Method for producing platelets

Abstract

The present invention relates to a method for producing platelets. The method of the present invention comprises the following steps: (S1) culturing pluripotent stem cells to obtain a culture containing hematopoietic stem cells; (S2) obtaining a population of suspended cells from the culture when cells in the culture that do not express CD41a make up at least 15% of the total cells; and (S3) differentiating the suspension cell population into megakaryocytes, whereby the method enables the production of high quality platelets in high yield.

Description

Technical Field

[0001] The present invention relates to a method for producing platelets. This specification was prepared with the support of the following national research project.

[0002] [Table 1]

[0003] Background Art

[0004] Platelet preparations are administered to patients suffering from major bleeding during surgery or injury, or bleeding associated with thrombocytopenia following chemotherapy, for the purpose of treating and preventing these conditions.

[0005] Currently, the production of platelet preparations relies on blood donations from healthy volunteers. Recently, the number of blood donors has decreased, and blood shortages are expected in the future. Therefore, a stable supply of platelets is an important issue in this technical field.

[0006] Traditional platelet preparations have a high risk of bacterial contamination, which may lead to serious infections after platelet transfusion. Therefore, there is a continuous need for safer platelet preparations in clinical settings.

[0007] To meet this need, methods for producing platelets from in vitro cultured megakaryocytes have recently been developed. However, platelets have not yet been obtained in high yields, and the process is not economically viable, requiring technical improvements. Summary of the invention

[0008] Problem that the invention aims to solve

[0009] It is an object of the present invention to provide a method for producing artificial platelets in high yield.

[0010] Another object of the present invention is to provide a method for producing platelets with high process efficiency.

[0011] Solutions for solving problems

[0012] 1. A method for producing platelets, comprising: (S1) culturing pluripotent stem cells to obtain a culture containing hematopoietic stem cells; (S2) when CD41a is expressed in the cells contained in the culture; - When the number of the cells is at least 15% of the total number of cells, obtaining a suspension cell population from the culture; and (S3) differentiating the suspension cell population into megakaryocytes in a first culture medium.

[0013] 2. The method for producing platelets according to 1 above, further comprising (S4) culturing megakaryocytes in a second culture medium containing thrombopoietin and maturing the megakaryocytes.

[0014] 3. A method for producing platelets according to 1 above, wherein (S1) includes the following steps: (S1a) culturing pluripotent stem cells in a third culture medium containing a GSK3 inhibitor; (S1b) culturing the cells cultured in the third culture medium in a fourth culture medium containing vascular endothelial growth factor and basic fibroblast growth factor; and (S1c) culturing the cells cultured in the fourth culture medium in a fifth culture medium containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor-β signaling inhibitor.

[0015] 4. The method for producing platelets according to 1 above, wherein the suspended cell population has CD45 or less of 50% of the total cell count. + Cell counting.

[0016] 5. The method for producing platelets according to 1 above, wherein the suspended cell population has CD34 of 45% or more of the total cell count. + Cell counting.

[0017] 6. The method for producing platelets according to 1 above, wherein the suspended cell population has CD41a of 85% or less of the total cell count. - Cell counting.

[0018] 7. The method for producing platelets according to 1 above, further comprising (S0) culturing the platelets at a density of 2,000 to 20,000 cells / cm 2 Pluripotent stem cells are seeded on the bottom of the culture container.

[0019] 8. The method for producing platelets according to 1 above, wherein the culture further contains hematopoietic progenitor cells and megakaryocyte progenitor cells.

[0020] 9. The method for producing platelets according to 1 above, wherein the first culture medium contains thrombopoietin, stem cell factor, interleukin-3, and interleukin-6.

[0021] 10. The method for producing platelets according to 1 above, wherein, in (S2), when CD41a + When the number of cells is 40 to 85% of the total number of cells, a suspension cell population is obtained from the culture.

[0022] 11. The method for producing platelets according to 1 above, wherein in (S2), when CD34 + When the number of cells is 45 to 80% of the total number of cells, a suspension cell population is obtained from the culture.

[0023] 12. The method for producing platelets according to 1 above, wherein in (S2), when CD45 + When the number of cells is 1 to 50% of the total number of cells, a suspension cell population is obtained from the culture.

[0024] 13. The method for producing platelets according to 1 above, wherein the pluripotent stem cells are human induced pluripotent stem cells.

[0025] 14. The method for producing platelets according to 4 above, wherein the CD45 + The proportion of cells was lower than CD41a + The proportion of cells.

[0026] 15. A method for producing a blood product, comprising the step of mixing the platelets produced by the method of any one of 1 to 14 above with other blood components.

[0027] Effects of the Invention

[0028] The method for producing platelets of the present invention does not require CD41a + Isolation and purification of megakaryocytes, since megakaryocytes are isolated from cells containing at least 15% CD41a - Production of cell suspension populations.

[0029] The method for producing platelets of the present invention enables rapid and economical platelet production because subsequent processes can be performed when the suspended cell population meets certain requirements.

[0030] The method for producing platelets of the present invention has excellent differentiation efficiency into megakaryocytes and platelet production efficiency.

[0031] The method for producing platelets of the present invention is simple, does not require complicated equipment, and is advantageous in terms of time and cost.

[0032] The platelets produced according to the method of the present invention can be activated by stimulants (eg, ADP (adenosine diphosphate), collagen, fibrinogen, etc.) to increase PAC-1 and CD62p expression. This confirms that the platelets of the present invention are platelets with normal functions.

[0033] The platelets produced according to the method of the present invention can show a therapeutic or preventive effect on diseases associated with thrombocytopenia or dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention is an embodiment of a process for culturing human induced pluripotent stem cells.

