KR102686850B1 - Method for producing platelets - Google Patents

Abstract

The present invention relates to a method for producing platelets. The manufacturing method of the present invention 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 not expressing cd41a among cells included in the culture medium is at least 15% of the total number of cells; and (s3) differentiating the floating cell population into megakaryocytes.

Description

Method for producing platelets {METHOD FOR PRODUCING PLATELETS}

The present invention relates to a method for producing platelets.

Platelet preparations are administered for the purpose of treating or preventing the symptoms of massive bleeding during surgery or injury, or to patients who exhibit a tendency to bleed due to thrombocytopenia following anticancer drug treatment.

Currently, the manufacture of platelet products relies on blood donations from healthy volunteers. The number of blood donors has been decreasing recently, and it is expected that there will be a blood shortage in the future. Therefore, stable supply of platelets is an important task in the field of technology.

Since conventional platelet products have a high risk of bacterial contamination, there is a possibility of causing serious infections after transplantation of platelet products. For this reason, safer platelet preparations are always required in clinical practice.

To meet that demand, a method for producing platelets from megakaryocytes cultured in vitro has recently been developed. However, platelets have not yet been obtained in high yield and the process cannot be performed economically, so technical improvement is needed.

Korean Patent Publication No. 10-2018-0063307

The purpose of the present invention is to provide a method for producing artificial platelets with high yield.

The purpose of the present invention is to provide a method for producing platelets with process efficiency.

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

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

3. In 1 above, (S1) is a method for producing platelets comprising the following steps: (S1a) culturing 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) cells cultured in the fourth medium containing vascular endothelial growth factor; basic fibroblast growth factor; and culturing in a fifth medium containing a transforming growth factor beta signaling inhibitor.

4. The method of producing platelets according to 1 above, wherein the floating cell population has a CD45 ⁺ cell number of 50% or less of the total cell number.

5. The method of producing platelets according to 1 above, wherein the floating cell population has a CD34 ⁺ cell number of 45% or more of the total cell number.

6. The method for producing platelets according to 1 above, wherein the floating cell population has a CD41a ^- cell number of 85% or less of the total cell number.

7. The method of producing platelets in 1 above, further comprising seeding (S0) gastric pluripotent stem cells at 2,000 to 20,000 cells/cm ² on the bottom of the culture medium.

8. The method of producing platelets according to 1 above, wherein the culture medium further contains hematopoietic progenitor cells and megakaryotic progenitor cells.

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

10. The method of producing platelets according to 1 above, wherein in (S2), a floating cell population is obtained from the culture medium when the number of CD41a ⁺ cells among the cells contained in the culture medium is 15% or more to less than 85% of the total number of cells. .

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

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

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

14. The method of producing platelets according to item 4 above, wherein the floating cell population has a lower proportion of CD45 ⁺ cells than the proportion of CD41a ⁺ cells.

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

The platelet production method of the present invention produces megakaryocytes from a suspended cell population containing at least 15% CD41a ^- cells, so there is no need to separate and purify CD41a ⁺ cells.

The method for producing platelets of the present invention allows for rapid and economical production of platelets because subsequent processes can be performed when the floating cell population satisfies predetermined requirements.

The platelet production method of the present invention has excellent differentiation efficiency into megakaryocytes and platelet production efficiency.

The method for producing platelets of the present invention has a simple process, does not require complex equipment, and is advantageous in terms of time and cost.

Platelets prepared according to the method of the present invention may be activated by stimulants (e.g., adenosine diphosphate (ADP), collagen, fibrinogen, etc.), resulting in increased expression of PAC-1 and CD62p. Through this, it can be confirmed that the platelets of the present invention are platelets that function normally.

Platelets prepared according to the method of the present invention can exhibit therapeutic or preventive effects in diseases related to thrombocytopenia or functional deficiency.

Figure 1 is an example of a culture process of human induced pluripotent stem cells.
Figure 2 shows the results of confirming surface markers (CD34, CD45, CD41a) of floating cell populations of Preparation Examples 1-1 to 1-4 and Comparative Preparation Example 1-1.
Figure 3 shows the results of confirming surface markers (CD34, CD45, CD41a) of floating cell populations of Preparation Examples 1-1 to 1-3 and Comparative Preparation Example 1-1.
Figure 4 shows the process for obtaining cell culture fluid in Examples 1 and 2.
Figure 5 is a photograph of the cell culture medium of Example 1.
Figure 6 shows the cell culture medium obtaining process of Comparative Example 1.
Figure 7 is a photograph of the cell culture fluid of Comparative Example 1.
Figure 8 shows that the cells of Example 1 express megakaryocyte-specific markers.
Figure 9 shows that cells of Example 2 express megakaryocyte-specific markers.
Figure 10 shows that the cells of Comparative Example 1 do not express megakaryocyte-specific markers.
Figure 11 shows that the cells of Example 1 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p.
Figure 12 shows that the cells of Example 2 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p.
Figure 13 shows the results of selecting CD41a ⁺ cells from the floating cell population of Preparation Example 1-2.
Figure 14 shows the ratio of CD41a ⁺ /CD42b ⁺ in Example 1 (Preparation Example 1-2) and Comparative Example 2.
Figure 15 shows that Example 1 shows a cell proliferation ability about 40 times higher than that of Comparative Example 2.
Figure 16 shows the results of confirming surface markers (CD34, CD45, CD41a) of floating cell populations of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2.
Figures 17-22 show that Examples 3-8 express megakaryocyte and platelet specific markers.
Figures 23 and 24 show that Comparative Examples 3 and 4 do not express megakaryocyte and platelet specific markers.

