Methods for producing red blood cells and platelets

Abstract

The present invention relates to a method for producing red blood cells, and more specifically, to (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, and (S2) obtaining CD41 from the culture medium. - and CD34 - The present invention relates to a method for producing red blood cells of superior quality in a short period of time, comprising the steps of (S3) obtaining a cell population and (S3) maturing the obtained cell population in a first medium containing erythropoietin.

Description

[Technical Field]

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

[0002] For patients suffering from blood deficiencies due to various reasons, blood transfusions are a crucial treatment option.

[0003] However, due to the aging population, the number of people eligible to donate blood has decreased, and the number of blood donors has declined. In addition, the recent spread of infectious diseases such as Covid-19 has resulted in an extreme shortage of blood supply.

[0004] Blood donation from blood donors carries the risk of transmission of infectious diseases and various other transfusion side effects. Therefore, ensuring a stable supply of red blood cells and platelets is a critical challenge in this field.

[0005] While hemoglobin solutions and oxygen carriers have been attempted as substitutes for red blood cells, which are used for oxygen transport among blood components, they have shown low oxygen-carrying 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 these demands, research into producing red blood cells and platelets outside the body has been ongoing in recent years. However, conventional methods for producing red blood cells or platelets have problems such as requiring long production times and being unable to produce in high yields. Furthermore, because they must be produced individually, process efficiency and economic viability are low. Therefore, technological improvements are needed to develop economical methods for mass-producing artificial red blood cells and platelets. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a method for producing red blood cells in a short period of time.

[0008] The present invention aims to provide a method for producing red blood cells in high yield.

[0009] The present invention aims to provide a method for simultaneously producing red blood cells and platelets.

[0010] The present invention aims to provide a method for producing red blood cells with process efficiency.

[0011] The present invention aims to provide a method for the simultaneous production of red blood cells and platelets with process efficiency. [Means for solving the problem]

[0012] 1. (S1) A step of culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, and (S2) a step of extracting CD41 from the culture medium. - and CD34 - A method for producing red blood cells, comprising the steps of (S3) obtaining a cell population and (S3) maturing the obtained cell population in a first medium containing erythropoietin (EPO).

[0013] 2. A method for producing red blood cells, further comprising the step of growing the cell population obtained in item 1 above in a second medium containing erythropoietin, stem cell factors, and interleukin-3.

[0014] 3. A method for producing red blood cells, further comprising the step of differentiating the cells obtained in item 1 above in a third medium containing erythropoietin and stem cell factors.

[0015] 4. A method for producing red blood cells, wherein the culture medium in item 1 above contains red blood cell precursors and megakaryocyte precursors.

[0016] 5. A method for producing red blood cells, wherein the maturation step in item 1 above is carried out for 4 to 14 days.

[0017] 6. In the above item 1, the method for producing red blood cells of (S1) includes the following steps: (S1a) culturing pluripotent stem cells in a fourth medium containing a GSK3 inhibitor; (S1b) culturing the cells cultured in the fourth medium in a fifth medium containing vascular endothelial growth factor and basic fibroblast growth factor; and (S1c) culturing the cells cultured in the fifth medium in a sixth medium containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor beta signal transduction inhibitor.

[0018] 7. In the above item 1, the method for producing red blood cells further includes the step of seeding pluripotent stem cells at 2,000 - 20,000 cells / cm 2 on the bottom of a culture vessel.

[0019] 8. In the above item 1, the method for producing red blood cells, wherein the pluripotent stem cells are human induced pluripotent stem cells.

[0020] 9. The method for producing a blood product includes the step of mixing red blood cells produced by any of the methods of items 1 - 8 with other blood components.

[0021] 10. (S1) culturing pluripotent stem cells to obtain a culture solution containing hematopoietic stem cells; (S2) classifying a cell population that is CD41 - and CD34 - and a cell population that is CD41a + or CD34 + from the culture solution; (S3) maturing the classified cell population that is CD41[[ID=u27]] - and CD34 - in a first medium containing erythropoietin; and (S4) differentiating the classified cell population that is CD41a + or CD34 + into megakaryocytes in a seventh medium. The method for producing red blood cells and platelets includes these steps.

[0022] 11. In the above item 10, the classified cell population that is CD41 - and CD34 -A method for producing red blood cells and platelets, further comprising the step of growing a cell population in a second medium containing erythropoietin, stem cell factors, and interleukin-3.

[0023] 12. In item 10 above, the classified CD41 - and CD34 - A method for producing erythrocytes and platelets, further comprising the step of differentiating a cell population in a third medium containing erythropoietin and stem cell factors.

[0024] 13. A method for producing red blood cells and platelets, wherein the culture medium in item 10 above includes red blood cell precursors and megakaryocyte precursors.

[0025] 14. In item 10 above, (S3) is a method for producing red blood cells and platelets, which is carried out for 4 to 14 days.

[0026] 15. In item 10 above, (S1) is a method for producing red blood cells and platelets, comprising the following steps: (S1a) culturing pluripotent stem cells in medium 4 containing a GSK3 inhibitor; (S1b) culturing the cells cultured in medium 4 in medium 5 containing vascular endothelial growth factor and basic fibroblast growth factor; and (S1c) culturing the cells cultured in medium 5 in medium 6 containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor beta signaling inhibitor.

[0027] 16. In item 10 above, (S0) pluripotent stem cells are placed at the bottom of the culture vessel at a rate of 2,000 to 20,000 cells / cm². 2 A method for producing red blood cells and platelets, further comprising the step of seeding.

[0028] 17. A method for producing red blood cells and platelets, further comprising the step of culturing megakaryotic cells in medium No. 8 containing thrombopoietin to mature them, as described in item 10 above.

[0029] 18. In item 10 above, the seventh medium is a method for producing red blood cells and platelets, wherein the medium contains thrombopoietin, stem cell factors, interleukin-3, and interleukin-6.

[0030] 19. In item 10 above, pluripotent stem cells are human induced pluripotent stem cells; a method for producing red blood cells and platelets.

[0031] 20. A method for producing a blood product, comprising the step of mixing red blood cells and platelets produced by any of the methods described in items 10 to 19 above. [Effects of the Invention]

[0032] The present invention's method for producing red blood cells is CD41 - and CD34 - Since red blood cells are produced from a population of cells, the time required for red blood cell production can be shortened.

[0033] The present invention's method for producing red blood cells is CD41 - and CD34 - These cell populations not only exhibit high erythrocyte differentiation potential, but also have high cell proliferation potential, enabling the mass production of erythrocytes.

[0034] The present invention provides a method for producing red blood cells that is simple in its process, does not require complex equipment, and is advantageous in terms of time and cost.

