Method for producing red blood cells and platelets

Abstract

The present invention relates to a method for producing erythrocytes, and more specifically, to a method comprising the steps of: (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) obtaining a CD41- / CD34-cell population from the cell culture medium; and (S3) maturing the obtained cell population in a first medium containing erythropoietin, and thus high-quality erythrocytes can be produced in a short time.

Description

Technical Field

[0001] This invention relates to a method for producing red blood cells and / or platelets. Background Technology

[0002] Blood transfusions are a vital treatment for patients suffering from blood deficiency due to various reasons.

[0003] However, the number of blood donors is decreasing as the aging population leads to a decline in the number of eligible blood donors. Furthermore, the recent spread of infectious diseases such as Covid-19 has resulted in extreme blood shortages.

[0004] The supply of blood from donors involves the risk of infectious disease transmission and other transfusion-related adverse reactions. Therefore, a stable supply of red blood cells and platelets is an important task in related technical fields.

[0005] Hemoglobin solutions or oxygen carriers have been tried as alternatives to red blood cells (which are used for oxygen transport between blood components), but they have shown low oxygen transport capacity or serious side effects. Therefore, there is a need to produce red blood cells and platelets that can eliminate immunological side effects and the risk of infection.

[0006] To meet this demand, recent research has focused on the in vitro production of red blood cells and platelets. However, conventional methods for producing red blood cells or platelets suffer from time-consuming processes and the inability to produce products at high yields. Furthermore, because these materials must be produced individually, the process efficiency and economic efficiency are low. Therefore, technological improvements are still needed for economical, large-scale production methods of artificial red blood cells and platelets. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] The purpose of this invention is to provide a method for producing red blood cells in a short time.

[0009] Another object of the present invention is to provide a method for producing red blood cells in high yield.

[0010] Another object of the present invention is to provide a method for simultaneously producing red blood cells and platelets.

[0011] Another object of the present invention is to provide a method for producing red blood cells with high process efficiency.

[0012] A further object of the present invention is to provide a method for simultaneously producing red blood cells and platelets with high process efficiency.

[0013] Solution for solving the problem

[0014] 1. A method for producing red blood cells, comprising: (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) obtaining CD41 from said culture medium. - / CD34 - Cell population; and (S3) maturate the obtained cell population in a first medium containing erythropoietin (EPO).

[0015] 2. The method according to 1 above further includes: proliferating the obtained cell population in a second culture medium containing erythropoietin, stem cell factor and interleukin-3.

[0016] 3. The method according to 1 above further includes: differentiating the obtained cells in a third culture medium containing erythropoietin and stem cell factors.

[0017] 4. According to the method of 1 above, wherein the culture medium contains erythrocyte precursor and megakaryocyte precursor.

[0018] 5. According to the method described in 1 above, the maturation step is carried out for 4 to 14 days.

[0019] 6. According to the method of 1 above, wherein (S1) comprises: (S1a) culturing pluripotent stem cells in a fourth culture medium containing a GSK3 inhibitor; (S1b) culturing the cells cultured in the fourth culture medium in a fifth culture medium containing vascular endothelial growth factor and basic fibroblast growth factor; and (S1c) culturing the cells cultured in the fifth culture medium in a sixth culture medium containing vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor β signaling inhibitors.

[0020] 7. The method according to 1 above further includes: (S0) dispensing pluripotent stem cells at a rate of 2,000 to 20,000 cells / cm³. 2 The inoculum is seeded at a density at the bottom of the culture container.

[0021] 8. The method according to 1 above, wherein the pluripotent stem cells include human induced pluripotent stem cells.

[0022] 9. A method for producing blood products, comprising a process of mixing red blood cells produced by any one of the methods described in 1 to 8 above with other blood components.

[0023] 10. A method for producing red blood cells and platelets, comprising: (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) sorting CD41 from said culture medium. - / CD34 - Cell population and CD41a+ Or CD34 + Cell population; (S3) to sort CD41 - / CD34 - The cell population matured in a first medium containing erythropoietin; and (S4) the sorted CD41a cells were then subjected to further treatment. + or CD34 + The cell population differentiated into megakaryocytes in the seventh culture medium.

[0024] 11. According to the method in 10 above, it further includes: making the sorted CD41 - / CD34 - The cell population proliferated in a secondary culture medium containing erythropoietin, stem cell factor, and interleukin-3.

[0025] 12. According to the method in 10 above, it further includes: making the sorted CD41 - / CD34 - The cell population differentiated in a third culture medium containing erythropoietin and stem cell factors.

[0026] 13. The method according to 10 above, wherein the culture medium contains erythrocyte precursor and megakaryocyte precursor.

[0027] 14. According to the method in 10 above, (S3) is carried out for 4 to 14 days.

[0028] 15. The method according to 10 above, wherein (S1) comprises: (S1a) culturing pluripotent stem cells in a fourth culture medium containing a GSK3 inhibitor; (S1b) culturing the cells cultured in the fourth culture medium in a fifth culture medium containing vascular endothelial growth factor and basic fibroblast growth factor; and (S1c) culturing the cells cultured in the fifth culture medium in a sixth culture medium containing vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor β signaling inhibitors.

[0029] 16. The method according to 10 above further includes: dispensing pluripotent stem cells (S0) at a rate of 2,000 to 20,000 cells / cm³. 2 The inoculum is seeded at a density at the bottom of the culture container.

[0030] 17. The method according to 10 above further includes: culturing and maturing megakaryocytes in an eighth culture medium containing thrombopoietin.

[0031] 18. According to the method of 10 above, wherein the seventh culture medium comprises thrombopoietin, stem cell factor, interleukin-3 and interleukin-6.

[0032] 19. The method according to 10 above, wherein the pluripotent stem cells include human induced pluripotent stem cells.

[0033] 20. A method for producing blood products, comprising mixing red blood cells and platelets produced by any one of the methods described in 10 to 19 above.

[0034] The effects of the invention

[0035] The red blood cell production method of the present invention is based on CD41 - / CD34 - Cell populations produce red blood cells, and thus can shorten the time required for red blood cell production.

[0036] The red blood cell production method of the present invention enables large-scale production of red blood cells because CD41 - / CD34 - The cell population not only exhibited a high potential for differentiation into erythrocytes, but also a high potential for cell proliferation.

[0037] The red blood cell production method of the present invention has a simple process, does not require complex equipment, and is advantageous in terms of time and cost.

[0038] The method of the present invention for producing red blood cells and platelets can produce red blood cells and platelets simultaneously, and thus provides high process efficiency.

[0039] The red blood cells and platelets produced according to the method of the present invention can exhibit therapeutic or preventive effects on diseases requiring blood transfusions. Attached Figure Description

[0040] Figure 1 This is an example of a process used to culture human induced pluripotent stem cells.

