 were introduced during the second stage of induction culture; this second stage lasted 2-4 days; and / or VEGF, bFGF, SCF, IL-6, TPO and IL-3 factors were introduced in the third stage of induction culture; the third stage lasted for 3-5 days.

Images