[0035] The present invention provides a method for producing red blood cells and platelets simultaneously, thus offering high process efficiency.

[0036] Red blood cells and platelets produced by the method of the present invention can exhibit therapeutic or preventive effects against diseases requiring blood transfusions. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1 shows one embodiment of the culture process for human induced pluripotent stem cells. [Figure 2]Figure 2 shows one embodiment of the process by which cells that are CD41a- and CD34- are differentiated into erythrocytes. [Figure 3] Figure 3 shows the results of checking the color of the culture medium pellets obtained in red blood cell production examples 1 and 2. [Figure 4] Figure 4 shows the results of blood cell staining of cells obtained in red blood cell production examples 1 and 2. [Figure 5] Figure 5 shows the results of immunostaining of cells obtained in red blood cell production examples 1 and 2. [Figure 6] Figure 6 shows the FACS analysis results of cells obtained in red blood cell production examples 1 and 2. [Figure 7] Figure 7 shows the results of confirming the proliferation rate of cells obtained in Red Blood Cell Production Example 1. [Figure 8] Figure 8 shows the results of hemoglobin ELISA performed on cells obtained in red blood cell production examples 1 and 2. [Figure 9] Figure 9 shows the results of observing cells obtained in red blood cell production examples 1 and 2 using a transmission electron microscope. [Figure 10] Figure 10 shows the results of observing cells obtained in red blood cell production examples 1 and 2 using a scanning electron microscope. [Figure 11] Figure 11 shows the results of identifying surface markers (CD34, CD45, CD41a) in the suspension cell populations of both the suspension cell production example and the comparative production example. [Figure 12] Figure 12 shows that the cell population from suspension cell production example 1 expresses megakaryocyte-specific markers. [Figure 13] Figure 13 shows that the cell population from suspension cell production example 1 expresses megakaryocyte-specific markers. [Figure 14] Figure 14 shows that the cell population obtained from comparative example 1 of suspension cells does not express megakaryocyte-specific markers. [Figure 15] Figure 15 shows that cells obtained from suspension cell production example 1 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p. [Figure 16]Figure 16 shows that cells obtained from suspension cell production example 3 were activated by ADP treatment, resulting in increased expression of PAC-1 and CD62p. [Figure 17] Figure 17 shows the results of selecting only CD41a+ cells from the cell population obtained in suspension cell production example 1. [Figure 18] Figure 18 shows the results of confirming the expression of platelet-specific markers in a cell population selected for CD41a+ cells and in the cell population from suspension cell production example 1. [Figure 19] Figure 19 shows the results confirming that the cell population from suspension cell production example 1 exhibits high cell proliferation ability when differentiated into platelets. [Modes for carrying out the invention]

[0038] The present invention relates to a method for producing red blood cells or platelets.

[0039] The manufacturing method of the present invention can produce red blood cells or platelets separately, or red blood cells and platelets together.

[0040] The present invention provides a method for producing red blood cells, comprising the steps of (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, and (S2) obtaining CD41 from the culture medium. - and CD34 - (S3) The step of obtaining a cell population, and (S3) the step of maturing the obtained cell population in a first medium containing erythropoietin (EPO).

[0041] The present invention provides a method for producing red blood cells, which involves (S0) placing the pluripotent stem cells at the bottom of a culture vessel at a rate of 2,000 to 20,000 cells / cm³. 2 This can further include a seeding step.

[0042] The present invention's method for producing red blood cells may further include the step of growing the obtained cell population in a second medium containing erythropoietin, stem cell factors, and interleukin-3.

[0043] The present invention's method for producing red blood cells may further include the step of differentiating the obtained cells in a third medium containing erythropoietin and stem cell factors.

[0044] The following describes the red blood cell manufacturing process in detail.

[0045] The present invention's method for producing red blood cells involves (S0) inoculating pluripotent stem cells at the bottom of a culture vessel at a rate of 2,000 to 20,000 cells / cm³. 2 This is the seeding step.

[0046] Pluripotent stem cells include induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0047] Induced pluripotent stem cells (iPSCs) are cells that did not originally possess pluripotency but acquired it through an artificial dedifferentiation process.

[0048] Induced pluripotent stem cells (iPSCs) may be derived from individuals selected from a group including humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, and cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (dolphins, whales, etc.).

[0049] Induced pluripotent stem cells may also be human induced pluripotent stem cells (hiPSCs).

[0050] Induced pluripotent stem cells can be generated using mouse and / or human cells. For example, induced pluripotent stem cells can be generated using embryonic, fetal, neonatal, and adult tissues.

[0051] Induced pluripotent stem cells can be obtained as a starting point from virtually any somatic cell at any developmental stage. Somatic cells can be obtained from embryos, fetuses, neonates, infants, or adult donors, but are not limited to these. Somatic cells may be fibroblasts, such as dermal fibroblasts obtained from skin samples or biopsies, synovial cells from synovial tissue, buccal cells, or lung fibroblasts, but are not limited to these.

[0052] Pluripotent stem cells are seeded at the bottom of the culture vessel and then cultured by adhesion.

[0053] The incubator can be any type used for cell culture in the field, without any restrictions. For example, it may be a large, medium, or small incubator. Alternatively, it may be a cell culture flask such as a T25, T75, T175, or T225.

[0054] Pluripotent stem cells have a density of 2,000 to 20,000 cells / cm³. 2 Seeding with this amount is preferable from the viewpoint of cell confluency. Seeding with this amount results in cell confluency of 70%-95%, 75%-95%, 80%-95%, or 85%-95%, allowing pluripotent stem cells to be properly cultured and differentiated, thereby obtaining a culture medium that sufficiently contains hematopoietic stem cells, hematopoietic progenitor cells, megakaryotic progenitor cells, etc. in the (S1) step described below.

[0055] Pluripotent stem cells are placed at the bottom of the culture vessel at a rate of 2,000 cells / cm². 2 If seeding is performed at a level less than (S2), the cell confluence may be 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, etc., when obtaining the suspension cell population (S2). Additionally, pluripotent stem cells are placed at the bottom of the culture vessel at a rate of 20,000 cells / cm³. 2When seeding is performed at excessive levels, cell confluence may exceed 100% before pluripotent stem cells have been sufficiently cultured and differentiated. This can cause undifferentiated cells to detach from the culture dish and die in suspension, or result in insufficient amounts of factors added to the culture medium. Consequently, intercellular signaling may be impaired, leading to insufficient differentiation.

[0056] Step (S1) in the present invention for producing red blood cells is a step of culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells. This step is a step of culturing the pluripotent stem cells seeded in step (S0) in order to obtain a suspension population of cells capable of differentiating into red blood cells. The culture medium obtained in this step contains red blood cell precursors and megakaryocyte precursors.