[0041] Figure 2 It makes CD41a - / CD34 - An example of the process of cell differentiation into erythrocytes.

[0042] Figure 3 shows the confirmation results of the pellet color of the culture medium obtained in Preparation Examples 1 and 2 for the production of red blood cells.

[0043] Figure 4 shows the results of blood cell staining of cells obtained in Preparation Examples 1 and 2 for the production of red blood cells.

[0044] Figure 5 The results of immunostained cells obtained in Preparation Examples 1 and 2 for the production of red blood cells are shown.

[0045] Figure 6 shows the results of FACS analysis of cells obtained in Preparation Examples 1 and 2 for the production of red blood cells.

[0046] Figure 7 The results confirming the cell proliferation rate obtained in Preparation Example 1 for the production of red blood cells are shown.

[0047] Figure 8 The results of hemoglobin ELISA performed on cells obtained in Preparation Examples 1 and 2 for the production of red blood cells are shown.

[0048] Figure 9 shows the results of observing the cells obtained in Preparation Examples 1 and 2 for the production of red blood cells using transmission electron microscopy.

[0049] Figure 10 shows the results of observing the cells obtained in Preparation Examples 1 and 2 for the production of red blood cells using scanning electron microscopy.

[0050] Figure 11 The results of confirming the surface markers (CD34, CD45, CD41a) of the floating cell populations in the floating cell preparation examples and comparative preparation examples are shown.

[0051] Figure 12 illustrates the expression of megakaryocyte-specific markers in the cell population of Floating Cell Preparation Example 1.

[0052] Figure 13 illustrates the expression of megakaryocyte-specific markers in the cell population of Floating Cell Preparation Example 1.

[0053] Figure 14 illustrates that the cell population obtained from comparative preparation example 1 of floating cells does not express megakaryocyte-specific markers.

[0054] Figure 15 illustrates that cells obtained from Floating Cell Preparation Example 1 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p.

[0055] Figure 16 This demonstrates that cells obtained from floating cell preparation example 3 were activated by ADP treatment, leading to increased expression of PAC-1 and CD62p.

[0056] Figure 17 This illustrates the selection of only CD41a cells from the cell population obtained in Floating Cell Preparation Example 1. + The results of the cells.

[0057] Figure 18 The selected CD41a is shown. + Confirmation results of the expression of platelet-specific markers in cell populations and floating cell preparation example 1.

[0058] Figure 19This demonstrates the confirmation that the cell population in Floating Cell Preparation Example 1 exhibited high cell proliferation capacity as it differentiated into platelets. Detailed Implementation

[0059] This invention relates to a method for producing red blood cells or platelets.

[0060] The production method of the present invention can produce red blood cells or platelets separately, or can produce red blood cells and platelets together.

[0061] The method for producing red blood cells according to the present invention may include: (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) obtaining CD41 from said culture medium. - / CD34 - Cell population; and (S3) maturate the obtained cell population in a first medium containing erythropoietin (EPO).

[0062] The red blood cell production method of the present invention may further include (S0) discharging pluripotent stem cells at a rate of 2,000 to 20,000 cells / cm³. 2 The inoculum is seeded at a density at the bottom of the culture container.

[0063] The red blood cell production method of the present invention may further include proliferating the obtained cell population in a second culture medium containing erythropoietin, stem cell factor and interleukin-3.

[0064] The red blood cell production method of the present invention may further include differentiating the obtained cells in a third culture medium containing erythropoietin and stem cell factors.

[0065] The process used to produce red blood cells will be described in detail below.

[0066] The red blood cell production method of the present invention (S0) involves discharging pluripotent stem cells at a rate of 2,000 to 20,000 cells / cm³. 2 The step of inoculating the culture container at a certain density.

[0067] Pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0068] Induced pluripotent stem cells (iPSCs) are differentiated cells that lack pluripotency but acquire pluripotent differentiation ability through artificial reprogramming (dedifferentiation).

[0069] Induced pluripotent stem cells (iPSCs) can be derived from organisms selected from the following groups: humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (pet dogs and wild dogs), cats (pet cats and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, dogs, horses, zebras, and marine mammals (dolphins, whales, etc.).

[0070] Induced pluripotent stem cells can be human induced pluripotent stem cells (hiPSCs).

[0071] Induced pluripotent stem cells can be generated using mouse and / or human cells. For example, induced pluripotent stem cells can be generated using embryonic tissue, fetal tissue, neonatal tissue, and adult tissue.

[0072] Induced pluripotent stem cells can be used as a starting point and are essentially derived from any somatic cell at any developmental stage. Somatic cells can be derived from, but are not limited to, embryonic donors, fetal donors, neonatal donors, juvenile donors, or adult donors. Somatic cells can be, but are not limited to, fibroblasts such as skin fibroblasts obtained from skin samples or biopsies, synovial cells from synovial tissue, buccal cells, or lung fibroblasts.

[0073] Pluripotent stem cells were seeded at the bottom of a culture vessel and then cultured under adherent conditions.

[0074] Incubators can be used without restriction, as long as they are used for cell culture in this field. For example, they can be large, medium, or small incubators. In addition, they can be cell culture flasks, such as T25, T75, T175, or T225.

[0075] In terms of cell confluency, pluripotent stem cells with a density of 2,000 to 20,000 cells / cm² are preferred. 2 The density of seeding is as follows. When seeded at this amount, the cell confluence can be 70% to 95%, 75% to 95%, 80% to 95%, or 85% to 95%, and the pluripotent stem cells can be appropriately cultured and differentiated so that a culture medium containing sufficient amounts of hematopoietic stem cells, hematopoietic progenitor cells, and megakaryocyte progenitor cells can be obtained in the following step (S1).

[0076] When pluripotent stem cells are present at a rate of less than 2,000 cells / cm² 2When seeded at the bottom of a culture dish, the cell confluence at the point of obtaining a floating cell population (S2) can be below 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30%. Furthermore, when pluripotent stem cells are present at a concentration exceeding 20,000 cells / cm², [further details are needed]. 2 When seeded at the bottom of a culture dish, the confluence of cells can exceed 100% when pluripotent stem cells are not fully cultured and differentiated. As a result, undifferentiated cells may detach from the culture dish and remain floating and die, or the amount of factors added to the culture medium may become insufficient, which can lead to poor signal transduction between cells and poor differentiation.

[0077] Step (S1) of the red blood cell production method of the present invention is a step of culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells. This step involves culturing the pluripotent stem cells seeded in step (S0) to obtain a floating cell population capable of differentiating into red blood cells. The culture medium obtained in this step contains red blood cell precursors and megakaryocyte precursors.

