Platelet production method

Abstract

The present invention relates to a method for producing platelets. The method comprises the steps of (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, (S2) obtaining a floating cell population from the culture medium when the number of cells that do not express CD41a accounts for at least 15% of the total number of cells, and (S3) differentiating the floating cell population into megakaryocytes, thereby enabling the production of high-quality platelets with a 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] [Background technology]

[0003] Platelet preparations are administered to patients who have experienced massive bleeding due to surgery or injury, or who have a tendency to bleed due to a decrease in platelets after anti-cancer drug treatment, for the purpose of treating and preventing the symptoms.

[0004] Currently, the production of platelet preparations relies on blood donations from healthy volunteers, but the number of blood donors has been declining in recent years, and blood shortages are predicted in the future. Therefore, a stable supply of platelets is an important issue in this field.

[0005] Conventional platelet preparations have a high risk of bacterial contamination, which can lead to serious infections after platelet transplantation. Therefore, safer platelet preparations are constantly being sought in clinical settings.

[0006] To meet this demand, 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, so technical improvements are needed. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for producing artificial platelets with a high yield.

[0008] Another object of the present invention is to provide a method for producing platelets that has process efficiency. [Means for solving the problem]

[0009] 1. (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) culturing cells containing CD41a - A method for producing platelets, comprising: (S1) obtaining a floating cell population from the culture medium when the cell number is at least 15% of the total cell number; and (S2) differentiating the floating cell population into megakaryocytes in a first culture medium.

[0010] 2. The method for producing platelets according to item 1 above, further comprising the step of (S4) culturing and maturing megakaryocytes in a second medium containing thrombopoietin.

[0011] 3. In the above item 1, (S1) is a method for producing platelets, comprising 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 transforming growth factor beta signaling inhibitor.

[0012] 4. In the above item 1, the floating cell population is CD45 + A method for producing platelets, in which the cell count is 50% or less of the total cell count.

[0013] 5. In the above item 1, the floating cell population is CD34 + A method for producing platelets, in which the cell count is 45% or more of the total cell count.

[0014] 6. In the above item 1, the floating cell population is CD41a - A method for producing platelets, in which the cell count is 85% or less of the total cell count.

[0015] 7. In the above item 1, (S0) the pluripotent stem cells are cultured at the bottom of a culture vessel at a density of 2,000 to 20,000 cells / cm. 2 The method for producing platelets further comprises the step of seeding with.

[0016] 8. The method for producing platelets according to item 1 above, wherein the culture medium further contains hematopoietic progenitor cells and megakaryoprogenitor cells.

[0017] 9. The method for producing platelets according to item 1 above, wherein the first culture medium contains thrombopoietin, stem cell factor, interleukin-3, and interleukin-6.

[0018] 10. In the above item 1, in (S2), among the cells contained in the culture medium, CD41a + A method for producing platelets, comprising obtaining a floating cell population from the culture medium when the cell number is 15% or more and less than 85% of the total cell number.

[0019] 11. In the above item 1, in (S2), among the cells contained in the culture medium, CD34 + A method for producing platelets, comprising obtaining a floating cell population from the culture medium when the cell number is 45 to 80% of the total cell number.

[0020] 12. In the above item 1, in (S2), among the cells contained in the culture medium, CD45 + A method for producing platelets, comprising obtaining a floating cell population from a culture medium when the cell number is 1 to 50% of the total cell number.

[0021] 13. The method for producing platelets according to item 1 above, wherein the pluripotent stem cells are human induced pluripotent stem cells.

[0022] 14. In the above item 4, the floating cell population is CD45 + The proportion of cells is CD41a + A method for producing platelets, which have a low ratio of cells to the total.

[0023] 15. A method for producing a blood product, comprising the step of mixing platelets produced by the method according to any one of items 1 to 14 with other blood components. [Effects of the Invention]

[0024] The method for producing platelets of the present invention is - To prepare megakaryocytes from a suspension cell population containing at least 15% CD41a + No cell isolation and purification is required.

[0025] The method for producing platelets of the present invention allows for rapid and economical production of platelets because subsequent steps can be carried out only when the suspended cell population meets predetermined requirements.

[0026] The method for producing platelets of the present invention is excellent in the efficiency of differentiation into megakaryocytes and the efficiency of platelet production.

[0027] The method for producing platelets of the present invention involves simple steps, does not require complicated equipment, and is advantageous in terms of time and cost.

[0028] Platelets produced by the method of the present invention can be activated by stimulants (e.g., adenosine diphosphate (ADP), collagen, fibrinogen, etc.) and can increase the expression of PAC-1 and CD62p. This confirms that the platelets of the present invention are platelets that perform normal functions.

