Method for differentiating induced pluripotent stem cells into megakaryocytes and platelets

Abstract

The invention discloses a method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, and relates to the technical field of induced pluripotent stem cell directional differentiation, and the method comprises the following steps: inoculating an iPSC single cell into a culture vessel, and carrying out adherent culture through a culture solution; after the cell adheres to the wall and before cloning is not formed, the liquid is changed into a first induction culture liquid, and the cell is induced and differentiated into a mesoderm cell; changing the solution into a second induction culture solution to induce and differentiate the mesoderm cells into hematopoietic endothelial cells; collecting the suspension cells, culturing the suspension cells in an amplification culture solution, and enriching megakaryocytes; inducing megakaryocyte polyploidy through a third induction culture solution; and culturing the polyploidy megakaryocyte through a fourth induction culture solution to promote platelet release. The method for inducing the pluripotent stem cells to differentiate into megakaryocytes and platelets is convenient and rapid, the megakaryocytes and platelets can be obtained through iPSC in a short time, and feeder cells, serum and additional gene editing are not needed; meanwhile, the yield is high.

Description

Technical Field

[0001] The present invention relates to the technical field of directed differentiation of induced pluripotent stem cells, and specifically provides a method for differentiating induced pluripotent stem cells into megakaryocytes and platelets. Background Art

[0002] As a key cellular component for maintaining hemostasis and vascular integrity, platelets are widely used in clinical practice for treating thrombocytopenia, postoperative bleeding, and blood system diseases. Currently, platelet supply completely relies on voluntary blood donation, which has problems such as supply-demand imbalance, short storage period, and risk of pathogen contamination. Especially in public health emergencies, it is difficult to ensure a stable supply. Based on this, developing an in vitro platelet preparation technology that is independent of blood donation and is safe and controllable has become an important research direction.

[0003] Induced pluripotent stem cells (iPSCs) provide a new path for large-scale in vitro production of functional platelets due to their self-renewal and multi-directional differentiation potential. Existing technologies induce the differentiation of iPSCs into megakaryocytes (MKs) through gene editing means and further release platelets. In theory, it can break through the limitation of blood donation dependence and achieve personalized treatment by combining gene editing technology. However, there are still the following key bottlenecks in this field: 1. Carcinogenic risk restricts clinical application: Currently, efficient differentiation systems generally rely on the overexpression of oncogenes (such as c-MYC) to construct immortalized megakaryocyte lines to increase platelet production. Such gene operations may lead to genomic instability, resulting in the presence of undifferentiated iPSCs or abnormal cell clones in the final differentiation products, posing a tumorigenic risk. In addition, if undifferentiated iPSCs are not completely removed in the in vitro culture system, teratomas may form after transplantation, seriously threatening the safety of treatment.

[0004] 2. Problems of safety and standardization in the introduction of exogenous substances: Existing differentiation systems mostly rely on mouse embryonic fibroblasts (Feeder cells) or fetal bovine serum (FBS) to provide growth support. Such exogenous components are prone to carrying pathogens (such as viruses, prion proteins), and xenogeneic proteins may cause immune rejection reactions. At the same time, batch-to-batch differences in exogenous substances make it difficult to standardize the process, and it is difficult to meet the requirements of clinical-grade cell products for clear composition and reproducible production.

[0005] 3. Process complexity and high production cost: The directed differentiation of iPSCs into platelets requires precise multi-stage regulation (such as iPSCs amplification, mesoderm induction, megakaryocyte maturation, and platelet release). Each stage requires strict optimization of cytokine combinations (such as TPO, SCF, FGF, etc.), oxygen concentration, and mechanical stimulation parameters. The operation is cumbersome and the cycle is as long as several weeks. In addition, the use of high-purity recombinant growth factors and special bioreactors significantly increases the cost, limiting the industrial application of the technology.