[0078] Step (S1) can consist of the following sub-steps:

[0079] (S1a) Pluripotent stem cells were cultured in a fourth medium containing an inhibitor of GSK3 (glycogen synthase kinase 3);

[0080] (S1b) Cells cultured in the fourth medium were then cultured in a fifth medium containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF); and

[0081] (S1c) Cells cultured in the fifth medium will be cultured in the sixth medium containing vascular endothelial growth factor; basic fibroblast growth factor; and a transforming growth factor β signaling inhibitor.

[0082] The fourth, fifth, and sixth culture media used in this step are culture media with different compositions and purposes.

[0083] The fourth culture medium includes 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 agonist, and may be represented by aminopyrimidine.

[0084] GSK3 inhibitors (such as CHIR99021) are not limited to a specific concentration, as long as it is an amount sufficient to culture pluripotent stem cells. GSK3 inhibitors can be included in the fourth culture medium at concentrations 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.

[0085] The fourth medium is the basal medium. The fourth medium may include RPMI 1640 medium or other types of basal media. In addition to the basal medium, the fourth medium may also contain antioxidants and / or B-27.

[0086] Antioxidants may include any one selected from the group consisting of: 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 (vitamin A), curcumin, edaravone, polyphenols, glutathione, hydroxytyrosol, L-carnitine, ladostigil, melatonin, mofegir, N-acetylcysteine ​​(NAC), N-acetylserotonin (NAS), oleocanthal, oleuropein, rasagiline, resveratrol, selegiline, selenium, tocopherol (vitamin E), tocotrienol, tyrosol, ubiquinone (coenzyme Q), and uric acid.

[0087] The fifth culture medium is the growth medium. The fifth culture medium may contain at least growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The fifth culture medium may also contain other growth factors used for culturing pluripotent stem cells.

[0088] Vascular endothelial growth factor (VEGF) is a member of the epidermal growth factor receptor (EGFR / ErbB). VEGF plays a crucial role in regulating the proliferation and differentiation of short-lived mesenchymal stem cells (SMS cells) and induces apoptosis, survival, or cell proliferation by activating various types of signal transduction pathways. VEGF includes, for example, human and non-human animal (e.g., mouse) VEGF.

[0089] Vascular endothelial growth factor is contained at a concentration that allows for proper culture of pluripotent stem cells. Vascular endothelial growth factor may be contained in the fifth culture medium at concentrations of, for example, 5 to 95 ng / ml, 10 to 90 ng / ml, 15 to 85 ng / ml, 20 to 80 ng / ml, 25 to 75 ng / ml, 30 to 70 ng / ml, 35 to 65 ng / ml, 40 to 60 ng / ml, 45 to 55 ng / ml, or 50 ng / ml.

[0090] Basic fibroblast growth factor (bFGF) is a protein belonging to the FGF family. It acts as a mitogen, angiogenic factor, osteogenic factor, and neurotrophic factor, and is also involved in cell proliferation and differentiation. Basic fibroblast growth factor (also known as FGF2) primarily activates receptor proteins, including FGFR1b, FGFR1c, FGFR2c, FGFR3c, and FGFR4c, and particularly strongly activates FGFR1c and FGFR3c.

[0091] Basic fibroblast growth factor is included in the fifth culture medium at a concentration that allows pluripotent stem cells to be cultured appropriately with other components. Basic fibroblast growth factor may be included in the fifth culture medium at concentrations, 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.

[0092] The sixth medium may not contain a transforming growth factor β (TGFβ) signaling inhibitor. When the sixth medium contains a TGFβ signaling inhibitor, differentiation may occur before sufficient cell proliferation takes place.

[0093] The sixth culture medium is a growth medium. Like the fifth culture medium, it may contain at least growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The sixth culture medium may also contain other growth factors used for culturing pluripotent stem cells.

[0094] The provisions regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the sixth culture medium shall apply to those provisions regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the fifth culture medium.

[0095] The sixth culture medium may contain an inhibitor of transforming growth factor β (TGFβ) signaling.

[0096] Transforming growth factor β (TGFβ) signaling inhibitors are substances that inhibit TGFβ signaling. TGFβ is a substance that regulates various physiological processes in the body, such as cell proliferation, differentiation, apoptosis, migration, extracellular matrix (ECM) production, angiogenesis, and development.

[0097] TGFβ signaling inhibitors can be used without restriction, as long as they are substances that can inhibit TGFβ signaling, and can be, for example, activin receptor-like kinase (ALK) receptor inhibitors.

[0098] Activin receptor-like kinase receptor inhibitors can be, but are not limited to, ALK5, ALK4, and ALK7 receptor inhibitors. For example, an ALK receptor inhibitor can be SB431542. SB431542 can be represented by the following chemical name: 4-[4-(2H-1,3-benzodioxolane-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide.

[0099] Transforming growth factor β (TGFβ) signaling inhibitors are contained at concentrations sufficient to allow pluripotent stem cells to be cultured together with other components. Transforming growth factor β (TGFβ) signaling inhibitors may be contained at concentrations of, for example, 1 to 20 μM, 5 to 15 μM, or 10 μM.

[0100] The culture medium obtained through the culture step (S1) contains hematopoietic stem cells (HSCs). The culture medium may also contain hematopoietic progenitor cells (HPCs) and megakaryocyte progenitor cells (MK-Ps).

[0101] In the red blood cell production method of the present invention, (S2) involves obtaining CD41 from cells contained in the culture medium of (S1). - / CD34 - The steps of cell population.

[0102] Any conventional techniques known in the art for selecting cells in a cell population based on marker expression may be used without limitation. For example, antibody-based selection methods or sorters may be used, and antibody-based selection methods may specifically be methods using microbeads.

[0103] Select CD41 in step (S2) - After cells, CD34 can be selected. - Cells, or cells can be selected in the reverse order.

[0104] In the red blood cell production method of the present invention, (S3) is the step of maturing the cell population obtained in (S2) in a first culture medium containing erythropoietin (EPO).

[0105] The first culture medium is a culture medium containing at least one growth factor and erythropoietin in the basal medium.

[0106] The maturation step (S3) can take 4 to 14 days, and through this process, the red blood cell production method of the present invention can produce red blood cells in a short time.

[0107] The red blood cell production method of the present invention may further include proliferating the cell population obtained in (S2) in a second culture medium before (S3) and differentiating the cell population in a third culture medium.

[0108] In addition to the basal culture medium, the secondary culture medium may also contain at least erythropoietin, stem cell factors, and interleukin-3. The above-mentioned proliferation phase can last 6 to 10 days, preferably about 8 days.