[0057] Step (S1) can be configured to include the following substeps: (S1a) A step in which pluripotent stem cells are cultured in medium IV containing a GSK3 (Glycogen Synthase Kinase 3) inhibitor; (S1b) A step of culturing cells cultured in medium 4 in medium 5 containing vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF); and, (S1c) A step in which cells cultured in medium 5 are cultured in medium 6 containing vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor beta signaling inhibitor.

[0058] The fourth, fifth, and sixth media used in this step are media with different compositions and purposes.

[0059] Medium 4 contains at least a GSK3 inhibitor. The GSK3 inhibitor may be, but is not limited to, CHIR99021, BIO(6-bromoindirubin-30-oxime), SB216763, CHIR-98014, CT98014, CT98023, CT99021, TWS119, SB41528, AR-A014418, AZD-1080, Alsterpaullone, Cazpaullone, or Kenpaullone. CHIR99021 is a GSK3 inhibitor and a Wnt signaling enhancer, and can be represented as an aminopyrimidine.

[0060] The concentration of the GSK3 inhibitor (e.g., CHIR99021) is not particularly limited, as long as it is sufficient to culture pluripotent stem cells. The GSK3 inhibitor may be included in Medium IV at concentrations of, for example, 2–10 μM, 2.5–9.5 μM, 3–9 μM, 3.5–8.5 μM, 4–8 μM, 4.5–7.5 μM, 5–7 μM, 5.5–6.5 μM, or 6 μM.

[0061] Medium 4 is a basal medium. Medium 4 may include RPMI1640 medium or other types of basal medium. In addition to the basal medium, Medium 4 may further contain antioxidants and / or B-27.

[0062] Antioxidants include 6-hydroxymelatonin, acetyl-L-carnitine (ALCAR), alpha-lipoic acid (ALA), ascorbic acid (e.g., AA2P (L-Ascorbic acid 2-phosphate), AA2G (L-Ascorbic acid 2-glucoside)), and carotenoids (vitamins). A) Curcumin, Edaravone, Polyphenols, Glutathione, Hydroxytyrosol, L-carnitine, Ladostigil, Melatonin, Mofegiline, N-Acetylcysteine ​​(NAC), N-Acetylserotonin (NAS), Oleocanthal, Oleuropein, Rasagiline, Resveratrol, Selegiline, Selenium, Tocopherols (vitamins) E) The following can be selected from the group consisting of tocotrienols, tyrosol, ubiquinone (coenzyme Q), and uric acid.

[0063] Medium V is a growth medium. Medium V contains at least growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Medium V may further contain other growth factors for culturing pluripotent stem cells.

[0064] Vascular endothelial growth factor (VEGF) is a member of the epidermal growth factor receptor (EGFR / ErbB) group. VEGF plays an essential role in regulating cell proliferation and differentiation, inducing apoptosis, survival, or cell proliferation by activating various forms of signaling pathways. Examples of VEGF include human and non-human animal (such as mouse) vascular endothelial growth factors.

[0065] Vascular endothelial growth factor is included in concentrations suitable for culturing pluripotent stem cells. Vascular endothelial growth factor may be included in Medium V at concentrations of, for example, 5-95 ng / ml, 10-90 ng / ml, 15-85 ng / ml, 20-80 ng / ml, 25-75 ng / ml, 30-70 ng / ml, 35-65 ng / ml, 40-60 ng / ml, 45-55 ng / ml, or 50 ng / ml.

[0066] Basic fibroblast growth factor (bFGF) is a protein belonging to the FGF family that functions as a cell proliferation and differentiation factor, as well as a mitogenic factor, angiogenic factor, bone morphogenetic factor, and nerve growth factor. Basic fibroblast growth factor, also known as FGF2, mainly activates receptor proteins including FGFR1b, FGFR1c, FGFR2c, FGFR3c, and FGFR4c, and is particularly potent in activating FGFR1c and FGFR3c.

[0067] Basic fibroblast growth factor is included in the medium at concentrations suitable for culturing pluripotent stem cells along with other components. Basic fibroblast growth factor may be included in Medium V at concentrations of, for example, 1–40 ng / ml, 5–35 ng / ml, 10–30 ng / ml, 15–25 ng / ml, or 20 ng / ml.

[0068] Medium 6 does not necessarily need to contain a transforming growth factor beta (TGFβ) signaling inhibitor. If Medium 6 contains a TGFβ signaling inhibitor, differentiation may occur in a state of insufficient cell proliferation.

[0069] Medium 6 is a growth medium. Like Medium 5, it contains at least growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Medium 6 may further contain other growth factors for culturing pluripotent stem cells.

[0070] The provisions regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in Medium 6 shall be the same as those regarding vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in Medium 5.

[0071] Medium 6 contains a transforming growth factor beta (TGFβ) signaling inhibitor.

[0072] Transforming growth factor beta (TGFβ) signaling inhibitors are substances that inhibit TGFβ signaling. TGFβ is a substance that regulates various physiological processes in the body, including cell proliferation, differentiation, apoptosis, migration, extracellular matrix (ECM) production, angiogenesis, and development.

[0073] TGFβ signaling inhibitors can be used without restriction as long as they are substances that can inhibit TGFβ signaling, for example, activin receptor-like kinase (ALK) receptor inhibitors.

[0074] Activin receptor-like kinase receptor inhibitors may be, but are not limited to, ALK5, ALK4, and ALK7 receptor inhibitors. For example, an ALK receptor inhibitor may be SB431542. SB431542 may be represented by the following compound name: 4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridine-2-yl)-1H-imidazole-2-yl]benzamide.

[0075] Transforming growth factor beta (TGFβ) signaling inhibitors are included along with other components at concentrations sufficient to culture pluripotent stem cells. For example, TGFβ signaling inhibitors may be included at concentrations of 1–20 μM, 5–15 μM, or 10 μM.

[0076] The culture medium obtained after culturing in step (S1) contains hematopoietic stem cells (HSCs). The culture medium may further contain hematopoietic progenitor cells (HPCs) and megakaryotic progenitor cells (MK-Ps).

[0077] (S2) of the present invention's method for producing red blood cells is a method in which, among the cells contained in the culture medium of (S1), CD41 - and CD34 - This is the step of obtaining a population of cells.

[0078] Conventional techniques in the field used to sort cells from a cell population based on marker expression can be used without limitation. For example, antibody-based sorting methods or sorter machines can be used. Specifically, antibody-based sorting methods may include methods using microbeads.

[0079] (S2) Step CD41 - After selecting the cells, CD34 - You may select cells in the order of [this], or you may select cells in the reverse order.