[0006] In summary, the existing iPSC-based platelet preparation technology is still limited by safety, standardization, and economic deficiencies. There is an urgent need to develop a new differentiation system that is exogenous-independent, cancer gene-free, and has a simplified process to achieve the safe and efficient production of clinical-grade platelet products. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, which does not rely on Feeder cells, serum, and additional gene editing to achieve the safe and efficient production of clinical-grade platelet products.

[0008] To achieve the above objectives, the present invention is realized through the following technical solutions: A method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, comprising the following steps: S1. Inoculate iPSC single cells into a culture vessel and perform adherent culture with a culture medium; S2. After the cells adhere and before clones are formed, change the medium to a first induction medium to induce the cells to differentiate into mesoderm cells; S3. Change the medium to a second induction medium to induce the mesoderm cells to differentiate into hematopoietic endothelial cells; S4. Collect the suspended cells and culture them in an amplification medium to enrich megakaryocytes; S5. Induce megakaryocyte polyploidization through a third induction medium; S6. Culture the polyploid megakaryocytes through a fourth induction medium to promote platelet release.

[0009] Further, step S1 includes: taking iPSC clones cultured in mTeSR1, enzymatically digesting them into single cells using Accutase, inoculating the single cells onto a culture plate or culture dish coated with GFR Matrigel, cell density: 35,000-40,000 cells / ml; adding a culture medium for adherent culture.

[0010] Further, the culture medium is mTeSR1 medium containing 4-6 μM / L of Y-27632.

[0011] Further, the first induction medium is custom tesr (05896) medium containing 35-45 ng / mL of BMP4, 40-60 ng / mL of Activin A, and 80-120 nM / L of Resminostat.

[0012] Further, the second induction medium is custom tesr (05896) medium containing 35-45 ng / mL of VEGF, 40-60 ng / mL of bFGF, and 1 μM / L of RepSox.

[0013] Furthermore, the amplification culture medium is Stem Span SFEMII medium containing 40 - 60 ng / mL of TPO, 17 - 23 ng / mL of SCF, 13 - 17 ng / mL of IL3, 13 - 17 ng / mL of IL6, and 17 - 23 ng / mL of IL11.

[0014] Furthermore, the third induction culture medium is StemSpan SFEMII medium containing 40 - 60 ng / mL of SCF, 180 - 220 ng / mL of TA316, 4 - 6 μM / L of myosin II inhibitor, 20 - 30 μM / L of Q-VD-Oph, 2 - 3 μM / L of harmine, and 20 - 30 ng / mL of CCL5.

[0015] Furthermore, the fourth induction culture medium is StemSpan SFEMII medium containing 40 - 60 ng / mL of SCF, 180 - 220 ng / mL of TA316, 13 - 17 μM / L of GM6001, 0.5 - 1 μM / L of SR1, 8 - 12 μM / L of Y-27632, 0.5 - 1.5 U of anticoagulant, and 0.8 - 1.2% of chemically defined lipid concentrate.

[0016] Furthermore, step S4 includes: controlling the suspension cell density of the amplification culture medium to be 230 - 270 K / mL.

[0017] Advantages Compared with the prior art, the present invention provides a method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, having the following advantages: 1. The method for differentiating induced pluripotent stem cells into megakaryocytes and platelets is convenient and fast, and megakaryocytes and platelets can be obtained from iPSCs in a relatively short time.

[0018] 2. The method for differentiating induced pluripotent stem cells into megakaryocytes and platelets can generate about 200 functional platelets from a single iPSC, with a high conversion efficiency, providing a feasible basis for clinical-scale mass production.