The present invention provides a method for producing platelets comprising 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 gastric culture when the number of cells that do not express CD41a among the cells contained in the gastric culture is at least 15% of the total number of cells; and (S3) differentiating the gastric floating cell population into megakaryocytes in the first medium.

The manufacturing method of the present invention may further include seeding (S0) gastric pluripotent stem cells at 2,000 to 20,000 cells/cm ² on the bottom of the incubator.

The production method of the present invention may further include the step (S4) of maturing megakaryocytes by culturing them in a second medium containing thrombopoietin.

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

The platelet manufacturing process from (S0) to (S5) will be described below.

(S0) stage

(S0) is a step of seeding pluripotent stem cells at 2,000 to 20,000 cells/cm ² on the bottom of the incubator.

Pluripotent stem cells include induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC).

Induced pluripotent stem cells (iPSCs) refer to cells that were differentiated cells that did not have pluripotency but gained pluripotency through an artificial dedifferentiation process.

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

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

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.

Induced pluripotent stem cells can use virtually any somatic cell at any developmental stage as a starting point. Somatic cells may be derived from, but are not limited to, embryonic, fetal, neonatal, juvenile, or adult donors. The somatic cells may be, but are not limited to, fibroblasts, such as skin fibroblasts obtained from a skin sample or biopsy, synoviocytes from synovial tissue, buccal cells, or lung fibroblasts.

Pluripotent stem cells are seeded on the bottom of the incubator and then attached and cultured.

The incubator can be used without limitation as long as it is used for cell culture in the art. For example, they can be large, medium and small incubators. It may also be a cell culture flask such as T25, T75, T175 or T225.

Pluripotent stem cells are preferably seeded at 2,000 to 20,000 cells/cm ² in terms of cell confluency. When seeded in this amount, 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. In the step (S1) described later, it is possible to obtain a culture medium containing a sufficient number of hematopoietic stem cells, hematopoietic progenitor cells, megakaryotic progenitor cells, etc.

If pluripotent stem cells are seeded on the bottom of the culture medium at less than 2,000 cells/cm ² , the cell confluency at the time of obtaining the floating cell population (S2) is 70% or less, 65% or less, 60% or less, or 55% or less. , 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, etc. Additionally, if pluripotent stem cells are seeded on the bottom of the incubator in excess of 20,000 cells/cm ² , cell confluency exceeds 100% at a time when the pluripotent stem cells have not been sufficiently cultured and differentiated, resulting in undifferentiated cells in the culture dish. They may fall off and become suspended and die, or the amount of factors added to the culture medium may become insufficient, which may result in poor signaling between cells, resulting in poor differentiation.

(S1) step

The (S1) step is a step of culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells. This step is to culture the pluripotent stem cells seeded in the (S0) step to obtain a floating cell population that can be differentiated into megakaryocytes.

The (S1) step may be composed of the following detailed steps:

(S1a) culturing pluripotent stem cells in a third medium containing 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) Cells cultured in medium 4 were treated with vascular endothelial growth factor; basic fibroblast growth factor; and culturing in a fifth medium containing a transforming growth factor beta signaling inhibitor.

The third, fourth, and fifth media used in this step are media with different compositions and purposes.

The third medium contains at least a GSK3 inhibitor. GSK3 inhibitors may be CHIR99021, BIO (6-bromoindirubin-30-oxime), SB216763, CHIR-98014, CT98014, CT98023, CT99021, TWS119, SB41528, AR-A014418, AZD-1080, Alsterpaullone, Cazpaullone or Kenpaullone. limited to this It doesn't work. CHIR99021 is a GSK3 inhibitor, a Wnt signaling agonist, and can be expressed as an aminopyrimidine.

The GSK3 inhibitor (eg CHIR99021) is not limited to a specific concentration as long as the amount is sufficient to cultivate pluripotent stem cells. The GSK3 inhibitor may be included in the third medium at a concentration, for example, of 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. .