[0080] Step (S3) of the present invention's method for producing red blood cells is a step of maturing the cell population obtained in (S2) in a first medium containing erythropoietin (EPO).

[0081] The first medium is a basal medium containing at least erythropoietin.

[0082] The maturation step (S3) can be carried out for 4 to 14 days, and this process allows the red blood cell production method of the present invention to produce red blood cells in a short period of time.

[0083] The present invention's method for producing red blood cells may further include, before (S3), the steps of growing the cell population obtained in (S2) in a second medium and differentiating it in a third medium.

[0084] The second culture medium further comprises erythropoietin, stem cell factors, and interleukin-3 in addition to the basal medium. The proliferation step can be carried out for 6 to 10 days, preferably for about 8 days.

[0085] The third medium further comprises at least erythropoietin and stem cell factors in addition to the basal medium. The differentiation step can be carried out for 3 to 7 days, preferably for about 5 days.

[0086] The present invention provides a method for producing red blood cells that selects cells that do not express specific markers (CD34, CD41a) and uses them for red blood cell production. This method increases not only the differentiation efficiency into red blood cells but also the cell proliferation capacity, enabling the efficient production of large quantities of red blood cells.

[0087] The efficiency of differentiation into red blood cells can be confirmed, for example, by the fact that when cells that do not express CD34 and CD41a are subsequently cultured, a large number of cells expressing CD235 (glycophorin A), a red blood cell surface marker, are produced.

[0088] The present invention provides a method for producing platelets, comprising the steps of (S1) culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, and (S2) obtaining CD41 from the culture medium. - and CD34 - The cell population and CD41a + or CD34 + (S4) The step of classifying the cell population, and (S4) the classified CD41a + or CD34 + The process includes the step of differentiating a cell population into megakaryotic cells in medium 7.

[0089] The aforementioned pluripotent stem cells may be within the range described above.

[0090] The above (S1) and (S2) may be within the range described above. The (S2) of the platelet production method of the present invention is CD41 selected in the above-described red blood cell production method (S2). - and CD34 - It may also be a cell population excluding the cell population that is specified.

[0091] Step (S4) is the CD41a classified in step (S2). + or CD34 + This step involves differentiating a population of cells into megakaryotic cells using medium 7.

[0092] Medium VII plays a role in differentiating the cell population into megakaryotic cells. Medium VII may contain cytokines. Medium VII may contain at least one of the following: thrombopoietin (TPO), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-6 (IL-6).

[0093] Thrombopoietin is a major growth factor that regulates hematopoiesis in megakaryocytes and platelets, and is mainly synthesized and secreted by hepatocytes.

[0094] Thrombopoietin does not need to be present at a specific concentration in Medium VII, but rather at a concentration sufficient to culture a suspension cell population along with other components. For example, thrombopoietin may be present at concentrations of 1–50 ng / ml, 5–45 ng / ml, 10–40 ng / ml, 15–35 ng / ml, 20–30 ng / ml, or 25 ng / ml.

[0095] Stem cell factors are matrix cell-derived cytokines synthesized by fibroblasts and other cell types. Stem cell factors do not need to be present at a specific concentration; they just need to be present in Medium VII at a concentration that allows for the culture of suspension cell populations along with other components. Thrombopoietin may be present at concentrations, for example, 1–50 ng / ml, 5–45 ng / ml, 10–40 ng / ml, 15–35 ng / ml, 20–30 ng / ml, or 25 ng / ml.

[0096] Interleukin-3 only needs to be present in Medium VII at a concentration sufficient to culture suspension cell populations together with other components; it does not need to be present at a specific concentration. Interleukin-3 may be present at concentrations such as 1–20 ng / ml, 5–15 ng / ml, or 10 ng / ml.

[0097] Interleukin-6 only needs to be present in Medium VII at a concentration sufficient to culture suspension cell populations together with other components; it does not need to be present at a specific concentration. Interleukin-6 may be present at concentrations such as 1–20 ng / ml, 5–15 ng / ml, or 10 ng / ml.

[0098] Medium VII is not limited to a specific medium. Medium VII may include IMDM (Iscove's Modified Dulbecco's Medium) as the basal medium. IMDM may further contain antioxidants (e.g., AA2P) and / or B-27.

[0099] The platelet production method of the present invention may further include the step of culturing megakaryotic cells in medium VIII containing thrombopoietin to mature them.

[0100] Thrombopoietin may, but is not limited to, be present in medium 8 at concentrations of, for example, 50-150 ng / ml, 60-140 ng / ml, 70-130 ng / ml, 80-120 ng / ml, 90-110 ng / ml, or 100 ng / ml.

[0101] Medium 8 can contain, in addition to thrombopoietin, further factors necessary for megakaryotic cell maturation.

[0102] Medium VIII is not limited to a specific medium. Medium VIII may include IMDM (Iscove's Modified Dulbecco's Medium) as the basal medium. IMDM may further contain antioxidants (e.g., Ascorbic Acid 2-Phosphate, AA2P, etc.) and / or B-27.

[0103] The result of this step may be mature megakaryotic cells, or a culture medium containing megakaryotic cells and / or mature megakaryotic cells.

[0104] In the method for producing platelets of the present invention, platelets may be separated and / or purified from the culture medium after further differentiation of the differentiated megakaryotic cells, or they may be separated and / or purified from the culture medium without further differentiation.

[0105] Platelet isolation and / or purification can be carried out by known isolation and / or purification methods. For example, this can be done by centrifugation of the culture medium or by passing the culture medium through a column.

[0106] When the culture medium is centrifuged, megakaryotic cells can be separated as a precipitate, and platelets as a suspension.

[0107] In the present invention's method for producing red blood cells and platelets, the aforementioned methods for producing red blood cells and platelets can be carried out together in a single routine.

[0108] The present invention relates to a method for producing red blood cells and platelets classified as (S3) CD41 - and CD34 - The steps include: maturing the cell population in medium 1 containing erythropoietin, and (S4) classifying CD41a + or CD34 + The step of differentiating a cell population into megakaryotic cells in medium 7 can be performed simultaneously or sequentially.

[0109] If performed sequentially, (S4) may be performed after (S3), or vice versa.

[0110] The present invention provides a method for producing red blood cells and platelets, which allows for the efficient use of cultured cells by producing red blood cells and platelets together in a single process. Furthermore, by selecting cells suitable for the characteristics of red blood cells and platelets and differentiating them accordingly, production efficiency can be increased.

[0111] The present invention will be described in more detail below with reference to examples. Example 1. Production and analysis of red blood cells 1. Red blood cell production example 1 1-1. Culture of human induced pluripotent stem cells (hiPSCs) To obtain a cell population capable of differentiating into red blood cells, human induced pluripotent stem cells were cultured as follows.