[0019] 3. The method for differentiating induced pluripotent stem cells into megakaryocytes and platelets does not require exogenous Feeder cells, serum, or additional gene editing, ensuring the safety of the product. Brief Description of the Drawings

[0020] Figure 1 It is the morphological diagram after passage of the present invention; Figure 2 It is the morphological diagram of mesoderm cells of the present invention; Figure 3Morphological diagram of hematopoietic endothelium of the present invention; Figure 4 Morphological diagram of megakaryocytes of the present invention (left) and flow cytometry detection of megakaryocytes (right); Figure 5 Morphology of megakaryocytes and platelets on day 15 of the present invention (left) and flow cytometry detection of platelets (right). Detailed implementation manners

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0022] Embodiment 1: A method for differentiating induced pluripotent stem cells into megakaryocytes and platelets: Subculture of iPSC single cells: Use the Accutase enzymatic digestion system to digest the iPSC clones cultured in mTeSR1 into single cells, and inoculate the single cells into a culture dish coated with GFR Matrigel. Cell density: 35,000 - 40,000 cells / ml; add culture medium for adherent culture. The culture medium is mTeSR1 medium containing 5 μM / L of Y-27632.

[0023] Specifically, during plating, the operation is carried out in a cold room to prevent GFR Matrigel from solidifying during the plating process. GFR Matrigel plating concentration: The ratio of GFR Matrigel to the buffer solution is 1:100, and the buffer solution uses DMEM; Plating volume: For a culture dish with a diameter of 10 cm, use 7 mL of GFR Matrigel dilution solution for plating. Before inoculating the cells, remove the residual GFR Matrigel dilution solution in the culture dish without damaging the formed gel layer. Then add the culture medium containing iPSC single cells to the culture dish for cell culture.

[0024] Mesoderm-directed induction: Day0: Before the cells adhere but do not form clones, change the medium to the first induction medium. The first induction medium is custom tesr(05896) medium containing 40 ng / mL of BMP4, 50 ng / mL of ActivinA, and 100 nM / L of Resminostat.

[0025] Among them, custom tesr (05896) medium is a medium produced by Stemcell Technologies, and its official catalog number is 05896.

[0026] Hematopoietic endothelium induction: Day2: At exactly 48 hours after the medium change on day0, change the medium to the second induction medium.

[0027] Day3 - 6: Change the medium every day to ensure that the second induction medium does not turn yellow.

[0028] Among them: At Day4, the cells are confluent, completely covering the bottom of the culture dish, forming an endothelium. The second induction medium is a custom tesr (05896) medium containing 40 ng / mL of VEGF, 50 ng / mL of bFGF, and 1 uM / L of RepSox.

[0029] Suspension cell sorting and megakaryocyte precursor amplification: Day6 - 8: Collect the suspension cells by centrifugation at 300 g for 8 minutes every day. The original adherent cells continue to be cultured with the second induction medium and the medium is changed every day. The collected suspension cells are cultured in suspension with the amplification medium.

[0030] During suspension culture, the volume of the amplification medium is dynamically supplemented to adjust the cell density, and the cell density in the amplification medium is controlled at 250K / mL. When collecting cells, use a pipette to blow the endothelium to make the blood cells fall off. The amplification medium is Stem Span SFEMII medium containing 50 ng / mL of TPO, 20 ng / mL of SCF, 15 ng / mL of IL3, 15 ng / mL of IL6, and 20 ng / mL of IL11.

[0031] Megakaryocyte maturation and polyploidization induction: Day9 - 12: Collect the suspension cells in the amplification medium by centrifugation at 300 g for 8 minutes, culture them with the third induction medium, and adjust the cell density to 250K / mL. Do not change the medium during this period, and shake the culture dish every half day to prevent cell aggregation.

[0032] Among them, the third induction medium is StemSpan SFEMII medium containing 50 ng / mL of SCF, 200 ng / mL of TA316, 5 μM / L of myosin II inhibitor, 25 μM / L of Q - VD - Oph, 2.5 μM / L of harmine, and 25 ng / mL of CCL5. The myosin II inhibitor is selected from diMF or Blebbistatin.

[0033] Platelet production and release: Day12 - 15: Collect the polyploid megakaryocytes by centrifugation at 250 g for 10 minutes, and culture them in suspension with the fourth induction medium at a volume ratio of 1:2. Do not change the medium during this period, and shake the culture dish every half day to prevent cell aggregation.