The third medium is Basic Medium. The third medium may include RPMI1640 medium or another type of basic medium. The third medium may further contain antioxidants and/or B-27 along with the base medium.

Antioxidants include 6-hydroxymelatonin, acetyl-L-carnitine (ALCAR), alpha-lipoic acid (ALA), and 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, Ladostigil, Melatonin, Mofegiline, N-Acetylcysteine (NAC), N-Acetyl Serotonin (N-Acetylserotonin, NAS), Oleocanthal, Oleuropein, Rasagiline, Resveratrol, Selegiline, Selenium, Tocopherols, vitamin E), tocotrienols, tyrosol, ubiquinone (coenzyme Q), and uric acid.

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

Vascular Endothelial Growth Factor (VEGF) is a member of the Epidermal Growth Factor Receptor (EGFR/ErbB). Vascular endothelial growth factorSMS plays an essential role in regulating cell proliferation and differentiation, resulting in the activation of various types of signaling pathways, leading to apoptosis, survival, or cell proliferation. Vascular endothelial growth factors include, for example, vascular endothelial growth factors of humans and non-human animals (mice, etc.).

Vascular endothelial growth factor is included at a concentration that can appropriately culture pluripotent stem cells. Vascular endothelial growth factor is, 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 70 ng/ml. It may be included in the fourth medium at a concentration of 65 ng/ml, 40 to 60 ng/ml, 45 to 55 ng/ml, or 50 ng/ml.

Basic Fibroblast Growth Factor (bFGF) is a protein belonging to the FGF family that functions as a mitogenic factor, angiogenesis factor, bone formation factor, and nerve growth factor, as well as cell proliferation and cell differentiation. Basic fibroblast growth factor, also called FGF2, mainly activates receptor proteins including FGFR1b, FGFR1c, FGFR2c, FGFR3c, and FGFR4c, and especially strongly activates FGFR1c and FGFR3c.

Basic fibroblast growth factor is included along with other ingredients at a concentration that allows for appropriate culturing of pluripotent stem cells. Basic fibroblast growth factor may be included in the fourth 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.

The fourth medium may not contain transforming growth factor beta (TGFβ) signaling inhibitor. If the fourth medium contains an inhibitor of TGFβ signaling, differentiation may occur without sufficient cell proliferation.

The fifth medium is the 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.

Matters regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) of medium 5 apply mutatis mutandis to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) of medium 4.

The fifth medium contains an inhibitor of transforming growth factor beta (TGFβ) signaling.

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

The TGFβ signaling inhibitor can be used without limitation as long as it is a substance that can inhibit TGFβ signaling. For example, it may be an Activin Receptor-like Kinase (ALK) receptor inhibitor.

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

Transforming growth factor beta (TGFβ) signaling inhibitor is included along with other ingredients at a concentration that allows for culturing pluripotent stem cells. Transforming growth factor beta (TGFβ) signaling inhibitor may be included at a concentration of, for example, 1 to 20 μM, 5 to 15 μM, or 10 μM.

The culture medium obtained through the (S1) stage of culture contains hematopoietic stem cells (HSC). The culture medium may further contain hematopoietic progenitor cells (HPC) and megakaryocyte progenitor cells (MK-P).

(S2) step

The (S2) step is a step of obtaining a floating cell population from the culture medium when the number of cells that do not express CD41a (CD41a ^- cells) among the cells included in the culture medium of (S1) is at least 15% of the total number of cells.

Floating cell population refers to cells and/or cell populations floating in a culture medium. The floating cell population contains a large number of cells that can be differentiated into megakaryocytes, such as hematopoietic stem cells, hematopoietic progenitor cells, and megakaryotic progenitor cells. Although they do not directly differentiate into megakaryocytes, they include hematopoietic stem cells, hematopoietic progenitor cells, and megakaryotic progenitor cells. It may contain cells that help it differentiate better than when it is alone.

This step determines the optimal time to obtain a floating cell population from the culture medium in (S1). Through this step, the optimal floating cell population for differentiation into megakaryocytes can be obtained, thereby increasing the differentiation efficiency into megakaryocytes and/or mature megakaryocytes and the efficiency of platelet production.

The point at which the floating cell population is obtained is when the number of CD41a ⁻ cells is at least 15% of the total cell number. That is, the number of cells expressing CD41a (CD41a ⁺ cells) is less than 85% of the maximum. When the number of CD41a ⁺ cells is less than 85%, the efficiency of differentiation into megakaryocytes and platelet production is excellent, and when the number of CD41a ⁺ cells is more than 85%, the efficiency of platelet production is rather low.