[0112] First, human induced pluripotent stem cells were cultured in mTeSR plus culture medium at a rate of 4000 cells / cm³ along with ROCK inhibitor (Y-27632) at a concentration of 10 μg / ml. 2 Cells were seeded into T75 flasks coated with vitronectin at a specific density and cultured at 37°C and 5% CO2. After 24 hours of culture, the flasks were washed once with PBS to remove Y-27632, and the mTeSR Plus culture medium was replaced with fresh medium. The culture medium was changed once daily for 3 days to secure cells for differentiation.

[0113] Subsequently, human induced pluripotent stem cells were cultured for 2 days at 37°C and 5% CO2 under conditions of RPMI1640 basal medium containing 1% P / S, 1% GlutaMAX, 300 μM AA2P, and 2% B-27, in a medium containing 6 μM CHIR99021, a GSK3 (glycogen synthase kinase 3) inhibitor, with the culture medium changed once daily (initiation stage).

[0114] To differentiate human induced pluripotent stem cells into cells capable of differentiating into megakaryotic cells, the RPMI1640 basal medium containing 1% P / S, 1% GlutaMAX, 300 μM AA2P, and 2% B-27 was modified to a medium containing 50 ng / ml VEGF and 20 ng / ml bFGF. The cells were then cultured for 3 days at 37°C in a 5% CO2 incubator, with the culture medium being changed once daily. This process was continued until the cells reached approximately 90% confluence.

[0115] Subsequently, RPMI1640 basal medium containing 1% P / S, 1% GlutaMAX, 300 μM AA2P, and 2% B-27, along with VEGF and bFGF, and 10 μM SB431542 were added, and the cultures were incubated for an additional 4 days, changing the culture medium every 2 days.

[0116] A cultured population of suspended cells was obtained, and CD41a and CD34 microbeads were used to extract CD41a from the suspended cell population. - CD34 - The cells and CD41a - CD34 + We selected cells that were such as [specific cells].

[0117] 1-2. Culture the selected cell population to induce differentiation into red blood cells. CD41a obtained by selecting from the suspension cell population acquired in "1-1. Culture of Human Induced Pluripotent Stem Cells (hiPSCs)" above. - CD34 - Cells and CD41a - CD34 + The cells were cultured.

[0118] CD41a - CD34 - Cells and CD41a - CD34 + Cells 1 × 10 5 Each cell was added to IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of cells / ml. 6 U / ml erythropoietin (EPO), 100 ng / ml SCF, and 10 ng / ml IL-3 were added to the medium, and the cells were cultured (grown) at 37°C and 5% CO2 for 8 days.

[0119] Subsequently, the culture medium composition was changed to an IMDM basal medium containing 300 μM AA2P and 2% B-27, with 6 U / ml EPO and 100 ng / ml SCF added, and the cells were cultured (differentiated) for an additional 5 days (in this invention, the proliferation and differentiation process is referred to as the differentiation method (Diff)). Then, the culture medium composition was changed to an IMDM basal medium containing 300 μM AA2P and 2% B-27, with 6 U / ml EPO added, and the cells were cultured (matured) for an additional 8 days to obtain the cell culture medium.

[0120] Red blood cells turn red due to hemoglobin during differentiation, so the color of the cell pellet was checked to confirm this.

[0121] The culture media of the two cell populations obtained above were placed in 15 ml conical tubes and centrifuged at 400 × g at room temperature (RT) for 5 minutes. The supernatant was removed, and the pellet was resuspended in 1 ml 1x PBS and transferred to a 1.7 ml tube, where it was centrifuged at 400 × g at room temperature (RT) for 5 minutes. The pellet was photographed with the tube held horizontally to allow for a clear view.

[0122] As a result, CD41a - CD34 - The cell pellet takes on a reddish tint, while CD41a - CD34 + The cells do not have a reddish tint, and CD41a - CD34 -We were able to confirm that the substance obtained by culturing cells was differentiated into red blood cells (Figure 3a).

[0123] 2. Red blood cell production example 2 2-1. Culture of human induced pluripotent stem cells (hiPSCs) Human induced pluripotent stem cells (hiPSCs) were cultured using the same method as described in "1-1. Culture of Human Induced Pluripotent Stem Cells (hiPSCs)" above, and CD41a - CD34 - The cells and CD41a - CD34 + We selected cells that were such as [specific cells].

[0124] 2-2. Culture the selected cell population and induce differentiation into erythrocytes. CD41a obtained by selecting from the suspension cell population acquired in "2-1. Culture of Human Induced Pluripotent Stem Cells (hiPSCs)" above. - CD34 - Cells and CD41a - CD34 + The cells were cultured.

[0125] CD41a - CD34 - Cells and CD41a - CD34 + Cells 1 × 10 5 Cells were placed in IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of cells / ml, and 6 U / ml EPO was added to the medium. The cells were then cultured at 37°C and 5% CO2 for 14 days (maturation) to obtain the cell culture medium.

[0126] The culture media of the two acquired cell populations were placed in 15 ml conical tubes and centrifuged at 400 x g at room temperature (RT) for 5 minutes. The supernatant was removed, and the pellet was resuspended in 1 ml 1x PBS and transferred to a 1.7 ml tube, where it was centrifuged at 400 x g at room temperature (RT) for 5 minutes. The pellet was photographed with the tube held horizontally to allow for clear visualization.

[0127] As a result, CD41a- CD34 - The cell pellet is reddish, while CD41a - CD34 + cells are not reddish, and CD41a - CD34 - It was confirmed that the substance obtained by culturing CD34 cells was differentiated into red blood cells (Figure 3b).

[0128] 3. Confirmation of being normal red blood cells 3-1. Blood cell staining (Wright-Giemsa staining) In the above "1-2" and "2-2", CD41a - CD34 - cells and CD41a - CD34 + To confirm whether the substance obtained by culturing CD34 cells was differentiated into red blood cells, Wright-Giemsa staining was performed.

[0129] Wright-Giemsa staining is a blood smear staining method, in which red blood cells are stained pink, platelets are stained light pink, the cytoplasm of lymphocytes is stained sky blue, and the cytoplasm of monocytes is stained blue.

[0130] The experimental method is as follows. The 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 centrifuging at 400×g for 5 minutes at 4°C, they were resuspended in 30 μl of 1xPBS, 5 μl was placed on a slide glass, and then dried on a heat block at 56°C. After the slide was completely cooled, it was reacted with 0.1% Triton X-100 at room temperature for 10 minutes. After washing with 1xPBS, it was stained with giemsa solution for 10 minutes. After washing with distilled water (DW), it was observed under a microscope (observation magnification 400x).