[0029] Platelets produced by the method of the present invention can exhibit therapeutic or preventive effects against diseases associated with thrombocytopenia or dysfunction. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 illustrates one embodiment of a culture process for human induced pluripotent stem cells. [Figure 2]FIG. 2 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-4 and Comparative Production Example 1-1. [Figure 3a] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-3 and Comparative Production Example 1-1. [Figure 3b] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-3 and Comparative Production Example 1-1. [Figure 3c] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-3 and Comparative Production Example 1-1. [Figure 3d] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-3 and Comparative Production Example 1-1. [Figure 3e] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-3 and Comparative Production Example 1-1. [Figure 3f] FIG. 3 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 1-1 to 1-3 and Comparative Production Example 1-1. [Figure 4] FIG. 4 shows the steps for obtaining the cell culture medium in Examples 1 and 2. [Figure 5] FIG. 5 is a photograph of the cell culture medium of Example 1. [Figure 6] FIG. 6 shows the steps of obtaining a cell culture medium in Comparative Example 1. [Figure 7] FIG. 7 is a photograph of the cell culture medium of Comparative Example 1. [Figure 8a] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers. [Figure 8b] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers. [Figure 8c] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers. [Figure 8d] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers. [Figure 8e] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers. [Figure 8f] FIG. 8 shows that the cells of Example 1 express megakaryocyte-specific markers. [Figure 9a] FIG. 9 shows that the cells of Example 2 express megakaryocyte-specific markers. [Figure 9b] FIG. 9 shows that the cells of Example 2 express megakaryocyte-specific markers. [Figure 9c] FIG. 9 shows that the cells of Example 2 express megakaryocyte-specific markers. [Figure 10a] FIG. 10 shows that the cells of Comparative Example 1 do not express megakaryocyte-specific markers. [Figure 10b] FIG. 10 shows that the cells of Comparative Example 1 do not express megakaryocyte-specific markers. [Figure 10c] FIG. 10 shows that the cells of Comparative Example 1 do not express megakaryocyte-specific markers. [Figure 11a] FIG. 11 shows that the cells of Example 1 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p. [Figure 11b] FIG. 11 shows that the cells of Example 1 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p. [Figure 11c] FIG. 11 shows that the cells of Example 1 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p. [Figure 12] FIG. 12 shows that the cells of Example 2 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p. [Figure 13] FIG. 13 shows the results of selecting CD41a+ cells from the floating cell population of Production Example 1-2. [Figure 14]FIG. 14 shows the CD41a+ / CD42b+ ratios in Example 1 (Production Example 1-2) and Comparative Example 2. [Figure 15] FIG. 15 shows that Example 1 exhibits approximately 40 times higher cell proliferation ability than Comparative Example 2. [Figure 16] FIG. 16 shows the results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2. [Figure 17a] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17b] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17c] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17d] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17e] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17f] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17g] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17h] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17i] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 17j] FIG. 17 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18a] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18b] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18c] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18d] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18e] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18f] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18g] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18h] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18i] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 18j] FIG. 18 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19a] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19b] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19c] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19d] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19e] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19f] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19g] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19h] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19i] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 19j] FIG. 19 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20a] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20b] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20c] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20d] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20e] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20f] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20g] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20h] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20i] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 20j] FIG. 20 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21a] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21b] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21c]FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21d] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21e] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21f] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21g] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21h] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21i] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 21j] FIG. 21 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22a] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22b] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22c] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22d] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22e] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22f] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22g] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22h]FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22i] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 22j] FIG. 22 shows that Examples 3 to 8 express megakaryocyte- and platelet-specific markers. [Figure 23a] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23b] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23c] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23d] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23e] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23f] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23g] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23h] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23i] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 23j] FIG. 23 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24a] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24b] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24c] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24d] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24e] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24f] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24g] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24h] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24i] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. [Figure 24j] FIG. 24 shows that comparative examples 3 and 4 do not express megakaryocyte and platelet specific markers. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a method for producing platelets, which comprises the following steps: (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, (S2) obtaining a floating cell population from the culture medium when the number of cells that do not express CD41a accounts for at least 15% of the total number of cells contained in the culture medium, and (S3) differentiating the floating cell population into megakaryocytes in a first medium.

[0032] The production method of the present invention comprises the steps of: (S0) inoculating the pluripotent stem cells into the bottom of a culture vessel at a density of 2,000 to 20,000 cells / cm 2 The method may further include seeding with

[0033] The production method of the present invention may further comprise the step of (S4) culturing and maturing megakaryocytes in a second medium containing thrombopoietin.

[0034] The production method of the present invention may further include the step (S5) of obtaining platelets from the culture medium obtained in (S4).

[0035] The platelet production process from (S0) to (S5) will be explained below.

[0036] (S0) Step (S0) is a method for culturing pluripotent stem cells at the bottom of a culture vessel at a density of 2,000 to 20,000 cells / cm. 2 This is the seeding step.

[0037] Pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0038] Induced pluripotent stem cells (iPSCs) are cells that originally lacked pluripotency but acquired it through an artificial reverse differentiation process.

[0039] The induced pluripotent stem cells (iPSCs) may be derived from an individual selected from the group including humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, and cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoon dogs, horses, zebras, and marine mammals (dolphins, whales, etc.).

[0040] The induced pluripotent stem cells may be human induced pluripotent stem cells (hiPSCs).

[0041] Induced pluripotent stem cells may be generated using mouse and / or human cells, for example, induced pluripotent stem cells may be generated using embryonic, fetal, neonatal, and adult tissues.