CD41a ^- cell count is, for example, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%, more than 21%, more than 22%, more than 23%, more than 24%, More than 25%, more than 26%, more than 27%, more than 28%, more than 29%, more than 30%, more than 31%, more than 32%, more than 33%, more than 34%, more than 35%, more than 36%, 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, More than 50%, more than 51%, more than 52%, more than 53%, more than 54%, more than 55%, more than 56%, more than 57%, more than 58%, more than 59%, more than 60%, more than 61%, 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 higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher, 82% or higher, 83% or higher, and 84% or higher.

CD41a ^- cell count 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%, 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, 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.

The percentage of CD41a ⁺ cells can be obtained by subtracting the percentage of CD41a ^- cells from 100%.

In (S2), the number of CD41a ⁺ cells among the cells contained in the culture medium is 15% or more to less than 85%, 20% or more to less than 85%, 30% or more to less than 85%, and 40% or more to 85% of the total cell number. It is preferable that it is less than 45% to less than 85% or more than 50% to less than 85%.

The number of cells expressing CD45, along with whether the cells in the culture express CD41a, can be taken into account to determine when to obtain a floating cell population. CD45 ⁺ cell count 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 of the total cell count. % or less, 39% or less, 38% or less, 37% or less, 36% or less, or 35% or less, a floating cell population can be obtained from the culture medium.

Additionally, the CD45 ⁺ 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, or 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 floating cell population can be obtained from the culture medium.

In one embodiment, the CD45 ⁺ cell count is 1-50%, 5-50%, 10-50%, or 15-50%.

The number of cells expressing CD34, along with whether the cells in the culture express CD41a, can be taken into account to determine when to obtain a floating cell population. CD34 ⁺ cell count, for example, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, 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 more than 75%, a floating cell population can be obtained from the culture medium.

In addition, the CD34 ⁺ 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 , a floating cell population can be obtained from the culture medium when the concentration is 54% or less, 53% or less, 52% or less, 51% or less, or 50% or less.

In addition to whether the cells in the culture express CD41a, a floating cell population can be obtained when the proportion of CD41a ⁺ cells is higher than the proportion of CD45 ⁺ cells.

Even if the percentage of CD41a ⁺ cells in the total cell population is low, below 50%, differentiation into megakaryocytes and platelets can easily occur if the percentage of CD45 ⁺ cells is lower than that.

By checking whether the cells in the culture medium express CD41a and, in addition, CD45 and CD34, there is no need to separate or purify only cells that have the potential to differentiate into megakaryocytes from the entire cell population.

The method for counting the number of cells can be any conventional technique in the art without limitation. For example, antibody-based selection methods or sorter machines can be used. An antibody-based selection method may, for example, use microbeads.

(S3) step

Step (S3) is a step of differentiating the floating cell population obtained in step (S2) into megakaryocytes in the first medium.

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

Thrombopoietin is a major growth factor that regulates megakaryocyte and platelet hematopoiesis and is mainly synthesized and secreted by hepatocytes.

Thrombopoietin can be included in the first medium along with other components at a concentration that allows for culturing a suspended cell population, and does not have to be included at a specific concentration. Thrombopoietin may be included in concentrations, for example, of 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. .

Stem cell factor is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. Stem cell factors may be included in the first medium along with other components at a concentration that allows for culturing a suspended cell population, and do not have to be included at a specific concentration. Thrombopoietin may be included in concentrations, for example, of 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. .

Interleukin-3 can be included in the first medium along with other components at a concentration that allows for culturing a suspended cell population, and does not have to be included at a specific concentration. Interleukin-3 may be included at a concentration of, for example, 1 to 20 ng/ml, 5 to 15 ng/ml or 10 ng/ml.

Interleukin-6 can be included in the first medium along with other components at a concentration that allows for culturing a suspended cell population, and does not have to be included at a specific concentration. Interleukin-6 may be included at a concentration of, for example, 1 to 20 ng/ml, 5 to 15 ng/ml or 10 ng/ml.

The first medium is not limited to a specific medium. The first medium is a basic medium and may include IMDM (Iscove's Modified Dulbecco's Medium) medium. IMDM medium may further include antioxidants (such as AA2P, etc.) and/or B-27.

(S4) step

Step (S4) is a step of maturing the megakaryocytes of (S3) by culturing them in a second medium containing thrombopoietin.

Thrombopoietin is added to the second medium at a concentration, for example, of 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. may be included, but is not limited thereto.

The second medium may further contain factors necessary for maturation of megakaryocytes in addition to thrombopoietin.

The second medium is not limited to a specific medium. The second medium is a basic medium and may include IMDM (Iscove's Modified Dulbecco's Medium) medium. IMDM medium may further contain antioxidants (e.g. Ascorbic Aacid 2-Phosphate, AA2P, etc.) and/or B-27.