[0131] As a result, CD41a - CD34 -In the substances obtained by culturing cells, orthochromatic normoblasts (red blood cells with a nucleus: white arrow) and reticulocytes (red blood cells without a nucleus: black arrow) were observed (Figure 4).

[0132] During the differentiation process of red blood cells, a phenomenon occurs where the nucleus shifts to one side before enucleation, and upon enucleation, the cell becomes composed solely of cytoplasm, but CD41a - CD34 - Since all of these cells were observed in the substance obtained by culturing cells, it was confirmed that this substance was red blood cells.

[0133] 3-2.Immunostaining To confirm that the substances obtained in "1-2" and "2-2" above were red blood cells, immunohistochemical staining was used to check for the presence or absence of CD235 expression, a red blood cell surface marker, and the presence or absence of nuclei.

[0134] The cells were centrifuged at 400×g for 5 minutes and resuspended in 200 μl of 1% BSA. CD235-PE antibody was then added and the cells were reacted at 4°C for 30 minutes. After reacting, the cells were centrifuged at 400×g at 4°C for 5 minutes, the supernatant was removed, and the cells were washed with 500 μl of 1x PBS. The washed cells were resuspended with 30 μl of 1x PBS, and then 1 μl of Hoechst 33342 was added, followed by observation under a fluorescence microscope.

[0135] To confirm CD235 expression, each cell was reacted with a CD235-PE antibody, and to confirm the presence or absence of a nucleus, it was reacted with Hoechst 33342.

[0136] As a result, as shown in Figure 5, CD41a - CD34 - In samples obtained from cultured cells, cells were observed in which only CD235 was stained while the nucleus remained unstained (white arrows). These can be considered reticulocytes that express CD235 during the differentiation process of erythrocytes and lack a nucleus. In contrast, CD41a -CD34 + In the substances obtained by culturing cells, almost no cells expressing CD235 were confirmed, and although CD235 was stained, no cells with unstained nuclei were found either.

[0137] 3-3. FACS Analysis To confirm whether the substances obtained in "1-2" and "2-2" express CD71 and CD235, which are erythrocyte-specific markers, FACS analysis was performed. For FACS analysis, a Lyric device from BD was used, and anti-CD71-APC and anti-CD235-PE antibodies were used. The antibodies were diluted 1:20 in PBS (FACS buffer) containing 1% BSA and reacted for 30 minutes to confirm the expression of the markers.

[0138] As a result, CD41a - CD34 - The substances obtained from the cells were positive for the expression of erythrocyte-specific markers (CD71, CD235) (Figure 6). From this, it was confirmed that this substance is erythrocytes.

[0139] On the other hand, CD41a - CD34 + The substances obtained from the cells had very low expression of CD71 and CD235, and it was confirmed that they were not erythrocytes (Figure 6).

[0140] 3-4. Cell Counting CD41a - CD34 - Cells and CD41a - CD34 + To confirm the cell proliferation ability of the cell population, during the induction of differentiation into erythrocytes in step 2 above, the growth rate of the cells was confirmed by cell count.

[0141] 20 μl of cells was mixed with 20 μl of 0.4% trypan blue, and after 1 minute, 10 μl was measured with a Countess 3 cell counter machine.

[0142] As a result, CD41a - CD34 - The cell population is CD41a - CD34 + It was shown that the cell proliferation rate is higher compared to the cell population, and CD41a - CD34 - We confirmed that a larger quantity of red blood cells can be produced when red blood cells are manufactured using cells (Figure 7).

[0143] 3-5. Hemoglobin ELISA CD41a - CD34 - Cells and CD41a - CD34 + An ELISA was performed to check the hemoglobin levels in the cell population.

[0144] 1 x 10 6 Cells were lysed in 30 μl of RIPA buffer at 4°C for 30 minutes, then centrifuged at 12000 rpm at 4°C for 30 minutes to obtain the supernatant. 25 μl of the supernatant was diluted in 225 μl of dilution buffer, and 100 μl was loaded per well. The experiment was conducted according to the protocol of the manufacturer of Ab157707, and the results were obtained (Figure 8).

[0145] 3-6. Transmission electron microscopy (TEM) CD41a - CD34 - To observe the cells, transmission electron microscopy was performed as follows.

[0146] CD41a - CD34 -Cells were pre-fixed with 2.5% glutaraldehyde (4°C, phosphate buffer, pH 7.2). Post-fixation was performed with 1% osmium tetraoxide (OsO4, 4°C, phosphate buffer, pH 7.2). After fixation, the samples were washed with the same buffer and then dehydrated in increasing order of alcohol concentration (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%, 100%). The samples were embedded and solidified in Epon 812 mixture. The embedded tissues were sectioned to a thickness of 1 μm, stained with 1% toluidine blue, and then the remaining portion was trimmed after identifying a specific area. Subsequently, 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 with a transmission electron microscope (JEM-1200EXII, JEOL) (Figure 9).

[0147] 3-7. Scanning Electron Microscopy (SEM) CD41a - CD34 - To observe the cells, scanning electron microscopy was performed as follows:

[0148] The cells were pre-fixed with 2.5% glutaraldehyde (4°C, phosphate buffer, pH 7.2). Post-fixation was performed with 1% osmium tetraoxide (OsO4, 4°C, phosphate buffer, pH 7.2). After fixation, the samples were washed with water using the same buffer, and then dehydrated in increasing order of alcohol concentration (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%, 100%). The samples were then treated with ethanoyl:HMDS (Hexamethyldisilazane) in ratios of 3:1, 1:1, and 1:3, and replaced with 100% HMDS. The samples were placed on a silicon wafer and dried. After coating with Pt (SMC12R-Plus (Semian, Korea): 15mA, 2-3 minutes), the cells were observed using Regulus 8220 (Hitachi, Japan) (Figure 10).

[0149] Example 2. Production and analysis of platelets 1. Culture of human induced pluripotent stem cells (hiPSCs) To obtain a cell population capable of differentiating into megakaryotic cells, human induced pluripotent stem cells were cultured as follows.

[0150] First, human induced pluripotent stem cells were cultured at a rate of 4000 cells / cm³ in mTeSR Plus culture medium along with ROCK inhibitor (Y-27632) at a concentration of 10 μg / ml. 2 The cells were placed in a T75 flask at a density and cultured at 37°C and 5% CO2. After 24 hours of culture, the cells were washed once with PBS to remove Y-27632, and the mTeSR Plus culture medium was replaced with fresh medium. The cells were cultured for 3 days, changing the culture medium once daily, to secure cells for differentiation.

[0151] Subsequently, human induced pluripotent stem cells were cultured for 2 days at 37°C and 5% CO2 in RPMI1640 basal medium containing 300 μM AA2P and 2% B-27, with 6 μM of CHIR99021, a GSK3 (glycogen synthase kinase 3) inhibitor (culture medium was changed once daily).