[0035] Figure 2 These are the results of confirming the surface markers (CD34, CD45, CD41a) of the suspension cell populations of Preparation Examples 1-1 to 1-4 and Comparative Preparation Example 1-1.

[0036] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the suspension cell populations of Preparation Examples 1-1 to 1-3 and Comparative Preparation Example 1-1.

[0037] Figure 4 This is the process used to obtain the cell cultures of Examples 1 and 2.

[0038] Figure 5 This is a photograph of the cell culture of Example 1.

[0039] Figure 6 This is the process for obtaining the cell culture of Comparative Example 1.

[0040] Figure 7 This is a photograph of the cell culture of Comparative Example 1.

[0041] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers.

[0042] FIG. 9 shows that the cells of Example 2 express megakaryocyte-specific markers.

[0043] FIG. 10 shows that the cells of Comparative Example 1 do not express megakaryocyte-specific markers.

[0044] FIG. 11 shows that the cells of Example 1 were activated by ADP treatment, and the expression of PAC-1 and CD62p increased.

[0045] Fig.12 It was shown that the cells of Example 2 were activated by ADP treatment, and the expression of PAC-1 and CD62p increased.

[0046] Fig.13 In the suspension cell population of Preparation Example 1-2, CD41a was selected + Cell results.

[0047] Fig.14 CD41a in Example 1 (Preparation Example 1-2) and Comparative Example 2 + / CD42b + proportion.

[0048] Fig.15 It was shown that Example 1 exhibited a cell proliferation ability about 40 times higher than that of Comparative Example 2.

[0049] Fig.16These are the results of confirming the surface markers (CD34, CD45, CD41a) of the suspension cell populations of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2.

[0050] Figures 17 to 22 show that Examples 3 to 8 express megakaryocyte- and platelet-specific markers.

[0051] FIG. 23 and FIG. 24 show that Comparative Examples 3 and 4 do not express megakaryocyte- and platelet-specific markers. DETAILED DESCRIPTION

[0052] The present invention provides a method for producing platelets, comprising the following steps: (S1) culturing pluripotent stem cells to obtain a culture containing hematopoietic stem cells; (S2) when the number of cells that do not express CD41a among the cells contained in the culture is at least 15% of the total number of cells, obtaining a suspension cell population from the culture; and (S3) differentiating the suspension cell population into megakaryocytes in a first culture medium.

[0053] The production method of the present invention may further include (S0) culturing the cells at a density of 2,000 to 20,000 cells / cm 2 Pluripotent stem cells are seeded on the bottom of the culture container.

[0054] The production method of the present invention may further include (S4) culturing megakaryocytes in a second culture medium containing thrombopoietin and maturing the megakaryocytes.

[0055] The production method of the present invention may further include (S5) obtaining platelets from the culture obtained in (S4).

[0056] Hereinafter, the platelet production process from (S0) to (S5) will be described.

[0057] (S0) Step

[0058] (S0) is 2,000 to 20,000 cells / cm 2 The step of seeding the bottom of a culture container with pluripotent stem cells.

[0059] Pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0060] Induced pluripotent stem cells (iPSCs) are cells that do not have pluripotency but have acquired pluripotency through an artificial reprogramming process.

[0061] Induced pluripotent stem cells (iPSCs) can be derived from an individual selected from the group consisting of humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), canines (domestic dogs and wild dogs), felines (domestic cats and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (dolphins, whales, etc.).

[0062] The induced pluripotent stem cells may be human induced pluripotent stem cells (hiPSC).

[0063] Induced pluripotent stem cells can be generated using mouse and / or human cells. For example, induced pluripotent stem cells can be generated using embryonic tissue, fetal tissue, neonatal tissue, and adult tissue.

[0064] Induced pluripotent stem cells can be derived from any somatic cell at any stage of development as a starting point. Somatic cells can be, but are not limited to, derived from embryonic donors, fetal donors, neonatal donors, adolescent donors, or adult donors. Somatic cells can be, but are not limited to, fibroblasts, such as skin fibroblasts obtained by skin samples or biopsies, synoviocytes from synovial tissue, buccal cells, or lung fibroblasts.

[0065] The pluripotent stem cells are attached and cultured after being seeded on the bottom of a culture container.

[0066] The culture vessel can be of any type without limitation, as long as it is used for cell culture in the art. For example, it can be a large, medium or small culture vessel. It can also be a cell culture flask such as T25, T75, T175, or T225.

[0067] 2,000 to 20,000 cells / cm 2 Inoculation of pluripotent stem cells is preferred in terms of cell confluency. When inoculated in this amount, the cell confluency can be 70% to 95%, 75% to 95%, 80% to 95%, or 85% to 95%, and the pluripotent stem cells are appropriately cultured and differentiated so that a culture containing a sufficient amount of hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte progenitor cells, etc. can be obtained in the (S1) step described later.

[0068] If pluripotent stem cells are grown at a density of less than 2,000 cells / cm 2Inoculated at the bottom of the culture container, the cell confluence when obtaining the suspended cell population (S2) can be 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, etc. In addition, if the pluripotent stem cells are grown at a density of more than 20,000 cells / cm 2 If seeded on the bottom of the culture vessel, the cell confluence may exceed 100% when the pluripotent stem cells have not been fully cultured and differentiated, and undifferentiated cells may fall off the culture dish and become floating and die, or the amount of factors added to the culture may be insufficient, resulting in poor signaling between cells and poor differentiation.

[0069] (S1) Step

[0070] (S1) is a step of culturing pluripotent stem cells to obtain a culture containing hematopoietic stem cells. This step is a step of culturing the pluripotent stem cells seeded in step (S0) to obtain a suspension cell population that can differentiate into megakaryocytes.