[0042] Induced pluripotent stem cells can be derived from virtually any somatic cell at any developmental stage as a starting point. Somatic cells can be derived from, but are not limited to, embryonic, fetal, neonatal, juvenile, or adult donors. Somatic cells can also be, but are not limited to, fibroblasts, such as skin fibroblasts obtained from skin samples or biopsies, synovial cells from synovial tissue, cheek cells, or lung fibroblasts.

[0043] Pluripotent stem cells are seeded on the bottom of a culture vessel and then cultured in an adherent manner.

[0044] The incubator can be any type commonly used for cell culture in the art, without limitation. For example, it may be a large-, medium-, or small-sized incubator. It may also be a cell culture flask such as a T25, T75, T175, or T225.

[0045] Pluripotent stem cells are 2,000-20,000 cells / cm 2 Seeding at this amount is preferable from the viewpoint of cell confluency. Seeding at this amount results in a cell confluency of 70% to 95%, 75% to 95%, 80% to 95%, or 85% to 95%, allowing pluripotent stem cells to be cultured and differentiated appropriately, thereby enabling a culture medium containing sufficient hematopoietic stem cells, hematopoietic progenitor cells, megakaryoprogenitor cells, etc. to be obtained in step (S1) described below.

[0046] Pluripotent stem cells were cultured at the bottom of the culture vessel at 2,000 cells / cm. 2When seeding is performed at less than 20,000 cells / cm, the cell confluency at the time of obtaining the floating cell population in (S2) may 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. Furthermore, pluripotent stem cells may be seeded at a density of 20,000 cells / cm at the bottom of the culture vessel. 2 If seeding is performed beyond this limit, the cell confluency may exceed 100% before the pluripotent stem cells are fully cultured and differentiated, causing undifferentiated cells to detach from the culture dish, become floating, and die, or the amount of factors added to the culture medium may be insufficient. This may result in poor intercellular signaling and poor differentiation.

[0047] (S1) Step Step (S1) is a step of culturing pluripotent stem cells to obtain a culture medium 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 capable of differentiating into megakaryocytes.

[0048] The step (S1) may be configured to include the following substeps: (S1a) culturing pluripotent stem cells in a third medium containing a GSK3 (Glycogen Synthase Kinase 3) inhibitor; (S1b) culturing the cells cultured in the third medium in a fourth medium containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF); and (S1c) A step of culturing the cells cultured in the fourth medium in a fifth medium containing vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor beta signaling inhibitor.

[0049] The third, fourth, and fifth media used in this step have different compositions and purposes.

[0050] The third culture medium contains at least a GSK3 inhibitor. The GSK3 inhibitor may 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 and a Wnt signaling enhancer, and may be expressed as an aminopyrimidine.

[0051] The concentration of the GSK3 inhibitor (e.g., CHIR99021) is not particularly limited, as long as it is an amount that allows pluripotent stem cells to be cultured. The GSK3 inhibitor may be contained in the third 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.

[0052] The third medium is a basal medium. The third medium may include RPMI 1640 medium or other types of basal medium. In addition to the basal medium, the third medium may further include antioxidants and / or B-27.

[0053] Antioxidants include 6-hydroxymelatonin, acetyl-L-carnitine (ALCAR), alpha-lipoic acid (ALA), ascorbic acid (e.g., L-ascorbic acid 2-phosphate (AA2P), L-ascorbic acid 2-glucoside (AA2G)), carotenoids (vitamins), and vitamin D3. A), Curcumin, Edaravone, Polyphenols, Glutathione, Hydroxytyrosol, L-carnitine, Ladostigil, Melatonin, Mofegiline, N-Acetylcysteine ​​(NAC), N-Acetylserotonin (NAS), Oleocanthal, Oleuropein, Rasagiline, Resveratrol, Selegiline, Selenium, Tocopherols, Vitamin E), tocotrienols, tyrosol, ubiquinone (coenzyme Q) and uric acid.

[0054] 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 for culturing pluripotent stem cells.

[0055] Vascular endothelial growth factor (VEGF) is a member of the epidermal growth factor receptor (EGFR / ErbB) family. VEGF plays an essential role in regulating cell growth and differentiation, leading to the activation of various signaling pathways to induce apoptosis, survival, or cell proliferation. VEGF includes, for example, human and non-human animal (e.g., mouse) VEGFs.

[0056] Vascular endothelial growth factor is contained in the fourth medium at a concentration that allows appropriate cultivation of pluripotent stem cells, 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.

[0057] Basic fibroblast growth factor (bFGF) is a protein belonging to the FGF family that functions as a mitogen, angiogenic factor, bone morphogenetic factor, and nerve growth factor, as well as regulating cell proliferation and differentiation. Basic fibroblast growth factor, also known as FGF2, activates receptor proteins, including FGFR1b, FGFR1c, FGFR2c, FGFR3c, and FGFR4c, with particular potent activation of FGFR1c and FGFR3c.

[0058] Basic fibroblast growth factor is included in the fourth medium at a concentration that allows appropriate cultivation of pluripotent stem cells together with other components, 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.

[0059] The fourth medium may not contain a transforming growth factor beta (TGFβ) signaling inhibitor, as containing a TGFβ signaling inhibitor in the fourth medium may result in differentiation under conditions of insufficient cell proliferation.