[0034] Among them, the fourth induction culture medium is StemSpan SFEMII medium containing 50 ng / mL of SCF, 200 ng / mL of TA316, 15 μM / L of GM6001, 10.75 μM / L of SR10, 10 μM / L of Y-27632, 1 U of enoxaparin, and 1% of chemically defined lipid concentrate.

[0035] Example 2: A method for differentiating induced pluripotent stem cells into megakaryocytes and platelets: iPSC single-cell passage: Use the Accutase enzymatic digestion system to digest the iPSC clones cultured in mTeSR1 into single cells, and inoculate the single cells into a culture dish coated with GFR Matrigel. Cell density: 35,000 - 40,000 cells / mL; add the culture medium for adherent culture. The culture medium is mTeSR1 medium containing 4 μM / L of Y-27632.

[0036] Specifically, when plating, operate in a cold room to prevent GFR Matrigel from solidifying during the plating process. GFR Matrigel plating concentration: The ratio of GFR Matrigel to the buffer solution is 1:100, and the buffer solution uses DMEM; Plating volume: For a culture dish with a diameter of 10 cm, use 7 mL of GFR Matrigel dilution for plating. Before inoculating the cells, remove the residual GFR Matrigel dilution in the culture dish without damaging the formed gel layer. Then add the culture medium containing iPSC single cells to the culture dish for cell culture.

[0037] Mesoderm-directed induction: Day0: Before the cells adhere but before clones are formed, change the medium to the first induction culture medium. The first induction culture medium is custom tesr (05896) medium containing 35 ng / mL of BMP4, 40 ng / mL of ActivinA, and 80 nM / L of Resminostat.

[0038] Hematopoietic endothelial induction: Day2: At exactly 48 hours after changing the medium on day0, change the medium to the second induction culture medium.

[0039] Day3 - 6: Change the medium every day to ensure that the second induction culture medium does not turn yellow.

[0040] Among them: On Day4, the cells are connected in sheets, completely covering the bottom of the culture dish, forming an endothelial layer. The second induction culture medium is custom tesr (05896) medium containing 35 ng / mL of VEGF, 40 ng / mL of bFGF, and 0.8 μM / L of RepSox.

[0041] Suspension cell sorting and megakaryocyte precursor amplification: Day6 - 8: Collect suspension cells by centrifugation at 300g for 8 minutes every day. The original adherent cells continue to be cultured with the second induction culture medium and the medium is changed every day. The collected suspension cells are cultured in suspension with the amplification culture medium.

[0042] During suspension culture, the volume of the amplification medium is dynamically supplemented to adjust the cell density, and the density of suspension cells in the amplification medium is controlled at 230K / mL. When collecting cells, use a pipette to blow the endothelium to make the blood cells fall off. The amplification culture medium is Stem Span SFEMII medium containing TPO 40ng / mL, SCF 17ng / mL, IL3 13ng / mL, IL6 13ng / mL, and IL11 17ng / mL.

[0043] Megakaryocyte maturation and polyploidization induction: Day9 - 12: Collect suspension cells in the amplification culture medium by centrifugation at 300g for 8 minutes, culture them with the third induction culture medium, and adjust the cell density to 250K / mL. Do not change the medium during this period, and shake the culture dish every half day to prevent cell aggregation.

[0044] Among them, the third induction culture medium is StemSpan SFEMII medium containing SCF 40ng / mL, TA316 180ng / mL, myosin II inhibitor 4μM / L, Q - VD - Oph 20μM / L, harmine 2μM / L, and CCL5 20ng / mL. The myosin II inhibitor is selected from diMF or Blebbistatin.

[0045] Platelet production and release: Day12 - 15: Collect polyploid megakaryocytes by centrifugation at 250g for 10 minutes, and culture them in suspension with the fourth induction culture medium at a volume ratio of 1:2. Do not change the medium during this period, and shake the culture dish every half day to prevent cell aggregation.