[0071] The (S1) step can be configured to include the following sub-steps:

[0072] (S1a) culturing pluripotent stem cells in a third culture medium containing a GSK3 (glycogen synthase kinase 3) inhibitor;

[0073] (S1b) culturing the cells cultured in the third culture medium in a fourth culture medium containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF); and

[0074] (S1c) The cells cultured in the fourth culture medium are cultured in a fifth culture medium containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor-β signaling inhibitor.

[0075] The third culture medium, the fourth culture medium, and the fifth culture medium used in this step are culture media having different compositions and purposes.

[0076] The third culture medium contains at least a GSK3 inhibitor. The GSK3 inhibitor can be, but is not limited to, CHIR99021, BIO (6-bromoindirubin-30-oxime), SB216763, CHIR-98014, CT98014, CT98023, CT99021, TWS119, SB41528, AR-A014418, AZD-1080, Alsterpaullone, Cazpaullone, or Kenpaullone. CHIR99021 is a GSK3 inhibitor (a Wnt signaling enhancer) and can be expressed as an aminopyrimidine.

[0077] GSK3 inhibitors (e.g., CHIR99021) are not limited to a specific concentration, as long as it is an amount capable of culturing pluripotent stem cells. GSK3 inhibitors can be included in the third culture medium at a concentration of, for example, 2 to 10 μM, 2.5 to 9.5 μM, 3 to 9 μM, 3.5 to 8.5 μM, 4 to 8 μM, 4.5 to 7.5 μM, 5 to 7 μM, 5.5 to 6.5 μM, or 6 μM.

[0078] The third culture medium is a basal culture medium. The third culture medium may include RPMI1640 culture medium or another type of basal culture medium. In addition to the basal culture medium, the third culture medium may further include an antioxidant and / or B-27.

[0079] The antioxidant can be selected from the group consisting of: 6-hydroxymelatonin, acetyl-L-carnitine (ALCAR), alpha-lipoic acid (ALA), ascorbic acid (e.g., AA2P (L-ascorbic acid 2-phosphate), AA2G (L-ascorbic acid 2-glucoside)), carotenoids (vitamin A), curcumin, edaravone, polyphenols, glutathione, hydroxytyrosol, L-carnitine, ladostingilvil, melatonin, mofeglin, N-acetylcysteine ​​(NAC), N-acetylserotonin (NAS), oleocanthal, oleuropein, rasagiline, resveratrol, selegiline, selenium, tocopherol (vitamin E), tocotrienols, tyrosol, ubiquinone (coenzyme Q), and uric acid.

[0080] The fourth culture medium is a growth medium. The fourth culture medium contains at least growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The fourth culture medium may further contain other growth factors to culture pluripotent stem cells.

[0081] Vascular endothelial growth factor (VEGF) is a member of the epidermal growth factor receptor (EGFR / ErbB). Vascular endothelial growth factor plays a crucial role in regulating cell proliferation and differentiation, and leads to activation of various forms of signal transduction pathways to induce apoptosis, survival, or cell proliferation. Vascular endothelial growth factor includes, for example, human and non-human animal (e.g., mouse) vascular endothelial growth factor.

[0082] The vascular endothelial growth factor is included in a concentration that can appropriately culture pluripotent stem cells. The vascular endothelial growth factor can be included in the fourth culture medium at a concentration of, for example, 5 to 95 ng / ml, 10 to 90 ng / ml, 15 to 85 ng / ml, 20 to 80 ng / ml, 25 to 75 ng / ml, 30 to 70 ng / ml, 35 to 65 ng / ml, 40 to 60 ng / ml, 45 to 55 ng / ml, or 50 ng / ml.

[0083] Basic fibroblast growth factor (bFGF) is a protein belonging to the FGF family, which functions as a mitogenic factor, angiogenic factor, bone formation factor, and nerve growth factor in cell proliferation and cell differentiation, etc. Basic fibroblast growth factor (also called FGF2) activates receptor proteins including FGFR1b, FGFR1c, FGFR2c, FGFR3c, and FGFR4c, and particularly strongly activates FGFR1c and FGFR3c.

[0084] Basic fibroblast growth factor is included with a concentration that can appropriately culture pluripotent stem cells together with other components. Basic fibroblast growth factor can be included in the fourth culture medium at a concentration of, for example, 1 to 40 ng / ml, 5 to 35 ng / ml, 10 to 30 ng / ml, 15 to 25 ng / ml, or 20 ng / ml.

[0085] The fourth culture medium may not contain a transforming growth factor β (TGFβ) signaling inhibitor. If the fourth culture medium contains a TGFβ signaling inhibitor, differentiation may occur without sufficient cell proliferation.

[0086] The fifth culture medium is a growth medium. Like the fourth culture medium, it contains at least growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The fifth culture medium may further contain other growth factors to culture pluripotent stem cells.

[0087] Matters concerning vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the fifth culture medium are the same as those for vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the fourth culture medium.

[0088] The fifth culture medium contains a transforming growth factor β (TGFβ) signaling inhibitor.

[0089] Transforming growth factor β (TGFβ) signaling inhibitor refers to a substance that inhibits TGFβ signaling. TGFβ is a substance that regulates various physiological processes in the body such as cell proliferation, differentiation, apoptosis, migration, production of extracellular matrix (ECM), angiogenesis, and development.

[0090] The TGFβ signaling inhibitor may be of any type without limitation as long as it is a substance capable of inhibiting TGFβ signaling, and may be, for example, an activin receptor-like kinase (ALK) receptor inhibitor.

[0091] The activin receptor-like kinase receptor inhibitor can be, but is not limited to, an ALK5, ALK4, and ALK7 receptor inhibitor. For example, the ALK receptor inhibitor can be SB431542. SB431542 can be represented by the following compound name: 4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide.