[0109] The third culture medium may also contain at least erythropoietin and stem cell factors in the basal medium. The above differentiation steps can be carried out for 3 to 7 days, preferably about 5 days.

[0110] The red blood cell production method of the present invention selects cells that do not express specific markers (CD34, CD41a) and uses them to produce red blood cells, thereby not only increasing the differentiation efficiency into red blood cells, but also increasing the cell proliferation capacity, thus effectively producing a large number of red blood cells.

[0111] When cells that do not express CD34 and CD41a were subsequently cultured, the efficiency of differentiation into erythrocytes was confirmed by the production of a large number of cells expressing CD235 (blood group glycoprotein A), a marker on the surface of erythrocytes.

[0112] The method for producing platelets according to the present invention may include the following steps: (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) sorting CD41 from the culture medium. - / CD34 - Cell population and CD41a + or CD34 + Cell population; and (S4) to sort CD41a + or CD34 + The cell population differentiated into megakaryocytes in the seventh culture medium.

[0113] The aforementioned pluripotent stem cells can be found within the above-mentioned range.

[0114] The above-described (S1) and (S2) are within the aforementioned range. In the platelet production method of the present invention, (S2) may be CD41, excluding the CD41 selected in (S2) of the above-described red blood cell production method. - / CD34 - Cell populations of cells.

[0115] Step (S4) is to sort CD41a in step (S2). + or CD34 + The steps involved in the differentiation of cell populations into megakaryocytes in the seventh culture medium.

[0116] The seventh medium is used to differentiate cell populations into megakaryocytes. The seventh medium may contain cytokines. The seventh medium may contain at least one of thrombopoietin (TPO), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-6 (IL-6).

[0117] Thrombopoietin is a major growth factor that regulates the hematopoiesis of megakaryocytes and platelets, and is mainly synthesized and secreted by hepatocytes.

[0118] Thrombopoietin can be included in the seventh medium at a concentration that allows the suspension of cell populations to be cultured together with other components, and does not necessarily need to be included at a specific concentration. Thrombopoietin can be included at concentrations such as 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.

[0119] Stem cell factors are stromal cell-derived cytokines synthesized by fibroblasts and other cell types. Stem cell factors can be included in the seventh medium at concentrations that allow for the culture of suspension cell populations with other components, and do not necessarily need to be included at specific concentrations. Thrombopoietin can be included at concentrations, 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.

[0120] Interleukin-3 can be included in the seventh medium at a concentration that allows floating cell populations to be cultured together with other components, and does not necessarily have to be included at a specific concentration. Interleukin-3 can be included at concentrations of, for example, 1 to 20 ng / ml, 5 to 15 ng / ml, or 10 ng / ml.

[0121] Interleukin-6 can be included in the seventh medium at a concentration that allows floating cell populations to be cultured together with other components, and does not necessarily have to be included at a specific concentration. Interleukin-6 can be included at concentrations of, for example, 1 to 20 ng / ml, 5 to 15 ng / ml, or 10 ng / ml.

[0122] The seventh medium is not limited to a specific medium. It may include Iscove's Modified Dulbecco's Medium (IMDM) as a basal medium. IMDM may also contain antioxidants (such as AA2P) and / or B-27.

[0123] The method of the present invention for producing platelets may further include maturing megakaryocytes by culturing them in an eighth medium containing thrombopoietin.

[0124] In the eighth culture medium, thrombopoietin may be contained at concentrations 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.

[0125] In addition to thrombopoietin, the eighth culture medium may also contain other factors necessary for the maturation of megakaryocytes.

[0126] The eighth medium is not limited to a specific medium. The eighth medium may include Iscove modified Duchenne broth (IMDM) as the basal medium. IMDM medium may also contain antioxidants (such as ascorbic acid 2-phosphate, AA2P, etc.) and / or B-27.

[0127] In this step, mature megakaryocytes or a culture medium containing megakaryocytes and / or mature megakaryocytes can be obtained.

[0128] In the method for producing platelets according to the present invention, platelets can be isolated and / or purified from the culture medium after further differentiation of megakaryocytes, or platelets can be isolated and / or purified from the culture medium without further differentiation.

[0129] Platelet isolation and / or purification can be carried out by any known isolation and / or purification method, such as by centrifuging the culture medium or passing the culture medium through a column.

[0130] When the culture medium is centrifuged, megakaryocytes can be separated as a precipitate, while platelets can be separated as a suspension.

[0131] The present invention relates to a method for producing red blood cells and platelets, which can be implemented together in one procedure as described above for both red blood cell production and platelet production.

[0132] The method of the present invention for producing red blood cells and platelets may include the following steps: (S3) making sorted CD41 - / CD34 -The cell population matured in a first medium containing erythropoietin, and (S4) the sorted CD41a cells were then subjected to [further treatment]. + or CD34 + The cell population differentiated into megakaryocytes simultaneously or sequentially in the seventh culture medium.

[0133] When proceeding sequentially, (S3) can be performed after (S4), or conversely, (S4) can be performed after (S3).

[0134] The method of the present invention for producing red blood cells and platelets can produce red blood cells and platelets together in a single process, thereby making economical use of cultured cells, and can improve production efficiency by selecting cells with characteristics suitable for red blood cells and platelets and differentiating them separately.

[0135] The invention will be described in more detail through the following embodiments.

[0136] Example 1. Preparation and analysis of red blood cells

[0137] 1. Preparation of Red Blood Cells Example 1

[0138] 1-1 Culture of Human Induced Pluripotent Stem Cells (hiPSCs)

[0139] The following method involves culturing human induced pluripotent stem cells to obtain a cell population capable of differentiating into erythrocytes.

[0140] First, human induced pluripotent stem cells were injected at a rate of 4000 cells / cm³. 2 Cells were seeded at a density in T75 flasks coated with fibronectin containing mTeSR plus medium and 10 μg / ml of the ROCK inhibitor (Y-27632) and cultured at 37°C and 5% CO2. After 24 hours of culture, Y-27632 was removed, cells were washed once with PBS, and the mTeSR plus medium was replaced with fresh medium. The medium was changed daily for 3 days to ensure the cells were ready for differentiation.

[0141] Then, human induced pluripotent stem cells were cultured for 2 days at 37°C and 5% CO2 in RPMI1640 basal medium containing 1% P / S, 1% GlutaMAX, 300 μM AA2P and 2% B-27 (supplemented with 6 μM CHIR99021 as a GSK3 (glycogen synthase kinase 3) inhibitor), with the medium being changed once a day (differentiation initiation stage).