What is obtained in this step may be mature megakaryocytes or a culture medium containing megakaryocytes and/or mature megakaryocytes.

(S5) step

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

This step may be to separate and/or purify platelets from the culture medium after further differentiating the culture medium of (S4), or to separate and/or purify platelets from the culture medium without further differentiating the culture medium of (S4).

Separation and/or purification of platelets may be performed by known separation and/or purification methods. For example, it can be performed by centrifuging the culture solution or passing the culture solution through a column.

When centrifuging the culture medium, megakaryocytes can be separated as sediment and platelets can be separated as suspension.

The present invention will be described in more detail through examples below.

Example

1. Example 1, Example 2 and Comparative Example 1

1-1. Preparation of human induced pluripotent stem cells (hiPSC)

To obtain a cell population that can be differentiated into megakaryocytes, human induced pluripotent stem cells were cultured as follows (Figure 1).

First, human induced pluripotent stem cells were placed in a T75 flask at a density of 4000 cells/cm ² in mTeSR plus culture medium with 10 ㎍/㎖ of ROCK inhibitor (Y-27632) and cultured at 37°C and 5% CO ₂ conditions. . 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 a new one. The culture medium was replaced once a day and cultured for 3 days to obtain human induced pluripotent stem cells.

1-2. Preparation of culture medium containing human hematopoietic stem cells (hHSC)

The obtained human induced pluripotent stem cells were cultured in RPMI1640 basic medium containing 300 μM AA2P and 2% B-27 and medium containing 6 μM CHIR99021, a GSK3 inhibitor, at a temperature of 37°C and 5% CO ₂ for 2 days ( (Replace culture medium once a day).

Afterwards, the medium composition was changed to RPMI1640 basic medium containing 300 μM AA2P and 2% B-27 and medium containing 50 ng/ml VEGF and 20 ng/ml bFGF, and incubated at 37°C and 5% CO ₂ incubator for 3 days. (culture medium changed once a day). Culture was continued until the cell confluency was approximately 70% or more. Afterwards, RPMI1640 basic medium containing 300 μM AA2P and 2% B-27 and 10 μM SB431542 along with VEGF and bFGF were added and cultured for an additional 4 days.

(1) Production Example 1-1

On the second day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was confirmed through flow cytometry (FACS). For flow cytometry, BD's Lyric equipment was used, and BD's anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies were used. Through this, the ratio of single or double positive cells of CD34 and CD41a and CD45 and CD41a were analyzed, respectively.

As a result, it was confirmed that the percentage of CD34 ⁺ cells compared to the total cells was 59.27% and the percentage of CD45 ⁺ cells was 5.21%, and a floating cell population was obtained from the culture medium at this time (day 7 and Figure 3).

(2) Production Example 1-2

On the fourth day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was confirmed in the same manner as Preparation Example 1-1. As a result, it was confirmed that the ratio of CD34 ⁺ cells was 57.55%, the ratio of CD41a ⁺ cells was 54.55%, and the ratio of CD45 ⁺ cells was 18.58% compared to the total cells. At this point, a floating cell population was collected from the culture medium. Obtained (day 9 in Figure 2 and Figure 3).

(3) Production Example 1-3

On the 6th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was confirmed in the same manner as Preparation Example 1-1. As a result, it was confirmed that the proportion of CD34 ⁺ cells was 61.59%, the proportion of CD41a ⁺ cells was 47.03%, and the proportion of CD45 ⁺ cells was 32.4% compared to the total cells. At this point, a floating cell population was obtained from the culture medium (Figure Day 11 of 2 and Figure 3).

(4) Production Example 1-4

On the 8th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was confirmed in the same manner as Preparation Example 1-1. As a result, it was confirmed that the proportion of CD34 ⁺ cells was 52.05%, the proportion of CD41a ⁺ cells was 18.88%, and the proportion of CD45 ⁺ cells was 45.56% compared to the total cells. At this point, a floating cell population was obtained from the culture medium (Figure Day 13 of 2).

(5) Comparative Manufacturing Example 1-1

On the 10th day of additional culture, the ratio of the number of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of floating cells was confirmed through flow cytometry. As a result, it was confirmed that the proportion of CD34 ⁺ cells was 91.2%, the proportion of CD41a ⁺ cells was 13%, and the proportion of CD45 ⁺ cells was 68.51% compared to the total cells. At this point, a floating cell population was obtained from the culture medium (Figure Day 15 of 2 and Figure 3).