[0092] The transforming growth factor β (TGFβ) signaling inhibitor is included at a concentration capable of culturing pluripotent stem cells together with other components. The transforming growth factor β (TGFβ) signaling inhibitor can be included at a concentration of, for example, 1 to 20 μM, 5 to 15 μM, or 10 μM.

[0093] The culture obtained by culturing in step (S1) contains hematopoietic stem cells (HSCs). The culture may further contain hematopoietic progenitor cells (HPCs) and megakaryocyte progenitor cells (MK-Ps).

[0094] (S2) Step

[0095] (S2) is when the culture contains cells that do not express CD41a (CD41a - The step of obtaining a suspension cell population from the culture of (S1) when the number of the suspension cells) is at least 15% of the total number of cells.

[0096] A suspension cell population refers to cells and / or cell populations suspended in culture. The suspension cell population contains a large number of cells that can differentiate into megakaryocytes, such as hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte progenitor cells, and may contain cells that do not directly differentiate into megakaryocytes but help hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte progenitor cells differentiate better than when they exist alone.

[0097] This step determines the optimal time to obtain the suspension cell population from the culture of (S1). By this step, the optimal suspension cell population for differentiation into megakaryocytes is obtained, and the differentiation efficiency into megakaryocytes and / or mature megakaryocytes and the platelet production efficiency can be improved.

[0098] When CD41a - When the number of cells is at least 15% of the total number of cells, a suspension cell population is obtained. + If the number of CD41a + If the number of cells is less than 85%, the differentiation efficiency into megakaryocytes and platelet production efficiency are excellent, and if CD41a + When the number of cells is above 85%, the platelet production efficiency decreases.

[0099] CD41a - The number of cells is, for example, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least , at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%.

[0100] CD41a -The number of cells is, for example, less than 85%, less than 84%, less than 83%, less than 82%, less than 81%, less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, less than 73%, less than 72%, less than 71%, less than 70%, less than 69%, less than 68%, less than 67%, less than 66%, less than 65%, less than 64%, less than 63%, less than 62%, less than 61%, less than 60%, less than 59%, less than 58%, less than 57%, less than 56%, less than 55%, less than 54%, less than 53%, less than 52%, less than 51%, less than 52%, less than 53%, less than 54%, less than 55%, less than 56%, less than 57%, less than 56%, less than 55%, less than 54%, less than 53%, less than 52%, less than 51%, less than 51%, less than 52%, less than 53 ... %, less than 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 44%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, or less than 15%.

[0101] CD41a + The percentage of cells can be calculated by subtracting CD41a from 100%. - The percentage of cells obtained.

[0102] In (S2), preferably, CD41a + The number of cells is 15% or more and less than 85%, 20% or more and less than 85%, 30% or more and less than 85%, 40% or more and less than 85%, 45% or more and less than 85%, or 50% or more and less than 85% of the total number of cells.

[0103] The timing of obtaining a suspension cell population can be determined by considering the number of cells expressing CD45 and whether the cells in the culture express CD41a. + A suspension cell population can be obtained from the culture when the number of cells is, for example, less than 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 44%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, or less than 35% of the total number of cells.

[0104] In addition, when CD45 +When the number of cells is, for example, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more, a suspension cell population can be obtained from the culture.

[0105] In one embodiment, CD45 + The number of cells is 1 to 50%, 5 to 50%, 10 to 50%, or 15 to 50%.

[0106] The timing of obtaining a suspension cell population can be determined by considering the number of cells expressing CD34 and whether the cells in the culture express CD41a. + The number of cells is, for example, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or When the content of the cell suspension in the culture medium is greater than 1%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, or greater than 75%, a suspension cell population can be obtained from the culture.

[0107] In addition, when CD34 + A population of suspended cells can be obtained from the culture when the number of cells is, for example, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, 85% or less, 84% or less, 83% or less, 82% or less, 81% or less, 80% or less, 79% or less, 78% or less, 77% or less, 76% or less, 75% or less, 74% or less, 73% or less, 72% or less, 71% or less, 70% or less, 69% or less, 68% or less, 67% or less, 66% or less, 65% or less, 64% or less, 63% or less, 62% or less, 61% or less, 60% or less, 59% or less, 58% or less, 57% or less, 56% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51% or less, or 50% or less.

[0108] When CD41a+ The percentage of cells with higher CD45 + When the percentage of cells is greater than 1%, a suspension cell population can be obtained.

[0109] Even though CD41a + The proportion of cells is as low as less than 50%, but if CD45 + The proportion of cells was lower than that of CD41a + If the ratio of cells is small, differentiation into megakaryocytes and platelets can occur well.

[0110] By determining whether cells in culture express CD41a as well as CD45 and CD34, it is not necessary to individually isolate or purify only cells capable of differentiating into megakaryocytes from the total cell population.

[0111] The method for counting the number of cells can use any conventional technique in the art without limitation. For example, an antibody-based selection method or a sorter can be used. An antibody-based selection method can be, for example, a method using microbeads.

[0112] (S3) Step

[0113] (S3) is a step of differentiating the suspension cell population obtained in step (S2) into megakaryocytes in a first culture medium.

[0114] The first culture medium is used to differentiate the suspended cell population into megakaryocytes. The first culture medium may include cytokines. The first culture medium may include at least one of thrombopoietin (TPO), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-6 (IL-6).

[0115] Thrombopoietin is the major growth factor regulating megakaryocyte and platelet hematopoiesis and is primarily synthesized and secreted by hepatocytes.

[0116] Thrombopoietin can be included in the first culture medium with a concentration that can culture a suspension cell population with other components, and need not be included with a specific concentration. Thrombopoietin can be included with a concentration of, for example, 1 to 50 ng / ml, 5 to 45 ng / ml, 10 to 40 ng / ml, 15 to 35 ng / ml, 20 to 30 ng / ml, or 25 ng / ml.