[0142] To induce human induced pluripotent stem cells to differentiate into megakaryocytes, the culture medium was modified to contain 1% P / S, 1% GlutaMAX, 300 μM AA2P, and 2% B-27 in RPMI 1640 basal medium, supplemented with 50 ng / ml VEGF and 20 ng / ml bFGF. The medium was changed daily, and the cells were cultured at 37°C and 5% CO2 for 3 days. Culture continued until cell confluence reached approximately 90%.

[0143] Then, 10 μM SB431542 was added together with VEGF and bFGF to RPMI1640 basal medium containing 1% P / S, 1% GlutaMAX, 300 μM AA2P and 2% B-27, and the medium was changed every 2 days while the culture was carried out for another 4 days.

[0144] A floating cell population was obtained by culturing, and CD41a was isolated from the floating cell population using CD41a and CD34 microbeads. - / CD34 - Cells and CD41a - / CD34 + cell.

[0145] 1-2. Culture the selected cell population to induce differentiation into erythrocytes.

[0146] CD41a was selected from the floating cell population obtained in the above-mentioned "1-1 Culture of Human Induced Pluripotent Stem Cells (hiPSCs)". - / CD34 - Cells and CD41a - / CD34 + Cells and culture.

[0147] CD41a - / CD34 - Cells and CD41a - / CD34 + Cells at 1×10 5 Cells were seeded at a density of 100 cells / ml in IMDM basal medium containing 300 μM AA2P and 2% B-27, and cultured (proliferated) for 8 days at 37°C and 5% CO2 in medium supplemented with 6 U / ml erythropoietin (EPO), 100 ng / ml SCF and 10 ng / ml IL-3.

[0148] Subsequently, the culture medium composition was changed to include IMDM basal medium containing 300 uM AA2P and 2% B-27 supplemented with 6 U / ml EPO and 100 ng / ml SCF, and the cells were further cultured (differentiated) for 5 days (this proliferation and differentiation process is referred to as the differentiation method (Diff) in this invention), and the culture medium composition was changed to include IMDM basal medium containing 300 uM AA2P and 2% B-27 supplemented with 6 U / ml EPO, and the cells were further cultured (matured) for 8 days to obtain cell culture.

[0149] Since red blood cells turn red due to hemoglobin during differentiation, the color of the cell deposits is examined to confirm this color.

[0150] The culture medium for the two cell populations obtained above was placed in a 15 ml conical tube and centrifuged at 400 × g for 5 minutes at room temperature (RT). The supernatant was removed, and the precipitate was resuspended in 1 ml of 1 × PBS, transferred to a 1.7 ml tube, and centrifuged at 400 × g for 5 minutes at room temperature (RT). The tube was laid flat so that the precipitate could be easily seen and photographed.

[0151] As a result, CD41a - / CD34 - The cell precipitate is red, while CD41a... - / CD34 + The cells are not red, confirmed by culturing CD41a. - / CD34 - The substances acquired by the cells have differentiated into red blood cells. Figure 3a ).

[0152] 2. Preparation of Red Blood Cells Example 2

[0153] 2-1 Culture of Human Induced Pluripotent Stem Cells (hiPSCs)

[0154] Human induced pluripotent stem cells were cultured using the same method as described in "1-1 Culture of Human Induced Pluripotent Stem Cells (hiPSCs)" above, and CD41a was selected. - / CD34 - Cells and CD41a - / CD34 + cell.

[0155] 2-2 Culture the selected cell population to induce differentiation into erythrocytes

[0156] CD41a was selected from the floating cell population obtained in the above-mentioned "2-1 Culture of Human Induced Pluripotent Stem Cells (hiPSCs)". - / CD34- Cells and CD41a - / CD34 + Cells and culture.

[0157] CD41a - / CD34 - Cells and CD41a - / CD34 + Cells at 1×10 5 Cells were seeded at a density of 100 cells / ml in IMDM basal medium containing 300 μM AA2P and 2% B-27, with 6 U / ml EPO added to the medium. Cells were cultured at 37°C and 5% CO2 (mature, Mat) for 14 days to obtain cell cultures.

[0158] The culture media from both obtained cell populations were placed in 15 ml conical tubes and centrifuged at 400 × g for 5 minutes at room temperature (RT). The supernatant was removed, and the precipitate was resuspended in 1 ml of 1 × PBS, transferred to a 1.7 ml tube, and centrifuged at 400 × g for 5 minutes at room temperature (RT). The tube was then placed down to allow easy visualization of the precipitate, and the precipitate was photographed.

[0159] As a result, CD41a - / CD34 - The cell precipitate is red, while CD41a... - / CD34 + The cells are not red, confirmed by culturing CD41a. - / CD34 - The substances acquired by the cells have differentiated into red blood cells. Figure 3b ).

[0160] 3. Confirm whether the red blood cell count is normal.

[0161] 3-1 Blood cell staining (Wright-Giemsa staining)

[0162] To confirm the efficacy of culturing CD41a in 1-2 and 2-2 above... - / CD34 - Cells and CD41a - / CD34 + To determine whether the substances obtained from the cells have differentiated into erythrocytes, Wright-Giemsa staining was performed.

[0163] Wright-Gymsa staining is a blood smear staining technique in which red blood cells are stained pink, platelets are stained light pink, lymphocyte cytoplasm is stained light blue, and monocyte cytoplasm is stained blue.

[0164] The experimental method is as follows. Cells were centrifuged at 400×g for 5 minutes at room temperature (RT), resuspended in 100 μl of 4% PFA (para-formaldehyde), and fixed at 4°C for 15 minutes. After centrifugation at 400×g for 5 minutes at 4°C, the cells were resuspended in 30 μl of 1x PBS, and 5 μl of the solution was placed on a glass slide and dried on a heating block at 56°C. After the slide had completely cooled, the cells were reacted with 0.1% Triton X-100 at room temperature for 10 minutes. After washing with 1x PBS, the cells were stained with Giemsa solution for 10 minutes. The cells were washed with distilled water (DW) and observed under a microscope (magnification 400×).

[0165] As a result, it was observed that culturing CD41a... - / CD34 - The cells obtained contain orthochromatic normoblasts (red blood cells with nuclei: white arrows) and reticulocytes (red blood cells without nuclei: black arrows), with the nuclei transferred to one side (Figure 4).

[0166] During erythrocyte differentiation, the nucleus migrates to one side before enucleation, and after enucleation, cells containing only cytoplasm are produced. This is observed through the culture of CD41a. - / CD34 - All these cells in the substance obtained from the cells were confirmed to be red blood cells.

[0167] 3-2 Immunostaining

[0168] To confirm whether the substances obtained in 1-2 and 2-2 above are erythrocytes, immunostaining was used to study the expression of CD235 (erythrocyte surface marker) and the presence of the cell nucleus.