[0152] To differentiate human induced pluripotent stem cells into cells capable of differentiating into megakaryotic cells, the culture medium composition was changed from RPMI1640 basal medium containing 300 μM AA2P and 2% B-27 to a medium containing 50 ng / ml VEGF and 20 ng / ml bFGF. The cells were cultured for 3 days at 37°C in a 5% CO2 incubator (the culture medium was changed once daily). During this process, the cells were cultured until their confluence reached approximately 70%. Subsequently, 10 μM SB431542 was added along with RPMI1640 basal medium containing 300 μM AA2P and 2% B-27, VEGF, and bFGF, and the cells were cultured for a predetermined period.

[0153] 1) Example of suspension cell production 1 On day 4 of additional culture, flow cytometry (FACS) was used to determine the ratio of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of suspended cells. BD's Lyric instrument was used for flow cytometry, employing BD's anti-CD34-FITC, anti-CD45-FITC, and anti-CD41a-APC antibodies. This allowed for the analysis of the ratio of single- or double-positive cells for CD34 and CD41a, and for CD45 and CD41a, respectively.

[0154] As a result, it was confirmed that the proportion of CD34+ cells was 57.55%, the proportion of CD41a+ cells was 54.55%, and the proportion of CD45+ cells was 18.58% of the total cells. When the proportion of CD34+ cells was 57.55%, the proportion of CD41a+ cells was 54.55%, and the proportion of CD45+ cells was 18.58% in the suspension cells, a population of floating cells (Figure 11, day 9) was obtained from the culture medium.

[0155] 2) Example of suspension cell production 2 On day 6 of additional culture, flow cytometry was used to determine the ratio of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of suspension cells. The results showed that 61.59% of the total cells were CD34+, 47.03% were CD41a+, and 32.4% were CD45+. When the proportion of suspension cells reached 61.59% CD34+, 47.03% CD41a+, and 32.4% CD45+, a suspension cell population (days 11-12 in Figure 11) was collected from the culture medium.

[0156] 3) Example of suspension cell production 3 On day 8 of additional culture, flow cytometry was used to determine the ratio of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of suspension cells. The results showed that 52.05% of the cells were CD34+, 18.88% were CD41a+, and 45.56% were CD45+. At the point when the proportion of suspension cells reached 52.05% CD34+, 18.88% CD41a+, and 45.56% CD45+, a suspension cell population (Day 13 in Figure 11) was obtained from the culture medium.

[0157] 4) Example of suspension cell production 4 On the second day of additional culture, flow cytometry was used to determine the ratio of cells expressing specific surface markers (CD34, CD45) to the total number of suspension cells. The results showed that 59.27% ​​of the cells were CD34+ and 5.21% were CD45+. When the proportion of suspension cells reached 59.27% ​​and 5.21%, a suspension cell population (Figure 11, day 7) was collected from the culture medium.

[0158] 5) Comparative manufacturing example of suspension cells 1 On day 10 of additional culture, flow cytometry was used to determine the ratio of cells expressing specific surface markers (CD34, CD41a, CD45) to the total number of suspension cells. The results showed that 91.2% of the cells were CD34+, 13% were CD41a+, and 68.51% were CD45+. When the proportion of suspension cells reached 91.2% CD34+, 13% CD41a+, and 68.51% CD45+, a suspension cell population (Day 15, Figure 11) was obtained from the culture medium.

[0159] 2. Culture the suspension cell population and induce differentiation into megakaryotic cells. To facilitate differentiation and maturation into megakaryotic cells, a population of floating cells obtained in "1. Culture of Human Induced Pluripotent Stem Cells (hiPSCs)" above was cultured.

[0160] For differentiation into megakaryotic cells from the suspension cell populations obtained in "1. Culture of Human Induced Pluripotent Stem Cells (hiPSCs)" above, each suspension cell population from suspension cell production examples 1-4 and comparative production example 1 was prepared in a 1 × 10⁻¹⁶ manner. 5 The cells were added to IMDM basal medium containing 300 μM AA2P and 2% B-27 at a density of cells / ml. 25 ng / ml TPO, 25 ng / ml SCF, 10 ng / ml IL-3, and 10 ng / ml IL-6 were added to the medium, and the cells were incubated at 37°C and 5% CO2 for 3 days.

[0161] Subsequently, the culture medium composition was changed to IMDM basal medium containing 300 μM AA2P and 2% B-27, with 100 ng / ml TPO added. The cells were cultured for an additional 5 days to perform the maturation step (Megakaryocyte maturation), and the cell culture medium was obtained.

[0162] 3. Confirmation of platelet-producing ability of megakaryocytes Platelets were obtained from the culture media of suspension cell production examples 1 and 3 and comparative production example 1, which were acquired in "2. Induction of differentiation of suspension cell populations into megakaryocytes" above. Specifically, all of the cell culture media from suspension cell production examples 1 and 3 and comparative production example 1, which were acquired in "2. Induction of differentiation of suspension cell populations into megakaryocytes" above, were collected and centrifuged at 300 × g for 3 minutes. The precipitate was separated as megakaryocytes and the suspension as platelets, and these were separated and analyzed for platelets.

[0163] The experiments described in (1) and (2) below confirmed that the final material obtained by the method described above was a platelet with normal activity.

[0164] (1) Confirmation of platelet-specific markers by FACS analysis The cell culture media of suspension cell production examples 1 and 3, and comparative production example 1 were centrifuged to remove the precipitated megakaryocytes, and the suspended platelets were centrifuged again at 2000 × g for 10 minutes. The platelets precipitated by centrifugation were subjected to FACS analysis to confirm the expression of surface markers. A BD Lyric instrument was used for the FACS analysis, and BD's anti-CD41a-FITC, anti-CD61-PE, and anti-CD42b-APC antibodies were used. The antibodies were diluted 1:20 in PBS (FACS buffer) containing 1% BSA and reacted for 30 minutes to confirm the expression of surface markers.

[0165] As a result, the substance obtained from suspension cell production example 1 was positive for the expression of platelet-specific markers (CD41a, CD42b, and CD61) (Figure 12), and the substance obtained from suspension cell production example 3 was also positive for the expression of platelet-specific markers (CD41a, CD42b, and CD61) (Figure 13). From this, it was confirmed that the cell populations from suspension cell production examples 1 and 3 are capable of generating platelets.

[0166] On the other hand, the substance obtained from comparative production example 1 of suspension cells showed very low expression of CD41a and CD61, and in particular, CD42b showed near-negative expression (Figure 14). From this, it was confirmed that the substance obtained from comparative production example 1 did not exhibit platelet surface marker characteristics, and that the cell population from comparative production example 1 hardly differentiated into megakaryotic cells and did not produce platelets.