[0117] Stem cell factor is a cytokine derived from stroma cells synthesized by fibroblasts and other cell types. Stem cell factor can be included in the first culture medium with a concentration that can cultivate a suspended cell population with other components, and does not need to be included with a specific concentration. Thrombopoietin can be included with a concentration of, for example, 1 to 50 ng / ml, 5 to 45 ng / ml, 10 to 40 ng / ml, 15 to 35 ng / ml, 20 to 30 ng / ml or 25 ng / ml.

[0118] Interleukin-3 can be included in the first culture medium at a concentration that enables the suspension cell population to be cultured with other components, and need not be included at a specific concentration. Interleukin-3 can be included at a concentration of, for example, 1 to 20 ng / ml, 5 to 15 ng / ml, or 10 ng / ml.

[0119] Interleukin-6 can be included in the first culture medium at a concentration that enables the suspension cell population to be cultured with other components, and need not be included at a specific concentration. Interleukin-6 can be included at a concentration of, for example, 1 to 20 ng / ml, 5 to 15 ng / ml, or 10 ng / ml.

[0120] The first culture medium is not limited to a specific culture medium. The first culture medium may include IMDM (Iscove's modified Dulbecco's medium) as a basal culture medium. IMDM may further include an antioxidant (e.g., AA2P, etc.) and / or B-27.

[0121] (S4) Step

[0122] (S4) is a step of culturing the megakaryocytes of (S3) in a second culture medium containing thrombopoietin and maturing the megakaryocytes of (S3).

[0123] Thrombopoietin may be included in the second culture medium at a concentration of, for example, 50 to 150 ng / ml, 60 to 140 ng / ml, 70 to 130 ng / ml, 80 to 120 ng / ml, 90 to 110 ng / ml, or 100 ng / ml, but is not limited thereto.

[0124] The second culture medium may further contain factors required for the maturation of megakaryocytes in addition to thrombopoietin.

[0125] The second culture medium is not limited to a specific culture medium. The second culture medium may include IMDM (Iscove's modified Dulbecco's medium) as a basal medium. IMDM may further include an antioxidant (e.g., ascorbic acid 2-phosphate AA2P, etc.) and / or B-27.

[0126] What is obtained in this step may be mature megakaryocytes, or a culture containing megakaryocytes and / or mature megakaryocytes.

[0127] (S5) Step

[0128] (S5) is a step of obtaining platelets from the culture obtained in (S4).

[0129] This step may be isolating and / or purifying platelets from the culture after further differentiating the culture of (S4), or isolating and / or purifying platelets from the culture of (S4) without further differentiating the culture of (S4).

[0130] The separation and / or purification of platelets can be performed by separation and / or purification methods known in the art. For example, it can be performed by centrifuging the culture or passing the culture through a column.

[0131] When the culture is centrifuged, the megakaryocytes can be separated as a pellet and the platelets as a supernatant.

[0132] Hereinafter, the present invention will be described in more detail with reference to Examples.

[0133] Example

[0134] 1. Example 1, Example 2, and Comparative Example 1

[0135] 1-1. Production of human induced pluripotent stem cells (hiPSCs)

[0136] In order to obtain a cell population capable of differentiating into megakaryocytes, human induced pluripotent stem cells were cultured as follows: Figure 1 ).

[0137] First, human induced pluripotent stem cells were cultured at 4000 cells / cm in mTeSR plus medium with 10 μg / ml ROCK inhibitor (Y-27632). 2 The density was placed in a T75 flask and incubated at 37 °C and 5% CO. 2 After 24 hours of culture, the cells were washed once with PBS to remove Y-27632, and the mTeSR plus medium was replaced with a fresh medium. While the medium was replaced once a day, the cells were cultured for 3 days to obtain human induced pluripotent stem cells.

[0138] 1-2. Production of culture medium containing human hematopoietic stem cells (hHSC)

[0139] The obtained human induced pluripotent stem cells were cultured in RPMI1640 basal medium with 6 μM CHIR99021 (GSK3 inhibitor), 300 μM AA2P and 2% B-27 at 37°C and 5% CO. 2 The cells were cultured for 2 days (the culture medium was replaced every day).

[0140] Then, the medium composition was changed to RPMI1640 basal medium with 50 ng / ml VEGF and 20 ng / ml bFGF, containing 300 μM AA2P and 2% B-27, and the cells were incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 3 days (the culture medium was changed once a day). The culture was continued until the cell confluence was about 70% or more. Then, 10 μM SB431542 was further added to the RPMI1640 basal medium with VEGF and bFGF, 300 μM AA2P and 2% B-27, and the cells were further cultured for 4 days.

[0141] (1) Preparation Example 1-1

[0142] On the second day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) relative to the total number of suspended cells was determined by flow cytometry (FACS). Lyric equipment from BD was used for flow cytometry, and anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies from BD were used. In this way, the ratio of single positive cells or double positive cells of CD34 and CD41a and CD45 and CD41a were analyzed respectively.

[0143] As a result, CD34 + The proportion of cells relative to the total cells was 59.27%, and CD45 + The ratio of cells to total cells was 5.21%. At this time, a suspension cell population was obtained from the culture ( Figure 2 and day 7 in Figure 3 ).

[0144] (2) Preparation Example 1-2

[0145] On the 4th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) relative to the total number of suspended cells was determined in the same manner as in Preparation Example 1-1. + The proportion of cells relative to total cells was 57.55%, CD41a + The proportion of cells relative to the total cells was 54.55%, and CD45 +The ratio of cells to total cells was 18.58%. At this time, a suspension cell population was obtained from the culture ( Figure 2 and day 9 in Figure 3 ).