[0046] Among them, the fourth induction culture medium is StemSpan SFEMII medium containing SCF 40ng / mL, TA316 180ng / mL, GM6001 13μM / L, SR10.5μM / L, Y - 27632 8μM / L, enoxaparin 0.5U, and chemically defined lipid concentrate 0.8%.

[0047] Example 3: A method for differentiating induced pluripotent stem cells into megakaryocytes and platelets: iPSC single - cell passage: Use the Accutase enzymatic digestion system to digest iPSC clones cultured in mTeSR1 into single cells. Seed the single cells into a culture plate coated with GFR Matrigel at a cell density of 35,000 - 40,000 cells / ml. Add the culture medium for adherent culture. The culture medium is mTeSR1 medium containing 5 μM / L of Y-27632.

[0048] Specifically, when plating, perform the operation in a cold room to prevent GFR Matrigel from solidifying during the plating process. The plating concentration of GFR Matrigel: The ratio of GFR Matrigel to the buffer solution is 1:100, and the buffer solution uses DMEM. The plating volume: For one well of a 12-well plate, use 700 μL of GFR Matrigel dilution solution. Before seeding the cells, remove the residual GFR Matrigel dilution solution in the culture dish without damaging the formed gel layer. Subsequently, add the culture medium containing iPSC single cells to the culture dish for cell culture.

[0049] Mesoderm-directed induction: Day0: Before the cells adhere but before clones are formed, change the medium to the first induction medium. The first induction medium is custom tesr (05896) medium containing 45 ng / mL of BMP4, 60 ng / mL of ActivinA, and 120 nM / L of Resminostat.

[0050] Hematopoietic endothelial induction: Day2: At exactly 48 hours after changing the medium on day0, change the medium to the second induction medium.

[0051] Day3 - 6: Change the medium every day to ensure that the second induction medium does not turn yellow.

[0052] Among them: On Day4, the cells are confluent, completely covering the bottom of the culture dish, forming an endothelial layer. The second induction medium is custom tesr (05896) medium containing 45 ng / mL of VEGF, 60 ng / mL of bFGF, and 1.2 μM / L of RepSox.

[0053] Suspension cell sorting and megakaryocyte precursor expansion: Day6 - 8: Collect the suspension cells by centrifugation at 300 g for 8 minutes every day. The original adherent cells continue to be cultured with the second induction medium and the medium is changed every day. The collected suspension cells are suspended and cultured with the expansion medium.

[0054] During suspension culture, the volume of the amplification medium was dynamically supplemented to adjust the cell density, and the density of suspended cells in the amplification medium was controlled at 270K / mL. When collecting cells, a pipette was used to blow and beat the endothelium to make the blood cells fall off. The amplification culture medium was Stem Span SFEMII medium containing 60 ng / mL of TPO, 23 ng / mL of SCF, 17 ng / mL of IL3, 17 ng / mL of IL6, and 23 ng / mL of IL11.

[0055] Megakaryocyte maturation and polyploidization induction: Day9 - 12: The suspended cells in the amplification culture medium were collected by centrifugation at 300g for 8 minutes, cultured with the third induction culture medium, and the cell density was adjusted to 250K / mL. During this period, the culture medium was not changed, and the culture dish was shaken every half day to prevent cell aggregation.

[0056] Among them, the third induction culture medium was StemSpan SFEMII medium containing 60 ng / mL of SCF, 200 ng / mL of TA316, 6 μM / L of myosin II inhibitor, 30 μM / L of Q - VD - Oph, 3 μM / L of harmine, and 30 ng / mL of CCL5. The myosin II inhibitor was selected from diMF or Blebbistatin.

[0057] Platelet production and release: Day12 - 15: The polyploid megakaryocytes were collected by centrifugation at 250g for 10 minutes and suspended and cultured in the fourth induction culture medium at a volume ratio of 1:2. During this period, the culture medium was not changed, and the culture dish was shaken every half day to prevent cell aggregation.