[0169] Cells were centrifuged at 400×g for 5 minutes and resuspended in 200 μl of 1% BSA. CD235-PE antibody was added and the mixture was incubated at 4°C for 30 minutes. The reacted cells were then centrifuged at 400×g for 5 minutes at 4°C, the supernatant was removed, and the cells were washed with 500 μl of 1×PBS. The washed cells were resuspended in 30 μl of 1×PBS, 1 μl of Hoechst 33342 was added, and the cells were observed under a fluorescence microscope.

[0170] To confirm CD235 expression, CD235-PE antibody was reacted with each cell, and to confirm the presence of the cell nucleus, Hoechst 33342 was reacted with each cell.

[0171] As a result, such Figure 5As shown, by culturing CD41a - / CD34 - Among the cells obtained, cells stained only with CD235 but not with the nucleus were observed (white arrows). These may be reticulocytes, which express CD235 during erythrocyte differentiation but do not possess a nucleus. On the other hand, cells obtained by culturing CD41a... - / CD34 + Among the substances obtained from cells, almost no cells expressing CD235 were found, nor were any cells stained with CD235 but not with the nucleus.

[0172] 3-3 FACS Analysis

[0173] FACS analysis was performed to confirm whether the substances obtained in steps 1-2 and 2-2 above expressed CD71 and CD235 (which are erythrocyte-specific markers). FACS analysis was performed using a BD Lyric instrument with anti-CD71-APC and anti-CD235-PE antibodies. The antibodies were diluted 1:20 in PBS (FACS buffer) containing 1% BSA and reacted for 30 minutes to confirm surface marker expression.

[0174] As a result, from CD41a - / CD34 - The substance acquired by the cells showed positive expression for erythrocyte-specific markers (CD71, CD235) (Figure 6). This confirmed that the substance was erythrocyte.

[0175] At the same time, it was confirmed that from CD41a - / CD34 + The cells did not obtain the substances that are red blood cells because the expression of CD71 and CD235 is very low (Figure 6).

[0176] 3-4 Cell count

[0177] To confirm CD41a - / CD34 - Cells and CD41a - / CD34 + The cell proliferation capacity of the cell population was determined by counting cells during the induced differentiation into erythrocytes in section 2 above.

[0178] 20 μl of cells were mixed with 20 μl of 0.4% trypan blue, and the 10 μl mixture was counted after 1 minute using a Countess 3 cell counter.

[0179] As a result, CD41a - / CD34 -The cell population showed a higher level of activity than CD41a. - / CD34 + The higher cell proliferation rate of the cell population confirms the effectiveness of using CD41a. - / CD34 - Cells can produce a larger number of red blood cells when they produce red blood cells. Figure 7 ).

[0180] 3-5 Hemoglobin ELISA

[0181] Perform ELISA to measure CD41a - / CD34 - Cells and CD41a - / CD34 + The amount of hemoglobin in a cell population.

[0182] 1x10 6 Cells were lysed in 30 μl RIPA buffer at 4 °C for 30 min, centrifuged at 12,000 rpm at 4 °C for 30 min, and the supernatant was obtained. 25 μl of the supernatant was diluted with 225 μl of dilution buffer, and 100 μl of the solution was loaded into each well. Results were obtained by performing experiments according to the manufacturer's protocol for Ab157707. Figure 8 ).

[0183] 3-6 Transmission Electron Microscopy (TEM)

[0184] To observe CD41a - / CD34 - Cells were subjected to transmission electron microscopy as follows.

[0185] CD41a was treated with 2.5% glutaraldehyde (4°C, phosphate buffer, pH 7.2). - / CD34 - Cells were pre-fixed. Then, they were post-fixed with 1% osmium tetroxide (OsO4, 4°C, phosphate buffer, pH 7.2). After fixation, the samples were washed with the same buffer and dehydrated with increasing alcohol concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%, 100%). The treated samples were embedded and solidified using Epon 812 mixture. After embedding, the tissue was cut into 1 μm thick sections, stained with 1% toluidine blue, and specific areas were designated. After trimming the remaining areas, ultrathin sections with a thickness of 50–70 nm were obtained using an ultramicrotome (EM UC7, Leica), double-stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM-1200EXⅡ, JEOL) (Figure 9).

[0186] 3-7 Scanning Electron Microscopy (SEM)

[0187] To observe CD41a - / CD34 - Cells were subjected to scanning electron microscopy as follows.

[0188] Cells were pre-fixed with 2.5% glutaraldehyde (4°C, phosphate buffer, pH 7.2). Then, they were post-fixed with 1% osmium tetroxide (OsO4, 4°C, phosphate buffer, pH 7.2). After fixation, the samples were washed with the same buffer and dehydrated with increasing alcohol concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%, 100%). Ethanol:HMDS (hexamethyldisilazane) ratios of 3:1, 1:1, and 1:3 were used to replace 100% HMDS. The samples were placed on silicon wafers and dried. After Pt coating (SMC12R-Plus (Semian, Korea): 15 mA, 2–3 min), the cells were observed using a Regulus 8220 (Hitachi, Japan).

[0189] Example 2. Platelet Production and Analysis

[0190] 1. Culture of human induced pluripotent stem cells (hiPSCs)

[0191] The following describes how to culture human induced pluripotent stem cells to obtain a cell population capable of differentiating into megakaryocytes.

[0192] First, human induced pluripotent stem cells were placed in mTeSRplus medium containing 10 μg / ml of the ROCK inhibitor (Y-27632) at a concentration of 4000 / cm³. 2 The cells were placed in T75 flasks at a density of 1000 μL and cultured at 37°C and 5% CO2. After 24 hours of culture, Y-27632 cells were removed by washing once with PBS and replacing the mTeSR plus medium with fresh medium. The medium was changed daily for 3 days to ensure sufficient cells for differentiation.

[0193] Then, human induced pluripotent stem cells were cultured for 2 days in RPMI1640 basal medium containing 300 μM AA2P and 2% B-27 (supplemented with 6 μM CHIR99021 as a GSK3 (glycogen synthase kinase 3) inhibitor) at 37°C and 5% CO2 (the medium was changed once a day).

[0194] To differentiate human induced pluripotent stem cells into megakaryocytes, the culture medium was modified to contain 300 μM AA2P and 2% B-27 in RPMI1640 basal medium (supplemented with 50 ng / mL VEGF and 20 ng / mL bFGF), and then cultured at 37°C in a 5% CO2 incubator for 3 days (with the medium changed daily). Culture was continued until cell confluence reached approximately 70%. Subsequently, 10 μM SB431542 was added along with VEGF and bFGF to the RPMI1640 basal medium containing 300 μM AA2P and 2% B-27, followed by further culture for a predetermined period.