[0146] (3) Preparation Example 1-3

[0147] On the 6th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) relative to the total number of suspended cells was determined in the same manner as in Preparation Example 1-1. + The proportion of cells relative to total cells was 61.59%, CD41a + The proportion of cells relative to the total cells was 47.03%, and CD45 + The ratio of cells to total cells was 32.4%. At this time, a suspension cell population was obtained from the culture ( Figure 2 and day 11 in Figure 3 ).

[0148] (4) Preparation Example 1-4

[0149] On the 8th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) relative to the total number of suspended cells was determined in the same manner as in Preparation Example 1-1. + The proportion of cells relative to total cells was 52.05%, CD41a + The proportion of cells relative to the total cells was 18.88%, and CD45 + The ratio of cells to total cells was 45.56%. At this time, a suspension cell population was obtained from the culture ( Figure 2 on the 13th day of the study).

[0150] (5) Comparative Preparation Example 1-1

[0151] On the 10th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) relative to the total number of suspended cells was determined by flow cytometry. + The proportion of cells relative to total cells was 91.2%, and CD41a + The proportion of cells relative to the total cells was 13%, and CD45 + The ratio of cells to total cells was 68.51%. At this time, a suspension cell population was obtained from the culture ( Figure 2 and day 15 in Figure 3 ).

[0152] 1-3. Inducing differentiation into megakaryocytes by culturing suspension cell populations

[0153] The suspension cell populations from Preparation Examples 1-2 and 1-4 were cultured and cultured at 1×10 5 The cells were introduced into IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of 10 cells / ml. Then, 25 ng / ml TPO, 25 ng / ml SCF, 10 ng / ml IL-3 and 10 ng / ml IL-6 were added to the medium, and the culture was incubated at 37°C and 5% CO. 2 The medium composition was then changed to IMDM basal medium with 100 ng / ml TPO, 300 μM AA2P and 2% B-27, and the culture was further incubated for 5 days to allow the megakaryocytes to mature and obtain the cell culture of Examples 1 and 2 ( Figure 4 ). The photo of the cell culture of Example 1 is Figure 5 Shown in.

[0154] The suspension cell population from Comparative Preparation Example 1-1 was cultured and 1×10 5 The cells were introduced into IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of 10 cells / ml. Then, 25 ng / ml TPO, 25 ng / ml SCF, 10 ng / ml IL-3 and 10 ng / ml IL-6 were added to the medium, and the culture was incubated at 37°C and 5% CO. 2 The medium composition was then changed to IMDM basal medium containing 300 μM AA2P and 2% B-27 with 100 ng / ml TPO, and the culture was further incubated for 5 days to allow the megakaryocytes to mature and obtain a cell culture (Comparative Example 1) ( Figure 6 and Figure 7 ).

[0155] 1-4. Confirmation of platelet production capacity of megakaryocytes

[0156] Cell cultures from Example 1, Example 2, and Comparative Example 1 were collected and centrifuged at 300×g for 3 minutes. The precipitate and supernatant were separated into megakaryocytes and platelets, respectively. Platelets were then analyzed and characterized by the following experiments.

[0157] (1-4-1) Identification of megakaryocyte-specific markers using FACS analysis

[0158] The cell cultures from Example 1, Example 2, and Comparative Example 1 were centrifuged to obtain pelleted cells (cells presumed to be megakaryocytes).

[0159] In order to confirm whether the precipitated cells are megakaryocytes, FACS analysis was performed to determine the surface marker expression of the precipitated cells. FACS analysis was performed using a Lyric device from BD, and anti-CD41a-FITC, anti-CD61-PE and anti-CD42b-APC antibodies from BD were used. The antibodies were diluted 1:20 in PBS (FACS buffer) containing 1% BSA, and used for a 30-minute reaction to confirm surface marker expression.

[0160] As a result, the precipitated cells from Example 1 were positive for the expression of megakaryocyte-specific markers (CD41a, CD42b, and CD61) (Figure 8). Further, the precipitated cells from Example 2 were also positive for the expression of megakaryocyte-specific markers (CD41a, CD42b, and CD61) (Figure 9). Therefore, it was confirmed that the suspension cell populations from Examples 1 and 2 differentiated into megakaryocytes and thus also produced platelets.

[0161] In contrast, the precipitated cells from Comparative Example 1 showed very low expression of CD41a and CD61, and, in particular, expression of CD42b was close to negative ( FIG. 10 ). This indicates that the suspension cell population from Comparative Example 1 hardly differentiated into megakaryocytes and hardly produced platelets.

[0162] (1-4-2) Confirmation of platelet function by ADP treatment

[0163] Example 1, Example 2, and Comparative Example 1 were centrifuged to remove the precipitated cells (megakaryocytes), and the suspended cells were centrifuged again at 2000 × g for 10 minutes. Then, the precipitated cells (presumed to be platelet cells) were collected by removing all supernatants and resuspended with a small amount of PBS. The following experiment was performed to confirm whether the cells obtained in this way were platelets with normal activity.

[0164] For PAC-1 analysis, cells were treated with 100 μM ADP and reacted at room temperature for 15 minutes, and then anti-PAC-1-FITC and anti-CD62p-PE antibodies from BD were reacted for 30 minutes. Then, the reacted cells were subjected to FACS analysis.

[0165] The cells from Examples 1 and 2 were activated by ADP treatment, and the expression of PAC-1 and CD62p increased (Figures 11 and Fig.12 ), confirming that the cells from Examples 1 and 2 are platelets with normal activity. PAC-1 is a complex formed by CD41a and CD61 when platelets are activated. CD62p (p-selectin) is a surface molecule whose expression increases when platelets are activated, and serves as a site to which leukocytes can bind to platelets.