[0058] Among them, the fourth induction culture medium was StemSpan SFEMII medium containing 60 ng / mL of SCF, 220 ng / mL of TA316, 17 μM / L of GM6001, 11 μM / L of SR, 12 μM / L of Y - 27632, 1.5 U of enoxaparin, and 1.2% of chemically defined lipid concentrate.

[0059] The differentiation system of the present invention significantly improves the conversion efficiency from iPSCs to platelets by optimizing the multi - stage induction strategy. Through quantitative detection, each single iPSC can be directed to differentiate into at least 18 hematopoietic stem cell / progenitor cells (HSPCs); in the stage of megakaryocyte directed differentiation, each HSPC can further generate 1.2 mature megakaryocytes (MKs); each mature MK releases at least 10 functional platelets (PLTs) through cytoplasmic fragmentation. Calculated comprehensively, a single iPSC can ultimately produce approximately 200 platelets.

[0060] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, characterized in that: The following steps are involved: S1. Inoculate iPSC single cells into culture vessels and culture them in culture medium for adherence; S2. After the cells adhere and before clones are formed, replace the medium with the first induction medium to induce the cells to differentiate into mesoderm cells; S3. Replace the medium with the second induction medium to induce the mesoderm cells to differentiate into hematopoietic endothelial cells; S4. Collect suspended cells and culture them in expansion medium to enrich megakaryocytes; S5. Induce polyploidy of megakaryocytes using the third induction medium; S6. Culture polyploidized megakaryocytes using the fourth induction medium to promote platelet release.

2. The method of claim 1, wherein: Step S1 includes: taking the iPSC clones cultured in mTeSR1, enzymatically treating them with Accutase to obtain single cells, inoculating the single cells into a culture plate or culture dish coated with GFR Matrigel at a cell density of 35,000 to 40,000 cells / ml; and adding culture medium for adherent culture.

3. The method of claim 1, wherein: The culture medium is mTeSR1 culture medium containing 4-6 μM / L of Y-27632.

4. The method of claim 1, wherein: The first induction culture medium is a custom tesr (05896) culture medium containing BMP4 35-45 ng / mL, ActivinA 40-60 ng / mL, and Resminostat 80-120 nM / L.

5. The method of claim 1, wherein: The second induction culture medium is a custom tesr (05896) culture medium containing VEGF 35-45 ng / mL, bFGF 40-60 ng / mL, and RepSox 1 μM / L.

6. The method of claim 1, wherein: The expansion culture medium is a Stem Span SFEMII culture medium containing TPO 40-60 ng / mL, SCF 17-23 ng / mL, IL3 13-17 ng / mL, IL6 13-17 ng / mL, and IL11 17-23 ng / mL.

7. The method of claim 1 for differentiating induced pluripotent stem cells into megakaryocytes and platelets, wherein: The third induction culture medium is StemSpan SFEMII medium containing SCF 40-60ng / mL, TA316 180-220ng / mL, myosin II inhibitor 4-6μM / L, Q-VD-Oph 20-30μM / L, harmine 2-3μM / L, and CCL5 20-30ng / mL.

8. The method of claim 1 for differentiating induced pluripotent stem cells into megakaryocytes and platelets, characterized in that: The fourth induction culture medium is a StemSpan SFEMII medium containing SCF 40-60 ng / mL, TA316 180-220 ng / mL, GM600113-17 μM / L, SR1 0.5-1 μM / L, Y-27632 8-12 μM / L, anticoagulant 0.5-1.5 U, and chemically defined lipid concentrate 0.8-1.2%.

9. The method of claim 1 for differentiating induced pluripotent stem cells into megakaryocytes and platelets, wherein: Step S4 includes: controlling the cell suspension density in the amplification culture medium to be 230-270K / mL.

Images