[0195] 1) Example 1 of preparation of floating cells

[0196] On the fourth day of additional culture, the proportion of cells expressing specific surface markers (CD34, CD41a, CD45) out of the total number of floating cells was studied by flow cytometry (FACS). For flow cytometry, a BD Lyric instrument was used, along with BD's anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies. The proportion of single-positive or double-positive cells for CD34 and CD41a, and CD45 and CD41a, was analyzed separately.

[0197] As a result, CD34 was confirmed. + The proportion of cells was 57.55% of the total cells, CD41a + The proportion of cells was 54.55% of the total cells, and CD45 + The proportion of cells was 18.58% of the total cells. CD34 was present in the floating cells. + The proportion of cells was 57.55%, CD41a + The proportion of cells was 54.55% and CD45 + At the time point when the cell percentage was 18.58%, a floating cell population was obtained from the culture medium. Figure 11 (Day 9 of the middle school term).

[0198] 2) Example 2 of preparation of floating cells

[0199] On the sixth day of additional culture, the proportion of cells expressing specific surface markers (CD34, CD41a, CD45) out of the total number of floating cells was studied by flow cytometry. As a result, CD34... + The proportion of cells was 61.59% of the total cells, CD41a + The proportion of cells was 47.03% of the total cells, and CD45 +The proportion of cells was 32.4% of the total cells. CD34 was present in the floating cells. + The proportion of cells was 61.59%, CD41a + The proportion of cells was 47.03% and CD45 + At the time point when the cell proportion was 32.4%, a floating cell population was obtained from the culture medium. Figure 11 (Days 11-12 of the middle period).

[0200] 3) Example 3 of floating cell preparation

[0201] On the eighth day of additional culture, the proportion of cells expressing specific surface markers (CD34, CD41a, CD45) out of the total number of floating cells was studied by flow cytometry. As a result, CD34... + The proportion of cells was 52.05% of the total cells, CD41a + The proportion of cells was 18.88% of the total cells, and CD45 + The proportion of cells was 45.56% of the total cells. CD34 was present in the floating cells. + The proportion of cells was 52.05%, CD41a + The proportion of cells was 18.88% and CD45 + At the time point when the cell percentage was 45.56%, a floating cell population was obtained from the culture medium. Figure 11 (Day 13 in the middle of the month).

[0202] 4) Example 4 of floating cell preparation

[0203] On the second day of further culture, the proportion of cells expressing specific surface markers (CD34, CD45) out of the total number of floating cells was studied by flow cytometry. As a result, CD34... + The proportion of cells was 59.27% ​​of the total cells, and CD45 + The proportion of cells was 5.21% of the total cells. CD34 was present in the floating cells. + The proportion of cells was 59.27% ​​and CD45 + At a time point when the cell percentage was 5.21%, a floating cell population was obtained from the culture medium. Figure 11 (Day 7 in the middle of the month).

[0204] 5) Comparative Preparation of Floating Cells Example 1

[0205] On the tenth day of culture, flow cytometry was used to investigate the proportion of cells expressing specific surface markers (CD34, CD41a, CD45) out of the total number of floating cells. As a result, CD34 was confirmed to be present. + The proportion of cells was 91.2% of the total cells, CD41a +The proportion of cells was 13% of the total cells, and CD45 + The proportion of cells was 68.51% of the total cells. CD34 was present in the floating cells. + The proportion of cells was 91.2%, CD41a + The proportion of cells was 13% and CD45 + At the time point when the cell percentage was 68.51%, a floating cell population was obtained from the culture medium. Figure 11 (Day 15 in the middle of the month).

[0206] 2. Culture floating cell populations and induce differentiation into megakaryocytes.

[0207] To differentiate and mature into megakaryocytes, a floating cell population obtained in the above-described "1. Culture of human induced pluripotent stem cells (hiPSCs)" was cultured.

[0208] To differentiate the floating cell populations obtained from “1. Culture of human induced pluripotent stem cells (hiPSCs)” above into megakaryocytes, the floating cell populations from floating cell preparation examples 1 to 4 and comparative preparation example 1 were cultured in IMDM basal medium containing 300 μM AA2P and 2% B-27 at a concentration of 1 × 10⁻⁶ mg / L. 5 Cells were placed at a density of 10 cells / ml, and then 25 ng / ml TPO, 25 ng / ml SCF, 10 ng / ml IL-3 and 10 ng / ml IL-6 were added to the culture medium. The cells were then cultured at 37°C and 5% CO2 for 3 days.

[0209] Then, the culture medium composition was changed to IMDM basal medium containing 300 uM AA2P and 2% B-27 (supplemented with 100 ng / ml TPO), and the cells were cultured for another 5 days to carry out the maturation (megakaryocyte maturation) step, thus obtaining the cell culture medium.

[0210] 3. Confirmation of platelet production capacity of megakaryocytes

[0211] Platelets were obtained from the culture media of floating cell preparation examples 1 and 3 obtained in "2. Induction of Differentiation of Floating Cell Population into Megakaryocytes" and comparative preparation example 1. Specifically, all the cell culture media of floating cell preparation examples 1 and 3 obtained in "2. Induction of Differentiation of Floating Cell Population into Megakaryocytes" and comparative preparation example 1 were collected and centrifuged at 300×g for 3 minutes. The precipitate was separated as megakaryocytes, and the supernatant was separated as platelets. The platelets in these separated products were then analyzed.

[0212] The following experiments (1) and (2) confirmed that the final substance obtained by the above method is platelets with normal activity.

[0213] (1) Identification of platelet-specific markers by FACS analysis

[0214] The cell culture medium used in Preparation Examples 1 and 3, and Comparative Preparation Example 1, was centrifuged to remove precipitated megakaryocytes, and the suspended platelets were centrifuged again at 2000×g for 10 minutes. FACS analysis was performed on the centrifuged platelets to confirm the expression of surface markers. FACS analysis was performed using a BD Lyric instrument, employing BD's anti-CD41a-FITC, anti-CD61-PE, and anti-CD42b-APC antibodies. The antibodies were diluted 1:20 in PBS (FACS buffer) containing 1% BSA and reacted for 30 minutes to confirm surface marker expression.

[0215] As a result, the substance obtained from floating cell preparation example 1 showed positive expression of platelet-specific markers (CD41a, CD42b, and CD61) (Figure 12), and the substance obtained from floating cell preparation example 3 also showed positive expression of platelet-specific markers (CD41a, CD42b, and CD61) (Figure 13). This confirms that the cell populations from floating cell preparation examples 1 and 3 are capable of producing platelets.

[0216] Meanwhile, the substance obtained from comparative preparation example 1 of floating cells showed very low expression of CD41a and CD61, and in particular, CD42b expression was almost negative (Fig. 14). This confirms that the substance obtained from comparative preparation example 1 does not show platelet surface marker characteristics, and the cell population of comparative preparation example 1 hardly differentiates into megakaryocytes and therefore does not produce platelets.