[0060] The fifth medium is a growth medium. Like the fourth medium, it contains at least growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The fifth medium may further contain other growth factors for culturing pluripotent stem cells.

[0061] The details regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in Medium 5 shall be the same as those regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in Medium 4.

[0062] The fifth medium contains a transforming growth factor beta (TGFβ) signaling inhibitor.

[0063] Transforming growth factor beta (TGFβ) signaling inhibitors are substances that inhibit TGFβ signaling, which regulates various physiological processes in the body, including cell proliferation, differentiation, apoptosis, migration, extracellular matrix (ECM) production, angiogenesis, and development.

[0064] The TGFβ signaling inhibitor can be any substance that can inhibit TGFβ signaling, and may be, for example, an activin receptor-like kinase (ALK) receptor inhibitor.

[0065] Activin receptor-like kinase receptor inhibitor can be, but not limited to, ALK5, ALK4 and ALK7 receptor inhibitor.For example, 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.

[0066] The transforming growth factor beta (TGFβ) signaling inhibitor is included together with other components at a concentration that allows pluripotent stem cells to be cultured, for example, at a concentration of 1 to 20 μM, 5 to 15 μM, or 10 μM.

[0067] The culture medium obtained through the culture in step (S1) contains hematopoietic stem cells (HSCs). The culture medium may further contain hematopoietic progenitor cells (HPCs) and megakaryoprogenitor cells (MK-Ps).

[0068] (S2) Step In step (S2), cells that do not express CD41a (CD41a - When the number of floating cells (floating cells) is at least 15% of the total cell number, a floating cell population is obtained from the culture medium.

[0069] The term "suspended cell population" refers to cells and / or cell populations suspended in a culture medium. The suspended cell population contains many cells that can differentiate into megakaryocytes, such as hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte precursor cells, and may also contain cells that do not directly differentiate into megakaryocytes but help hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte precursor cells to differentiate better than if they existed alone.

[0070] In this step, the optimal time point for obtaining a floating cell population from the culture medium in (S1) is determined. By obtaining a floating cell population optimal for differentiation into megakaryocytes in this step, the efficiency of differentiation into megakaryocytes and / or mature megakaryocytes and the efficiency of platelet production can be increased.

[0071] The time to obtain the floating cell population was CD41a - The cell number is at least 15% of the total cell number, i.e., cells expressing CD41a (CD41a + The number of CD41a cells is less than 85%. + When the number of cells is less than 85%, the differentiation efficiency into megakaryocytes and platelet production efficiency is excellent, and CD41a + If the number of cells is 85% or more, the efficiency of platelet production actually decreases.

[0072] CD41a - The cell count may be, for example, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 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 9% 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.

[0073] CD41a -The cell count may be, for example, 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, or 51% or less of the total cell count. , 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, or 15% or less.

[0074] CD41a + The percentage of cells ranged from 100% to CD41a - It is obtained by subtracting the ratio of cells.

[0075] In (S2), among the cells contained in the culture medium, CD41a + The cell number is preferably 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 cell number.

[0076] The time point for obtaining the floating cell population can be determined by considering whether the cells in the culture medium express CD41a and the number of cells expressing CD45. + A floating cell population can be obtained from the culture medium when the cell number is, for example, 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, or 35% or less of the total cell number.

[0077] Also, CD45 + A floating cell population can be obtained from the culture medium when the cell count 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.

[0078] In one embodiment, CD45 + The cell count is 1-50%, 5-50%, 10-50%, or 15-50%.

[0079] The time point for obtaining the floating cell population can be determined by considering whether the cells in the culture medium express CD41a and the number of cells expressing CD34. + For example, if the cell count is 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, or 75% or more, a floating cell population can be obtained from the culture medium.

[0080] Also, CD34 +A suspension cell population can be obtained from the culture medium when the cell count 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.

[0081] Whether the cells in the culture express CD41a or not, as well as CD41a + The proportion of cells is CD45 + When the ratio of cells is higher than that of the total cells, a floating cell population can be obtained.

[0082] CD41a in the total cell population + Even if the percentage of cells is low (less than 50%), CD45 + If the proportion of cells is lower than that, differentiation into megakaryocytes and platelets can occur well.

[0083] By determining whether the cells in the culture medium express CD41a and, in addition, whether they express CD45 and CD34, it is not necessary to separately separate or purify only the cells capable of differentiating into megakaryocytes from the entire cell population.

[0084] The cell number can be counted using techniques commonly used in the art without limitation. For example, an antibody-based selection method or a sorter machine can be used. The antibody-based selection method may be, for example, a method using microbeads.

[0085] (S3) Step Step (S3) is a step in which the floating cell population obtained in step (S2) is differentiated into megakaryocytes in a first medium.

[0086] The first culture medium serves to differentiate the floating cell population into megakaryocytes. The first culture medium may contain cytokines. The first culture medium may contain at least one of thrombopoietin (TPO), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-6 (IL-6).

[0087] Thrombopoietin is the major growth factor regulating hematopoiesis of megakaryocytes and platelets, and is synthesized and secreted primarily by hepatocytes.