1-3. Inducing differentiation into megakaryocytes by culturing floating cell populations

The floating cell populations of Preparation Examples 1-2 and 1-4 were cultured and added to IMDM basic medium containing 300uM AA2P and 2% B-27 at a density of 1 × 10 ⁵ cells/ml, and 25 ng/ml was added to the medium. TPO, 25 ng/ml SCF, 10 ng/ml IL-3, and 10 ng/ml IL-6 were added and cultured at 37°C, 5% CO ₂ for 3 days. Afterwards, the medium composition was changed to a medium containing 100 ng/mL TPO in IMDM basic medium containing 300uM AA2P and 2% B-27, and cultured for additional 5 days to perform a megakaryocyte maturation step according to Examples 1 and 2. A cell culture medium was obtained (Figure 4). A photo of the cell culture medium of Example 1 is shown in Figure 5.

The floating cell population of Comparative Preparation Example 1-1 was cultured and added to IMDM basic medium containing 300uM AA2P and 2% B-27 at a density of 1×10 ⁵ cells/ml, and the medium was supplemented with 25 ng/ml TPO, 25 ng/ml SCF, 10 ng/ml IL-3 and 10 ng/ml IL-6 were added and cultured at 37°C and 5% CO ₂ for 3 days. Afterwards, the medium composition was changed from IMDM basic medium containing 300uM AA2P and 2% B-27 to medium containing 100 ng/ml TPO and further cultured for 5 days to perform the megakaryocyte maturation step and cell culture medium (comparative example) 1) was obtained (Figures 6 and 7).

1-4. Confirmation of platelet production ability of megakaryocytes

The cell culture fluids of Example 1, Example 2, and Comparative Example 1 were collected and centrifuged at 300 xg for 3 minutes. The sediment was separated into megakaryocytes and the suspended matter was separated into platelets, and analysis of the platelets was performed. The characteristics of the isolated platelets were confirmed through the following experiment.

(1-4-1) Confirmation of megakaryocyte-specific markers through FACS analysis

The cell cultures of Examples 1, 2, and Comparative Example 1 were centrifuged to obtain precipitated cells (cells believed to be megakaryocytes).

To confirm whether the precipitated cells were megakaryocytes, FACS analysis was performed to confirm the expression of surface markers of the precipitated cells. For FACS analysis, BD's Lyric equipment was used, and BD's anti-CD41a-FITC, anti-CD61-PE, and anti-CD42b-APC antibodies were used. The antibody was diluted 1:20 in PBS (FACS buffer) containing 1% BSA, reacted for 30 minutes, and surface marker expression was confirmed.

As a result, the precipitated cells of Example 1 had positive expression of megakaryocyte-specific markers (CD41a, CD42b, and CD61) (FIG. 8). In addition, the precipitated cells of Example 2 also showed positive expression of megakaryocyte-specific markers (CD41a, CD42b, and CD61) (FIG. 9). Therefore, it can be seen that the floating cell populations of Examples 1 and 2 were well differentiated into megakaryocytes and accordingly produced platelets well.

On the other hand, the sediment cells of Comparative Example 1 showed very low expression of CD41a and CD61, and in particular, CD42b expression was close to negative (Figure 10). From this, it can be seen that the floating cell population of Comparative Example 1 was hardly differentiated into megakaryocytes and hardly produced platelets.

(1-4-2) Confirmation of platelet function through ADP treatment

Example 1, Example 2, and Comparative Example 1 were centrifuged to remove precipitated cells (megakaryocytes), and the suspended cells were centrifuged again at 2000 xg for 10 minutes. To collect the cells precipitated by centrifugation (cells thought to be platelets), all suspended matter was removed and resuspended in a small amount of PBS. The following experiment was performed to confirm that the cells obtained in this way were platelets with normal activity.

For PAC-1 analysis, cells were treated with 100 μM of ADP, incubated at room temperature for 15 minutes, and then incubated with BD's anti-PAC-1-FITC and anti-CD62p-PE antibodies for 30 minutes. Afterwards, FACS analysis was performed on the reacted cells.

The cells of Examples 1 and 2 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p (Figures 11 and 12), confirming that the cells of Examples 1 and 2 were platelets with normal activity. PAC-1 appears as a complex of 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 for white blood cells to bind to platelets.

Based on the above-mentioned experimental results, it can be seen that after culturing hiPSCs, the cell population satisfies a predetermined condition (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 predetermined value). It was confirmed that the differentiation efficiency into megakaryocytes and platelet production efficiency can be increased when performing the subsequent culture step.

2. Comparative Example 2: CD41a in suspended cell population ⁺ Comparison of platelet production efficiency when only cells were selected and subsequently cultured

Only CD41a ⁺ cells were selected from the floating cell population obtained in Preparation Example 1-2 (Comparative Example 2) and subsequently cultured to compare the platelet production efficiency with Example 1 (FIG. 13).