[0217] (2) Platelet function was confirmed by ADP treatment.

[0218] To confirm whether the substances obtained from floating cell preparations in Examples 1 and 3 were platelets with normal activity, the following experiments were conducted.

[0219] The substances obtained from the floating cell preparations in Examples 1 and 3 were centrifuged to remove the precipitated megakaryocytes, and the suspended platelets were centrifuged again at 2000×g for 10 minutes. To collect the precipitated platelets, all supernatant was removed due to centrifugation, and the platelets were resuspended in a small amount of PBS.

[0220] For PAC-1 analysis, platelets were treated with 100 μM ADP for 15 minutes at room temperature and then reacted with BD's anti-PAC-1-FITC and anti-CD62p-PE antibodies for 30 minutes. The reacted platelets were immediately subjected to FACS analysis. The materials obtained from Preparation Examples 1 and 3 were activated by ADP treatment, and the expression of PAC-1 and CD62p increased (Figures 15 and 16), confirming that the substances obtained from Preparation Examples 1 and 3 were platelets with normal activity. PAC-1 is a complex of CD41a and CD61 that appears when platelets are activated. CD62p (p-selectin) is a surface molecule whose expression increases when platelets are activated; it serves as a site where leukocytes can bind to platelets.

[0221] The above experimental results confirm that when subsequent culture steps are performed after culturing hiPSCs, the differentiation efficiency into megakaryocytes and platelet production efficiency can be improved if the cell population meets a certain condition (the proportion of cells expressing specific surface markers (e.g., CD41a, CD34, CD45) is greater than or less than a predetermined value).

[0222] 4. Comparison of platelet production efficiency when only CD41a+ cells are selected and subsequently cultured.

[0223] From the floating cell population obtained in Example 1 of the above-mentioned floating cell preparation, only CD41a+ cells were selected. Figure 17 They were then cultured to compare platelet production efficiency with that of the floating cell population in Example 1 of floating cell preparation.

[0224] Using the same method as the FACS analysis in 3. (1) above, the expression of platelet-specific markers was studied in both cases. As a result, CD41a was confirmed. + / CD42b + The proportions are similar, therefore the platelet differentiation rates are similar. Figure 18 ).

[0225] However, as a result of examining cell proliferation capacity in both cases, it was confirmed that the cell proliferation capacity was not limited to CD41a. + Cells and also contain non-CD41a - Cells (e.g., CD41a) - When the cell population differentiates into platelets, as in Preparation Example 1, the cell proliferation capacity is higher than that in the case of CD41a selection. + The rate of differentiation into platelets is about 40 times higher. Figure 19 ).

[0226] Based on the above results, it was confirmed that when CD41a is further included... -When cell populations are used for platelet production, as in Preparation Example 1 of this invention, platelets can be produced more efficiently in terms of both quantity and quality, rather than by selecting only CD41a. + Cells use them for platelet production.

Claims

1. A method for producing red blood cells, comprising: (S1) Culture pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) Obtain CD41 from the culture medium. - / CD34 - Cell population; and (S3) Mature the obtained cell population in a first culture medium containing erythropoietin (EPO).

2. The method according to claim 1, further comprising: The obtained cell population was proliferated in a secondary culture medium containing erythropoietin, stem cell factor, and interleukin-3.

3. The method according to claim 1, further comprising: The obtained cells were then differentiated in a third culture medium containing erythropoietin and stem cell factors.

4. The method according to claim 1, wherein the culture medium comprises erythrocyte precursor and megakaryocyte precursor.

5. The method of claim 1, wherein the maturation step is performed for 4 to 14 days.

6. The method according to claim 1, wherein (S1) comprises: (S1a) Pluripotent stem cells were cultured in a fourth medium containing GSK3 inhibitors; (S1b) Cells cultured in the fourth medium will be cultured in the fifth medium containing vascular endothelial growth factor and basic fibroblast growth factor. and (S1c) Cells cultured in the fifth medium will be cultured in the sixth medium containing vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor β signaling inhibitors.

7. The method according to claim 1, further comprising: (S0) Pluripotent stem cells were injected at a rate of 2,000 to 20,000 cells / cm³. 2 The inoculum is seeded at a density at the bottom of the culture container.

8. The method according to claim 1, wherein the pluripotent stem cells include human induced pluripotent stem cells.

9. A method for producing blood products, comprising a process of mixing red blood cells produced by the method according to any one of claims 1 to 8 with other blood components.

10. A method for producing red blood cells and platelets, comprising: (S1) Culture pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells; (S2) Sorting CD41 from the culture medium - / CD34 - Cell population and CD41a + or CD34 + Cell population; (S3) Make the sorted CD41 - / CD34 - The cell population matured in a primary culture medium containing erythropoietin; and (S4) Make the sorted CD41a + or CD34 + The cell population differentiated into megakaryocytes in the seventh culture medium.

11. The method of claim 10, further comprising: The sorted CD41 - / CD34 - The cell population proliferated in a secondary culture medium containing erythropoietin, stem cell factor, and interleukin-3.

12. The method of claim 10, further comprising: The sorted CD41 - / CD34 - The cell population differentiated in a third culture medium containing erythropoietin and stem cell factors.

13. The method of claim 10, wherein the culture medium comprises erythrocyte precursor and megakaryocyte precursor.

14. The method of claim 10, wherein (S3) is performed for 4 to 14 days.

15. The method of claim 10, wherein (S1) comprises: (S1a) The pluripotent stem cells are cultured in a fourth culture medium containing a GSK3 inhibitor; (S1b) Cells cultured in the fourth medium will be cultured in the fifth medium containing vascular endothelial growth factor and basic fibroblast growth factor. and (S1c) Cells cultured in the fifth medium will be cultured in the sixth medium containing vascular endothelial growth factor, basic fibroblast growth factor and transforming growth factor β signaling inhibitors.

16. The method of claim 10, further comprising: The pluripotent stem cells (S0) were processed at a rate of 2,000 to 20,000 cells / cm³. 2 The inoculum is seeded at a density at the bottom of the culture container.

17. The method of claim 10, further comprising: Megakaryocytes were cultured and matured in medium VIII containing thrombopoietin.

18. The method of claim 10, wherein the seventh culture medium comprises thrombopoietin, stem cell factor, interleukin-3 and interleukin-6.

19. The method of claim 10, wherein the pluripotent stem cells comprise human induced pluripotent stem cells.

20. A method for producing blood products, comprising mixing red blood cells and platelets produced by the method according to any one of claims 10 to 19.

Images