[0166] The above experimental results confirmed that when hiPSCs are cultured and the cell population meets certain conditions (the ratio of the number of cells expressing specific surface markers (such as CD41a, CD34, CD45) to the total number of cells is higher or lower than a certain value), when subsequent culture steps are performed, the differentiation efficiency into megakaryocytes and the platelet production efficiency can be improved.

[0167] 2. Comparative Example 2: When selecting only CD41a from a suspension cell population + Comparison of platelet production efficiency in cells and subsequent culture

[0168] From the suspension cell population obtained in Preparation Example 1-2 (Comparative Example 2), only CD41a + Cells were cultured and the platelet production efficiency was compared with that of Example 1 ( Fig.13 ).

[0169] The expression of platelet-specific markers in both cases was confirmed by the same method as the above FACS analysis. As a result, CD41a + / CD42b + The ratios were similar, 57.58 and 56.20, confirming that there was no significant difference in platelet differentiation rate ( Fig.14 ).

[0170] However, when the cells were tested for their proliferation capacity, they were found to be significantly lower than those containing only CD41a + Compared with the differentiation of the cell population into platelets, as in Example 1, the cell population containing CD41a + cells and not CD41a + Cells (e.g., CD41a - ) showed a 40-fold higher cell proliferation capacity in the differentiation of platelets ( Fig.15 ).

[0171] As a result of the above, it was confirmed that when the CD41a - Cell populations of cells are used for platelet production rather than selecting for CD41a only + When the cells are used for platelet production, platelets can be produced quantitatively and qualitatively with high efficiency.

[0172] 3. Examples 3 to 8 and Comparative Examples 3 and 4

[0173] 3-1. Production of human induced pluripotent stem cells (hiPSCs)

[0174] In order to obtain a cell population capable of differentiating into megakaryocytes, human induced pluripotent stem cells were cultured as follows.

[0175] First, human induced pluripotent stem cells were cultured at 4500 cells / cm in mTeSR plus medium with 10 μg / ml ROCK inhibitor (Y-27632). 2 The density was placed in a T75 flask and incubated at 37 °C and 5% CO. 2 After 24 hours of culture, the cells were washed once with PBS to remove Y-27632, and the mTeSR plus medium was replaced with a fresh medium. While the medium was replaced once a day, the cells were cultured for 3 days to obtain human induced pluripotent stem cells.

[0176] 3-2. Production of a culture containing human hematopoietic stem cells (hHSC)

[0177] The obtained human induced pluripotent stem cells were cultured in RPMI1640 basal medium with 6 μM CHIR99021 (GSK3 inhibitor), 300 μM AA2P and 2% B-27 at 37°C and 5% CO. 2 Culture for 2 days.

[0178] Then, the medium composition was changed to RPMI1640 basal medium with 50 ng / ml VEGF and 20 ng / ml bFGF, containing 300 μM AA2P and 2% B-27, and the cells were incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 3 days. The culture was continued until the cell confluence was about 90% or more. Then, 10 μM SB431542 was further added to the RPMI1640 basal medium with VEGF and bFGF, containing 300 μM AA2P and 2% B-27, and the cells were further cultured for 4 days.

[0179] Cultures were obtained on days 7, 9, 11, 13, 15, 17, 19, and 21 of culture, and the number of cells expressing specific surface markers (CD34, CD41a, and CD45) relative to the total number of cells in suspension was determined by flow cytometry (FACS). Lyric instrument from BD and anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies from BD were used for flow cytometry ( Fig.16 ).

[0180] 3-3. CD41a in culture + 、CD45 + , and CD34 + The percentage of cells

[0181] CD41a in cultures on days 7, 9, 11, 13, 15, 17, 19, and 21 of culture + 、CD45 + , and CD34 + The percentages of cells are shown in Table 2 below.

[0182] [Table 2]

[0183] category(%) Cultivation days <![CDATA[CD41a + Cell percentage (%)]]> <![CDATA[CD45 + Cell percentage (%)]]> <![CDATA[CD34 + Cell percentage (%)]]> Preparation Example 3-1 7 61.13 1.22 56.32 Preparation Example 3-2 9 82.74 8.57 77.01 Preparation Example 3-3 11 84.44 19.13 78.83 Preparation Example 3-4 13 51.68 34.72 50.56 Preparation Example 3-5 15 29.86 49.26 47.14 Preparation Example 3-6 17 20.00 46.20 46.38 Comparative Preparation Example 3-1 19 14.63 37.72 29.07 Comparative Preparation Example 3-2 21 9.68 47.09 18.16

[0184] 3-4. Differentiation of the Suspension Cell Populations of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 into Megakaryocytes

[0185] The suspension cell populations of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 were respectively prepared at 0.5×10 5 The cells were introduced into IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of 10 cells / ml, and after adding 25 ng / ml SCF, 10 ng / ml IL-3, and 10 ng / ml IL-6 to the medium, they were incubated at 37°C and 5% CO. 2 Culture for 3 days.

[0186] Then, the medium composition was changed to IMDM basal medium with 100 ng / ml TPO containing 300 μM AA2P and 2% B-27, and the cells were further cultured for 5 days to allow megakaryocyte maturation and obtain cell culture.

[0187] 3-5. Confirmation of platelet production capacity of Examples 3 to 8 and Comparative Examples 3 and 4

[0188] The cell cultures of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 that have undergone the megakaryocyte maturation step were collected and centrifuged at 300×g for 3 minutes. The precipitate believed to be megakaryocytes was removed, and the following experiment was performed on the supernatant containing platelets to confirm whether the final product was a platelet with normal activity. The materials obtained from the cell cultures of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 were named Examples 3 to 8 and Comparative Examples 3 and 4, respectively, in the order described.