[0088] Thrombopoietin does not need to be present at a specific concentration in the first medium, as long as it is present at a concentration that allows the suspension cell population to be cultured together with other components. Thrombopoietin may be present at 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.

[0089] Stem cell factor is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. Stem cell factor does not need to be present at a specific concentration in the first medium, as long as it is present at a concentration that allows the suspension cell population to be cultured with other components. Thrombopoietin may be present at concentrations of, for example, 1-50 ng / ml, 5-45 ng / ml, 10-40 ng / ml, 15-35 ng / ml, 20-30 ng / ml, or 25 ng / ml.

[0090] Interleukin-3 does not need to be present at a specific concentration in the first medium, as long as it is present at a concentration that allows the suspension cell population to be cultured together with other components. Interleukin-3 may be present at a concentration of, for example, 1 to 20 ng / ml, 5 to 15 ng / ml, or 10 ng / ml.

[0091] Interleukin-6 does not need to be present at a specific concentration in the first medium, as long as it is present at a concentration that allows the suspension cell population to be cultured together with other components. Interleukin-6 may be present at a concentration of, for example, 1 to 20 ng / ml, 5 to 15 ng / ml, or 10 ng / ml.

[0092] The first medium is not limited to a specific medium. The first medium may contain IMDM (Iscove's Modified Dulbecco's Medium) as a basal medium. The IMDM medium may further contain an antioxidant (e.g., AA2P) and / or B-27.

[0093] (S4) Step Step (S4) is a step in which the megakaryocytes of step (S3) are cultured in a second medium containing thrombopoietin to mature them.

[0094] Thrombopoietin may be contained in the second culture medium at a concentration of, for example, but not limited to, 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.

[0095] The second medium may further contain, in addition to thrombopoietin, factors necessary for megakaryocyte maturation.

[0096] The second medium is not limited to a specific medium. The second medium may contain IMDM (Iscove's Modified Dulbecco's Medium) as a basal medium. The IMDM medium may further contain an antioxidant (e.g., ascorbic acid 2-phosphate, AA2P, etc.) and / or B-27.

[0097] What is obtained in this step may be matured megakaryocytes, or megakaryocytes and / or a culture medium containing matured megakaryocytes.

[0098] (S5) Step Step (S5) is a step of obtaining platelets from the culture medium obtained in step (S4).

[0099] In this step, platelets may be separated and / or purified from the culture medium after further differentiation of the culture medium in (S4), or platelets may be separated and / or purified from the culture medium without further differentiation of the culture medium in (S4).

[0100] Platelets can be separated and / or purified by known separation and / or purification methods, for example, by centrifuging the culture medium or passing the culture medium through a column.

[0101] When the culture medium is centrifuged, megakaryocytes can be separated as a precipitate and platelets can be separated as a floating matter.

[0102] The present invention will be described in more detail below with reference to examples.

[0103] Example 1. Example 1, Example 2 and Comparative Example 1 1-1. Production of human induced pluripotent stem cells (hiPSCs) To obtain a cell population capable of differentiating into megakaryocytes, human induced pluripotent stem cells were cultured as follows (Figure 1).

[0104] First, human induced pluripotent stem cells were cultured in mTeSR plus culture medium at a density of 4000 cells / cm with 10 μg / ml of ROCK inhibitor (Y-27632). 2 The cells were placed in a T75 flask at a density of 100 μg / ml and cultured at 37°C in 5% CO2. After 24 hours of culture, the cells were washed once with PBS to remove Y-27632, and the mTeSR plus culture medium was replaced with fresh medium. The culture medium was changed once daily for three days, yielding human induced pluripotent stem cells.

[0105] 1-2. Preparation of culture medium containing human hematopoietic stem cells (hHSCs) The obtained human induced pluripotent stem cells were cultured for two days at 37°C in 5% CO2 in RPMI1640 basal medium containing 300 μM AA2P, 2% B-27, and 6 μM of the GSK3 inhibitor CHIR99021 (the culture medium was changed once a day).

[0106] The medium was then changed to RPMI 1640 basal medium containing 300 μM AA2P, 2% B-27, 50 ng / ml VEGF, and 20 ng / ml bFGF, and cultured at 37°C in a 5% CO2 incubator for 3 days (the culture medium was changed once daily). The cells were cultured until they reached approximately 70% confluency. After that, RPMI 1640 basal medium containing 300 μM AA2P, 2% B-27, VEGF, and bFGF were added, along with 10 μM SB431542, and the cells were cultured for an additional 4 days.

[0107] (1) Manufacturing Example 1-1 On day 2 of the additional culture, the ratio of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was determined by flow cytometry (FACS). Flow cytometry was performed using a BD Lyric instrument using BD anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies. The ratios of single- and double-positive cells for CD34 and CD41a, and CD45 and CD41a, respectively, were analyzed.

[0108] As a result, CD34 + The percentage of cells was 59.27%, CD45 + The cell ratio was confirmed to be 5.21%, and at this point, a floating cell population was obtained from the culture medium (day 7 in Figure 2 and Figure 3).