As a result of confirming the expression of platelet-specific markers in the two cases using the same method as the FACS analysis above, the ratios of CD41a ⁺ /CD42b ⁺ were similar at 57.58 and 56.20, confirming that there was no significant difference in platelet differentiation rate (Figure 14) .

However, as a result of checking the cell proliferation ability in both cases, as in Example 1, in the case of differentiating a cell population including CD41a ⁺ cells as well as non-CD41a cells (e.g., CD41a ^- ) into platelets, only CD41a ⁺ cells were used. It was confirmed that the cell proliferation ability was approximately 40 times higher than when differentiated into platelets by selection (FIG. 15).

As a result of the above, rather than selecting only CD41a ⁺ cells and using them for platelet production, platelets can be produced quantitatively and qualitatively more efficiently by using a cell population that also includes CD41a ^- cells as in Preparation Example 1-2 for platelet production. It was confirmed that it was possible.

3. Examples 3 to 8 and Comparative Examples 3 and 4

3-1. Preparation of human induced pluripotent stem cells (hiPSC)

To obtain a cell population that can be differentiated into megakaryocytes, human induced pluripotent stem cells were cultured as follows.

First, human induced pluripotent stem cells were placed in a T75 flask at a density of 4500 cells/cm ² in mTeSR plus culture medium with 10 ㎍/㎖ of ROCK inhibitor (Y-27632) and cultured at 37°C and 5% CO ₂ conditions. . 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 a new one. The culture medium was replaced once a day and cultured for 3 days to obtain human induced pluripotent stem cells.

3-2. Preparation of culture medium containing human hematopoietic stem cells (hHSC)

The obtained human induced pluripotent stem cells were cultured in RPMI1640 basic medium containing 300 μM AA2P and 2% B-27 and medium containing 6 μM CHIR99021, a GSK3 inhibitor, at a temperature of 37°C and 5% CO ₂ for 2 days.

Afterwards, the medium composition was changed to RPMI1640 basic medium containing 300 μM AA2P and 2% B-27 and medium containing 50 ng/ml VEGF and 20 ng/ml bFGF, and incubated at 37°C and 5% CO ₂ incubator for 3 days. It was cultured in . Culture was continued until the cell confluency was approximately 90% or more. Afterwards, RPMI1640 basic medium containing 300 μM AA2P and 2% B-27 and 10 μM SB431542 along with VEGF and bFGF were added and cultured for an additional 4 days.

Culture media were obtained on days 7, 9, 11, 13, 15, 17, 19, and 21 of culture, and specific surface markers (CD34, CD41a) were compared to the total number of floating cells through flow cytometry (FACS). and CD45) were confirmed. For flow cytometry, BD's Lyric equipment was used and BD's anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies were used (FIG. 16).

3-3. CD41a in culture ⁺ , CD45 ⁺ and CD34 ⁺ proportion of cells

The proportions of CD41a ⁺ , CD45 ⁺ and CD34 ⁺ cells in the culture medium on days 7, 9, 11, 13, 15, 17, 19 and 21 of culture are shown in Table 1 below.

division(%) Culture date CD41a ⁺ cell ratio CD45 ⁺ cell ratio CD34 ⁺ cell ratio Manufacturing Example 3-1 7 61.13 1.22 56.32 Manufacturing Example 3-2 9 82.74 8.57 77.01 Manufacturing Example 3-3 11 84.44 19.13 78.83 Manufacturing Example 3-4 13 51.68 34.72 50.56 Manufacturing Example 3-5 15 29.86 49.26 47.14 Manufacturing Example 3-6 17 20.00 46.20 46.38 Comparative Manufacturing Example 3-1 19 14.63 37.72 29.07 Comparative Manufacturing Example 3-2 21 9.68 47.09 18.16

3-4. Differentiation of floating cell populations of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 into megakaryocytes

The suspended cell populations of Preparation Examples 3-1 to 3-6 and Comparative Preparation Examples 3-1 and 3-2 were cultured in IMDM base containing 300 μM AA2P and 2% B-27, respectively, at a density of 0.5 × 10 ⁵ cells/ml. It was added to the medium, 25 ng/ml SCF, 10 ng/ml IL-3, and 10 ng/ml IL-6 were added to the medium, and cultured at 37°C and 5% CO ₂ for 3 days.

Afterwards, the medium composition was changed to IMDM basic medium containing 300 μM AA2P and 2% B-27 and medium containing 100 ng/ml TPO, and cultured for additional 5 days to perform the megakaryocyte maturation step to obtain cell culture fluid. did.

3-5. Confirmation of platelet production ability of Examples 3 to 8 and Comparative Examples 3 and 4

Preparation examples 3-1 to 3-6 following megakaryocyte maturation step and Comparative Preparation Examples 3-1 and 3-2, respectively, were collected and centrifuged at 300 xg for 3 minutes. The precipitate that appears to be megakaryocytes was removed, and the suspension containing platelets was subjected to the following experiment to confirm whether the final product was platelets with normal activity. The materials obtained from the cell culture fluids 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 in the order of description.