[0189] (1) FACS analysis to identify platelet-specific markers

[0190] The cell cultures from Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 that had undergone the megakaryocyte maturation step were centrifuged. The precipitated megakaryocytes were removed. The suspended platelets were centrifuged again at 2000×g for 15 minutes.

[0191] FACS analysis was performed on cells presumed to be megakaryocytes and platelets to confirm the expression of surface markers. Lyric equipment from BD was used, and anti-CD41a-FITC, anti-CD61-PE and anti-CD42b-APC antibodies from BD were used. The antibodies were diluted 1:20 in PBS (FACS buffer) containing 1% BSA and used for a 30-minute reaction to confirm surface marker expression.

[0192] As a result, Examples 3 to 8 were positive for the expression of megakaryocyte- and platelet-specific markers ( FIGS. 17 to 22 ). This confirmed that the suspension cell populations of Preparation Examples 3-1 to 3-6 were able to efficiently produce megakaryocytes and platelets.

[0193] In contrast, the expression of surface markers of megakaryocytes and platelets was hardly shown in Comparative Examples 3 and 4. As a result, it was confirmed that the suspension cell populations of Comparative Preparation Examples 3-1 and 3-2 hardly showed differentiation into megakaryocytes and were substantially unable to produce platelets (Figs. 23 and 24).

[0194] (2) Confirmation of platelet function by ADP treatment

[0195] The following experiment was conducted to confirm whether Examples 3 to 8 and Comparative Examples 3 and 4 that had undergone the megakaryocyte maturation step were platelets having normal functions.

[0196] The cell cultures from Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 that had undergone the megakaryocyte maturation step were centrifuged. The precipitated megakaryocytes were removed. The suspended platelets were centrifuged again at 2000×g for 15 minutes. To collect the platelets precipitated by centrifugation, the supernatant was removed and a small amount of PBS was used to resuspend the cells.

[0197] For PAC-1 analysis, 500 μM ADP was added to platelets to react at room temperature for 15 minutes, and then reacted with anti-PAC-1-FITC and anti-CD62p-PE antibodies from BD for 30 minutes. The reacted platelets were immediately subjected to FACS analysis.

[0198] PAC-1 is present when CD41a and CD61 form a complex when platelets are activated.CD62p (p-selectin) is a surface molecule whose expression increases when platelets are activated, and serves as a site to which leukocytes can bind to platelets.

[0199] Examples 3 to 8 were activated by treatment with ADP, and the expression of PAC-1 and CD62p increased. This confirmed that Examples 3 to 8 were platelets with normal functions (Figures 17 to 22). In contrast, Comparative Examples 3 and 4 showed almost no platelet activation (Figures 23 and 24).

[0200] As mentioned above, it was confirmed that when used in CD41a - When the number of cells is at least 15% of the total number of cells, when the suspension cell population obtained from the culture is subjected to the subsequent steps, the efficiency of differentiation into megakaryocytes and the efficiency of platelet production are improved.

Claims

1. A method for producing platelets, wherein include: (S1) culturing pluripotent stem cells to obtain a culture containing hematopoietic stem cells; (S2) When CD41a - When the number of cells is at least 15% of the total number of cells, obtaining a suspension cell population from the culture; and (S3) Differentiating the suspended cell population into megakaryocytes in a first culture medium. 2 . The method for producing platelets according to claim 1 , further comprising (S4) culturing the megakaryocytes in a second culture medium containing thrombopoietin and maturing the megakaryocytes.

3. The method for producing platelets according to claim 1, wherein (S1) The following steps are involved: (S1a) culturing the pluripotent stem cells in a third culture medium containing a GSK3 inhibitor; (S1b) culturing the cells cultured in the third culture medium in a fourth culture medium containing vascular endothelial growth factor and basic fibroblast growth factor; and (S1c) culturing the cells cultured in the fourth culture medium in a fifth culture medium containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor-β signaling inhibitor.

4. The method for producing platelets according to claim 1, wherein the suspended cell population has less than 50% of the total cell count of CD45 + Cell counting.

5. The method for producing platelets according to claim 1, wherein the suspended cell population has more than 45% of the total cell count of CD34 + Cell counting.

6. The method for producing platelets according to claim 1, wherein the suspension cell population has less than 85% of the total cell count of CD41a - Cell counting.

7. The method for producing platelets according to claim 1, further comprising (S0) culturing the platelets at a density of 2,000 to 20,000 cells / cm 2 The pluripotent stem cells are seeded on the bottom of a culture container.

8. The method for producing platelets according to claim 1, wherein the culture further comprises hematopoietic progenitor cells and megakaryocyte progenitor cells.

9. The method for producing platelets according to claim 1, wherein the first culture medium comprises thrombopoietin, stem cell factor, interleukin-3, and interleukin-6.

10. The method for producing platelets according to claim 1, in, In (S2), when the cells contained in the culture medium express CD41a + When the number of cells is 40 to 85% of the total number of cells, a suspension cell population is obtained from the culture.

11. The method for producing platelets according to claim 1, in, In (S2), when the cells contained in the culture are CD34 + When the number of cells is 45 to 80% of the total number of cells, a suspension cell population is obtained from the culture.

12. The method for producing platelets according to claim 1, in, In (S2), when the cells contained in the culture medium have CD45 + When the number of cells is 1 to 50% of the total number of cells, a suspension cell population is obtained from the culture.

13. The method for producing platelets according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells.

14. The method for producing platelets according to claim 4, wherein the CD45 + The proportion of cells was lower than CD41a + The proportion of cells.

15. A method for producing a blood product, comprising the step of mixing the platelets produced by the method of any one of claims 1 to 14 with other blood components.

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