[0109] (2) Manufacturing Example 1-2 On the fourth day of the additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was determined using the same method as in Production Example 1-1. +The percentage of cells was 57.55%, CD41a + The percentage of cells was 54.55%, CD45 + The cell ratio was confirmed to be 18.58%, and at this point, a floating cell population was obtained from the culture medium (day 9 in Figure 2 and Figure 3).

[0110] (3) Manufacturing Example 1-3 On the sixth day of the additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was determined using the same method as in Production Example 1-1. + The percentage of cells was 61.59%, CD41a + The percentage of cells was 47.03%, CD45 + The cell ratio was confirmed to be 32.4%, and at this point, a floating cell population was obtained from the culture medium (day 11 in Figure 2 and Figure 3).

[0111] (4) Manufacturing Example 1-4 On the 8th day of the additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was determined using the same method as in Production Example 1-1. + The percentage of cells was 52.05%, CD41a + The percentage of cells was 18.88%, CD45 + The cell ratio was confirmed to be 45.56%, and at this point, a floating cell population was obtained from the culture medium (day 13 in Figure 2).

[0112] (5) Comparative Manufacturing Example 1-1 On the 10th day of the additional culture, the ratio of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was determined by flow cytometry. + The percentage of cells was 91.2%, CD41a + The percentage of cells is 13%, CD45 + The cell ratio was confirmed to be 68.51%, and at this point, a floating cell population was obtained from the culture medium (day 15 in Figure 2 and Figure 3).

[0113] 1-3. Induction of megakaryocyte differentiation by culturing floating cell populations The floating cell populations of Production Examples 1-2 and 1-4 were cultured to give 1×10 5 The cells were placed in IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of 100 μg / ml. 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 cells were cultured for 3 days at 37°C and 5% CO2. The medium composition was then changed to IMDM basal medium containing 300 μM AA2P, 2% B-27, and 100 ng / ml TPO. The cells were cultured for an additional 5 days for megakaryocyte maturation, yielding the cell cultures of Examples 1 and 2 (Figure 4). A photograph of the cell culture medium of Example 1 is shown in Figure 5.

[0114] The floating cell population of Comparative Production Example 1-1 was cultured to obtain 1×10 5 The cells were placed in IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of 100 μg / ml. 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 cells were cultured for 3 days at 37°C and 5% CO. The medium composition was then changed to IMDM basal medium containing 300 μM AA2P, 2% B-27, and 100 ng / ml TPO. The cells were cultured for an additional 5 days for megakaryocyte maturation, yielding a cell culture solution (Comparative Example 1) (FIGS. 6 and 7).

[0115] 1-4. Confirmation of megakaryocyte platelet production ability The cell culture media from Examples 1, 2, and Comparative Example 1 were collected and centrifuged at 300 x g for 3 minutes. The precipitate was separated as megakaryocytes, and the supernatant was separated as platelets. These were then separated and analyzed for platelet analysis. The characteristics of the separated platelets were investigated using the following experiments.

[0116] (1-4-1) Confirmation of megakaryocyte-specific markers by FACS analysis The cell culture solutions of Examples 1, 2 and Comparative Example 1 were centrifuged to obtain precipitated cells (cells thought to be megakaryocytes).

[0117] To confirm whether the precipitated cells were megakaryocytes, surface marker expression of the precipitated cells was confirmed by FACS analysis. For FACS analysis, a BD Lyric instrument was used, using BD anti-CD41a-FITC, anti-CD61-PE, and anti-CD42b-APC antibodies. The antibodies were diluted 1:20 in PBS containing 1% BSA (FACS buffer) and incubated for 30 minutes to confirm surface marker expression.

[0118] As a result, the precipitated cells of Example 1 were positive for the expression of megakaryocyte-specific markers (CD41a, CD42b, and CD61) (Figure 8). The precipitated cells of Example 2 were also positive for the expression of megakaryocyte-specific markers (CD41a, CD42b, and CD61) (Figure 9). These results confirmed that the floating cell populations of Examples 1 and 2 were successfully differentiated into megakaryocytes, and that platelets were also successfully produced accordingly.

[0119] In contrast, the precipitated cells from Comparative Example 1 had very low expression of CD41a and CD61, and in particular, CD42b expression was nearly negative (Figure 10). This indicates that the floating cell population from Comparative Example 1 hardly differentiated into megakaryocytes and hardly produced platelets.

[0120] (1-4-2) Confirmation of platelet function by ADP treatment Examples 1, 2, and Comparative Example 1 were centrifuged to remove precipitated cells (megakaryocytes), and the suspended cells were centrifuged again at 2000 x g for 10 minutes. Then, to recover the precipitated cells (cells considered to be platelets), all the suspended matter was removed and the cells were resuspended in a small amount of PBS. The following experiment was performed to confirm whether the cells thus obtained were platelets with normal activity.

[0121] For PAC-1 analysis, cells were treated with 100 μM ADP and incubated at room temperature for 15 minutes, then incubated with anti-PAC-1-FITC and anti-CD62p-PE antibodies (BD) for 30 minutes, followed by FACS analysis of the reacted cells.