(1) Confirmation of platelet-specific markers through FACS analysis

The cell cultures of 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 to remove precipitated megakaryocytes, and the suspended platelets were centrifuged at 2000 xg for 15 minutes. Centrifuged.

FACS analysis was performed on cells thought to be megakaryocytes and platelets to confirm the expression of surface markers. For FACS analysis, BD's Lyric equipment was used, and BD's anti-CD41a-FITC, anti-CD61-PE, and anti-CD42b-APC antibodies were used. The antibody was diluted 1:20 in PBS (FACS buffer) containing 1% BSA, reacted for 30 minutes, and surface marker expression was confirmed.

As a result, Examples 3 to 8 showed positive expression of megakaryocyte and platelet specific markers (FIGS. 17 to 22). Through this, it was confirmed that the suspended cell populations of Preparation Examples 3-1 to 3-6 were capable of producing megakaryocytes and platelets well.

On the other hand, Comparative Examples 3 and 4 showed almost no surface marker characteristics of megakaryocytes and platelets. Accordingly, it was confirmed that the floating cell populations of Comparative Preparation Examples 3-1 and 3-2 were hardly differentiated into megakaryocytes and were substantially unable to produce platelets (FIGS. 23 and 24).

(2) Confirmation of platelet function through ADP treatment

The following experiment was performed to confirm whether Examples 3 to 8 and Comparative Examples 3 and 4, which had undergone the megakaryocyte maturation step, were platelets with normal activity.

The cell cultures of 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 to remove precipitated megakaryocytes, and the suspended platelets were centrifuged at 2000 xg for 15 minutes. Centrifuged. To collect platelets precipitated by centrifugation, all suspended solids were removed and resuspended in a small amount of PBS.

For PAC-1 analysis, platelets were treated with 500 μM of ADP, reacted at room temperature for 15 minutes, and incubated with BD's anti-PAC-1-FITC and anti-CD62p-PE antibodies for 30 minutes. Reacted platelets were immediately subjected to FACS analysis.

PAC-1 appears as a complex of 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 for white blood cells to bind to platelets.

Examples 3 to 8 were activated by ADP treatment and increased PAC-1 and CD62p expression, confirming that Examples 3 to 8 were platelets with normal activity (FIGS. 17 to 22). On the other hand, Comparative Examples 3 and 4 showed little platelet activity (Figures 23 and 24).

As described above, it was confirmed that the differentiation efficiency into megakaryocytes and platelet production efficiency were increased when the subsequent steps were performed with the floating cell population obtained from the culture medium when the number of CD41a ^- cells was at least 15% of the total cell number.

Claims (15)

(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 CD41a ^- cells among the cells contained in the culture medium is at least 15% and 85% or less of the total number of cells; and
(S3) A method for producing platelets comprising differentiating the floating cell population into megakaryocytes in the first medium.
The method of claim 1, further comprising (S4) maturing the megakaryocytes by culturing them in a second medium containing thrombopoietin.
The method of 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) Cells cultured in the fourth medium were treated with vascular endothelial growth factor; basic fibroblast growth factor; and culturing in a fifth medium containing a transforming growth factor beta signaling inhibitor.
The method of claim 1, wherein the number of CD45 ⁺ cells in the floating cell population is 50% or less of the total number of cells.
The method of claim 1, wherein the floating cell population has a CD34 ⁺ cell count of 45% or more of the total cell count.
delete The method of claim 1, further comprising seeding the pluripotent stem cells at 2,000 to 20,000 cells/cm ² on the bottom of the culture medium (S0).
The method of claim 1, wherein the culture medium further contains hematopoietic progenitor cells and megakaryotic progenitor cells.
The method of claim 1, wherein the first medium contains thrombopoietin, stem cell factor, interleukin-3, and interleukin-6.
The method of claim 1, wherein in (S2), the floating cell population is obtained from the culture medium when the number of CD41a ⁺ cells among the cells included in the culture medium is 40 to 85% of the total number of cells.
The method of claim 1, wherein in (S2), the floating cell population is obtained from the culture medium when the number of CD34 ⁺ cells among the cells included in the culture medium is 45 to 80% of the total number of cells.
The method of claim 1, wherein in (S2), the floating cell population is obtained from the culture medium when the number of CD45 ⁺ cells among the cells included in the culture medium is 1 to 50% of the total number of cells.
The method of producing platelets according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells.
The method of claim 4, wherein the floating cell population has a ratio of CD45 ⁺ cells lower than the ratio of CD41a ⁺ cells.
delete