[0167] (2) Confirmation of platelet function by ADP treatment To confirm whether the substances obtained from suspension cell production examples 1 and 3 were platelets with normal activity, the following experiments were performed.

[0168] The substances obtained from suspension cell production 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 recover the platelets precipitated by centrifugation, all suspended matter was removed and the platelets were resuspended in a small amount of PBS.

[0169] For PAC-1 analysis, platelets were treated with 100 μM ADP and reacted at room temperature for 15 minutes. BD's anti-PAC-1-FITC and anti-CD62p-PE antibodies were then reacted for 30 minutes. The reacted platelets were immediately subjected to FACS analysis. The substances obtained from Production Examples 1 and 3 were activated by ADP treatment, and the expression of PAC-1 and CD62p increased (Figures 15 and 16). This confirmed that the substances obtained from Production Examples 1 and 3 were platelets with normal activity. PAC-1 appears when platelets are activated, forming a complex of CD41a and CD61. CD62p (p-selectin) is a surface molecule whose expression increases when platelets are activated, and it plays a role as a site where leukocytes can bind to platelets.

[0170] The experimental results described above confirmed that when subsequent culture steps are performed after culturing hiPSCs, provided that the cell population meets predetermined conditions (the ratio of cells expressing specific surface markers (e.g., CD41a, CD34, CD45) to the total number of cells is above or below a predetermined value), the differentiation efficiency into megakaryotic cells and the platelet production efficiency are enhanced.

[0171] 4. Comparison of platelet generation efficiency when only CD41a+ cells are selected and subsequently cultured. From the suspension cell population obtained in the aforementioned suspension cell production example 1, only CD41a+ cells were selected (Figure 17) and subsequently cultured, and the platelet generation efficiency was compared with that of the suspension cell population from production example 1.

[0172] Using the same method as the FACS analysis described in "3.(1)" above, we confirmed the expression of platelet-specific markers in the two cases and found that the ratio of CD41a+ / CD42b+ was similar, indicating that the platelet differentiation rates were similar (Figure 18).

[0173] However, after examining the cell proliferation capacity in both cases, we confirmed that differentiating a cell population that includes not only CD41a+ cells but also non-CD41a+ cells (e.g., CD41a-) into platelets, as in Production Example 1, showed approximately 40 times higher cell proliferation capacity compared to selecting CD41a+ cells and differentiating them into platelets (Figure 19).

[0174] From the results described above, it was confirmed that using a cell population that includes CD41a- cells and other cells, as in Production Example 1 of this application, for platelet production is more efficient in terms of both quantity and quality than selecting only CD41a+ cells for platelet production.

Claims

1. (S1) A step of culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, (S2) CD41 from the culture medium － and CD34 － The steps include obtaining a population of cells, A method for producing red blood cells, comprising the step of (S3) maturing the acquired cell population in a first medium containing erythropoietin (EPO).

2. The method for producing red blood cells according to claim 1, further comprising the step of growing the obtained cell population in a second medium containing erythropoietin, stem cell factors, and interleukin-3.

3. A method for producing red blood cells according to claim 1, further comprising the step of differentiating the obtained cells in a third medium containing erythropoietin and stem cell factors.

4. The method for producing red blood cells according to claim 1, wherein the culture medium comprises a red blood cell precursor and a megakaryocyte precursor.

5. The method for producing red blood cells according to claim 1, wherein the maturation step is carried out for 4 to 14 days.

6. The method for producing red blood cells according to claim 1, comprising the following steps (S1): (S1a) A step of culturing the pluripotent stem cells in a fourth medium containing a GSK3 inhibitor; (S1b) A step of culturing the cells cultured in the fourth medium in a fifth medium containing vascular endothelial growth factor and basic fibroblast growth factor; and, (S1c) A step of culturing the cells cultured in the fifth medium in a sixth medium containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor beta signaling inhibitor.

7. (S0) The pluripotent stem cells are placed at the bottom of the culture vessel at a rate of 2,000 to 20,000 cells / cm². ２ A method for producing red blood cells according to claim 1, further comprising the step of seeding.

8. The method for producing red blood cells according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells.

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

10. (S1) A step of culturing pluripotent stem cells to obtain a culture medium containing hematopoietic stem cells, (S2) CD41 from the culture medium － and CD34 － A population of cells and CD41a ＋ or CD34 ＋ The step of classifying the cell population, (S3) maturing the classified CD41 － and CD34 － cell population, which is, in a first medium containing erythropoietin; (S4) The classified CD41a ＋ or CD34 ＋ A method for producing red blood cells and platelets, comprising the step of differentiating a cell population into megakaryotic cells in medium 7.

11. The aforementioned classified CD41 － and CD34 － A method for producing red blood cells and platelets according to claim 10, further comprising the step of growing a cell population in a second medium containing erythropoietin, stem cell factors, and interleukin-3.

12. The aforementioned classified CD41 － and CD34 － A method for producing red blood cells and platelets according to claim 10, further comprising the step of differentiating a cell population in a third medium containing erythropoietin and stem cell factors.

13. The method for producing red blood cells and platelets according to claim 10, wherein the culture medium comprises red blood cell precursors and megakaryocyte precursors.

14. The method for producing red blood cells and platelets according to claim 10, wherein (S3) is carried out for 4 to 14 days.

15. The method for producing red blood cells and platelets according to claim 10, comprising the following steps (S1): (S1a) A step of culturing the pluripotent stem cells in a fourth medium containing a GSK3 inhibitor; (S1b) A step of culturing the cells cultured in the fourth medium in a fifth medium containing vascular endothelial growth factor and basic fibroblast growth factor; and, (S1c) A step of culturing the cells cultured in the fifth medium in a sixth medium containing vascular endothelial growth factor, basic fibroblast growth factor, and a transforming growth factor beta signaling inhibitor.

16. (S0) The pluripotent stem cells are placed at the bottom of the culture vessel at a rate of 2,000 to 20,000 cells / cm². ２ A method for producing red blood cells and platelets according to claim 10, further comprising the step of seeding.

17. A method for producing red blood cells and platelets according to claim 10, further comprising the step of culturing the megakaryocytes in a medium containing thrombopoietin to mature them.

18. The method for producing red blood cells and platelets according to claim 10, wherein the seventh culture medium comprises thrombopoietin, stem cell factor, interleukin-3, and interleukin-6.

19. The method for producing red blood cells and platelets according to claim 10, wherein the pluripotent stem cells are human induced pluripotent stem cells.

20. A method for producing a blood product, comprising the step of mixing red blood cells and platelets produced by the method described in any one of claims 10 to 19.

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