[0122] The cells of Examples 1 and 2 were activated by ADP treatment, and the expression of PAC-1 and CD62p increased (FIGS. 11 and 12). This confirmed that the cells of Examples 1 and 2 were platelets with normal activity. PAC-1 is a complex formed by CD41a and CD61 upon platelet activation. CD62p (p-selectin) is a surface molecule whose expression increases upon platelet activation, and functions as a site for leukocytes to bind to platelets.

[0123] The above experimental results confirmed that the efficiency of differentiation into megakaryocytes and platelet production can be improved by culturing hiPSCs and performing subsequent culturing steps when the cell population meets certain conditions (i.e., the ratio of the number of cells expressing specific surface markers (e.g., CD41a, CD34, CD45) to the total number of cells is above or below a certain value).

[0124] 2. Comparative Example 2: CD41a from a floating cell population + Comparison of platelet production efficiency when only cells are selected and subsequently cultured CD41a was isolated from the floating cell population obtained in Production Example 1-2. + The platelet production efficiency when only the cells were selected (Comparative Example 2) and subsequently cultured was compared with that in Example 1 (FIG. 13).

[0125] The expression of platelet-specific markers in both cells was confirmed by the same method as the FACS analysis described above. + / CD42b + The ratios were similar at 57.58 and 56.20, confirming that there was no significant difference in the platelet differentiation rate (Figure 14).

[0126] However, as a result of examining the cell proliferation ability of both, as in Example 1, CD41a + Not only cells that are CD41a but also cells that are not CD41a - When a cell population containing CD41a was differentiated into platelets, + The cell proliferation ability was approximately 40 times higher than when only the cells were selected and differentiated into platelets (FIG. 15).

[0127] From the above results, CD41a + Rather than selecting only cells, we selected CD41a as in Example 1-2. - It was confirmed that platelets can be produced quantitatively and qualitatively more efficiently when a cell population containing both erythrocytes and other cells is used for the production of platelets.

[0128] 3. Examples 3 to 8 and Comparative Examples 3 and 4 3-1. Production of human induced pluripotent stem cells (hiPSCs) To obtain a cell population capable of differentiating into megakaryocytes, human induced pluripotent stem cells were cultured as follows.

[0129] First, human induced pluripotent stem cells were cultured in mTeSR plus culture medium at a density of 4500 cells / cm with 10 μg / ml of ROCK inhibitor (Y-27632). 2 The cells were placed in a T75 flask at a density of 100 μg / ml and cultured at 37°C in 5% CO2. After 24 hours of culture, the cells were washed once with PBS to remove Y-27632, and the mTeSR plus culture medium was replaced with fresh medium. The culture medium was changed once daily for three days, yielding human induced pluripotent stem cells.

[0130] 3-2. Preparation of culture medium containing human hematopoietic stem cells (hHSCs) The obtained human induced pluripotent stem cells were cultured for two days at 37°C and 5% CO2 in RPMI1640 basal medium containing 300 μM AA2P, 2% B-27, and 6 μM CHIR99021, a GSK3 inhibitor.

[0131] The medium was then changed to RPMI 1640 basal medium containing 300 μM AA2P, 2% B-27, 50 ng / ml VEGF, and 20 ng / ml bFGF, and cultured at 37°C in a 5% CO2 incubator for 3 days. Culture was continued until cell confluency reached approximately 90%. After that, RPMI 1640 basal medium containing 300 μM AA2P, 2% B-27, VEGF, and bFGF were added, along with 10 μM SB431542, and the cells were cultured for an additional 4 days.

[0132] Culture media was collected on days 7, 9, 11, 13, 15, 17, 19, and 21, and the number of cells expressing specific surface markers (CD34, CD41a, and CD45) relative to the total number of floating cells was determined by flow cytometry (FACS). Flow cytometry was performed using a BD Lyric instrument and BD anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies (Figure 16).

[0133] 3-3.CD41a in culture medium + , CD45 + and CD34 + Cell ratio CD41a in culture medium on days 7, 9, 11, 13, 15, 17, 19, and 21 of culture + , CD45 + and CD34 + The cell ratios are shown in Table 2 below.

[0134] [Table 2]

[0135] 3-4. Differentiation of the floating cell populations of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2 into megakaryocytes The floating cell populations of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2 were diluted to 0.5 × 10 5 The cells were placed in IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of 1 / ml, and 25 ng / ml SCF, 10 ng / ml IL-3, and 10 ng / ml IL-6 were added to the medium, followed by culturing at 37°C and 5% CO2 for 3 days.

[0136] The medium composition was then changed to IMDM basal medium containing 300 μM AA2P, 2% B-27, and 100 ng / ml TPO, and the cells were cultured for an additional 5 days to carry out the megakaryocyte maturation step, and cell culture medium was obtained.

[0137] 3-5. Confirmation of platelet production capacity in Examples 3 to 8 and Comparative Examples 3 and 4 The cell culture media of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2 that had undergone the megakaryocyte maturation step were each collected and centrifuged at 300 x g for 3 minutes. The precipitate, believed to be megakaryocytes, was removed, and the supernatant containing platelets was subjected to the following experiment to confirm whether the final product contained platelets with normal activity. The substances obtained from the cell culture media of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2 were named Examples 3 to 8 and Comparative Examples 3 and 4, in the order of their appearance.

[0138] (1) Confirmation of platelet-specific markers by FACS analysis The cell culture media of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2 that had undergone the megakaryocyte maturation step were centrifuged. The precipitated megakaryocytes were removed, and the suspended platelets were centrifuged again at 2000×g for 15 minutes.

[0139] FACS analysis was performed on megakaryocytes and potential platelets to confirm the expression of surface markers. For FACS analysis, BD Lyric instruments were used with BD anti-CD41a-FITC, anti-CD61-PE, and anti-CD42b-APC antibodies. The antibodies were diluted 1:20 in PBS containing 1% BSA (FACS buffer) and incubated for 30 minutes to confirm the expression of surface markers.

[0140] As a result, the expression of megakaryocyte- and platelet-specific markers was positive in Examples 3 to 8 (FIGS. 17 to 22). This confirmed that the floating cell populations of Production Examples 3-1 to 3-6 were capable of producing megakaryocytes and platelets well.

[0141] In contrast, surface marker characteristics of megakaryocytes and platelets were hardly observed in Comparative Examples 3 and 4. This confirmed that the floating cell populations of Comparative Production Examples 3-1 and 3-2 hardly differentiated into megakaryocytes and were substantially unable to produce platelets (FIGS. 23 and 24).

[0142] (2) Confirmation of platelet function by ADP treatment In order to confirm whether the platelets of Examples 3 to 8 and Comparative Examples 3 and 4 that had undergone the megakaryocyte maturation step had normal activity, the following experiment was carried out.

[0143] The cell culture media of Production Examples 3-1 to 3-6 and Comparative Production Examples 3-1 and 3-2 that had undergone the megakaryocyte maturation step were centrifuged. The precipitated megakaryocytes were removed, and the suspended platelets were centrifuged again at 2000 x g for 15 minutes. To recover the precipitated platelets by centrifugation, all the suspended matter was removed, and the platelets were resuspended in a small amount of PBS.

[0144] For PAC-1 analysis, platelets were treated with 500 μM ADP and incubated at room temperature for 15 minutes, followed by incubation with anti-PAC-1-FITC and anti-CD62p-PE antibodies (BD) for 30 minutes. The incubated platelets were immediately subjected to FACS analysis.

[0145] PAC-1 is a complex formed by CD41a and CD61 upon platelet activation. CD62p (p-selectin) is a surface molecule whose expression is increased upon platelet activation and serves as a site for leukocyte binding to platelets.

[0146] In Examples 3 to 8, platelets were activated by ADP treatment, and the expression of PAC-1 and CD62p increased. This confirmed that the platelets in Examples 3 to 8 had normal activity (Figs. 17 to 22). In contrast, in Comparative Examples 3 and 4, almost no platelet activity was observed (Figs. 23 and 24).

[0147] As mentioned above, CD41a - It was confirmed that the efficiency of differentiation into megakaryocytes and platelet production was improved when subsequent steps were performed using a floating cell population obtained from the culture medium when the cell number was at least 15% of the total cell number.

Claims

1. (S1) culturing pluripotent stem cells to obtain a culture solution containing hematopoietic stem cells; (S2) Among the cells contained in the culture medium, CD41a － harvesting a suspension cell population from the culture when the cell number is at least 15% of the total cell number; (S3) differentiating the floating cell population into megakaryocytes in a first culture medium.

2. The method for producing platelets according to claim 1, further comprising the step of (S4) culturing the megakaryocytes in a second medium containing thrombopoietin to mature them.

3. The method for producing platelets according to claim 1, wherein (S1) comprises the following steps: (S1a) culturing the pluripotent stem cells in a third medium containing a GSK3 inhibitor; (S1b) culturing the cells cultured in the third medium in a fourth 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 transforming growth factor beta signaling inhibitor.

4. The floating cell population is CD45 ＋ The method for producing platelets according to claim 1, wherein the number of cells is 50% or less of the total number of cells.

5. The floating cell population is CD34 ＋ The method for producing platelets according to claim 1, wherein the number of cells is 45% or more of the total number of cells.

6. The floating cell population is CD41a － The method for producing platelets according to claim 1, wherein the number of cells is 85% or less of the total number of cells.

7. (S0) The pluripotent stem cells are cultured at the bottom of a culture vessel at a density of 2,000 to 20,000 cells / cm ２ 2. The method of claim 1, further comprising the step of seeding with

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

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

10. In (S2), among the cells contained in the culture medium, CD41a ＋ 2. The method for producing platelets according to claim 1, wherein the floating cell population is obtained from the culture medium when the cell number is 40 to 85% of the total cell number.

11. In (S2), among the cells contained in the culture medium, CD34 ＋ 2. The method for producing platelets according to claim 1, wherein the floating cell population is obtained from the culture medium when the cell number is 45 to 80% of the total cell number.

12. In (S2), among the cells contained in the culture medium, CD45 ＋ 2. The method for producing platelets according to claim 1, wherein the floating cell population is obtained from the culture medium when the number of cells is 1 to 50% of the total number of cells.

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

14. The floating cell population is ＋ The ratio of cells to CD41a ＋ The method for producing platelets according to claim 4, wherein the ratio of platelets to cells is low.

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

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