Medium composition for inducing differentiation of stem cells into platelet precursor cells and differentiation method using the same

Abstract

The present invention relates to a medium composition for inducing differentiation of stem cells into platelet precursor cells and a method for inducing differentiation of stem cells into platelet precursor cells in the presence of the medium composition. The medium composition for inducing differentiation of stem cells into platelet precursor cells according to one embodiment has the effect of increasing the efficiency of differentiation into platelet precursor cells and enabling differentiation into platelet precursor cells without genetic manipulation.

Description

[Technical Field]

[0001] The present invention relates to a medium composition for inducing differentiation of stem cells into platelet precursor cells. [Background technology]

[0002] Treatment of blood-related diseases and surgical procedures requires a large number of blood cells. Among blood cells, platelets, which are essential for blood clotting and hemostasis, are particularly important. Platelet preparations are administered to patients who experience massive bleeding during surgery or injury, or who show a tendency to bleed due to thrombocytopenia after anticancer drug treatment, for the purpose of treating and preventing these symptoms. Currently, the production of platelet preparations relies on blood donations from healthy volunteers, but the existing blood supply system and transfusions are reaching their limits.

[0003] In vivo, megakaryocytes form pseudopodial formations called proplatelets (platelet precursors), fragment their cytoplasm, and release platelets. It is believed that megakaryocytes undergo multinucleation by endomitosis before releasing platelets. Endomitosis in megakaryocytes is a multipolar mitosis caused by abnormalities in karyokinesis and cytokinesis without schizosphere formation or spindle elongation, resulting in the formation of cells containing several lobulated nuclei. Repeated endomitosis induces megakaryocyte multinucleation.

[0004] In recent years, research has been conducted into techniques for inducing differentiation of pluripotent stem cells, such as ES cells and iPS cells, in vitro to prepare blood cells such as platelets. However, the efficiency of inducing differentiation into megakaryocytes is low, so there is no differentiation technique that can mass-produce artificial platelets. Therefore, there is a need for a technique that can induce differentiation into highly pure megakaryocytes without gene transfer. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect is a medium composition for inducing differentiation of stem cells into platelet precursor cells, comprising: (i) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of CHIR99021, Bone Morphogenetic Protein 4 (BMP4), Vascular Endothelial Growth Factor (VEGF), Basic Fibroblast Growth Factor (bFGF), Retinoic acid, SB-431542, Polyvinyl Alcohol (PVA), Stem Cell Factor (SCF), Interleukin 3 (IL3), Valproic acid (VPA), and combinations thereof; and (ii) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of Stem Cell Factor (SCF), Thrombopoietin (TPO), Polyvinyl Alcohol (PVA), Basic Fibroblast Growth Factor (bFGF), Butyzamide, Interleukin 6 (IL6), and combinations thereof. and (iii) a third medium composition for maturing megakaryocyte precursor cells into platelet precursor cells, the third medium composition comprising one or more selected from the group consisting of stem cell factor (SCF), interleukin 6 (IL6), valproic acid (VPA), fasudil, butyzamide, basic fibroblast growth factor (bFGF), and combinations thereof.

[0006] Another aspect is to provide a method for inducing differentiation of stem cells into platelet progenitor cells, comprising the steps of inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells in the presence of the first medium composition, inducing differentiation of hematopoietic progenitor cells or hematopoietic stem cells into megakaryocyte progenitor cells in the presence of the second medium composition, and inducing maturation of megakaryocyte progenitor cells into platelet progenitor cells in the presence of the third medium composition. [Means for solving the problem]

[0007] One aspect is a medium composition for inducing differentiation of stem cells into platelet precursor cells, comprising: (i) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of CHIR99021, Bone Morphogenetic Protein 4 (BMP4), Vascular Endothelial Growth Factor (VEGF), Basic Fibroblast Growth Factor (bFGF), Retinoic acid, SB-431542, Polyvinyl Alcohol (PVA), Stem Cell Factor (SCF), Interleukin 3 (IL3), Valproic acid (VPA), and combinations thereof; and (ii) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of Stem Cell Factor (SCF), Thrombopoietin (TPO), Polyvinyl Alcohol (PVA), Basic Fibroblast Growth Factor (bFGF), Butyzamide, Interleukin 6 (IL6), and combinations thereof. and (iii) a third medium composition for maturing megakaryocyte precursor cells into platelet precursor cells, the third medium composition comprising one or more selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), interleukin 6 (IL6), fasudil, butyzamide, basic fibroblast growth factor (bFGF), and combinations thereof.

[0008] As used herein, the term "megakaryocyte" can be used interchangeably with the term "platelet precursor cell" and refers to a large (e.g., diameter ≥ 10 μm) polyploid hematopoietic cell with the propensity to generate proplatelets and / or platelets. They are characterized by positivity for the cell surface markers CD41a, CD42a, and CD42b, and may also express markers selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. One morphological characteristic of mature megakaryocytes is the development of large, multilobed nuclei. Mature megakaryocytes can cease proliferation but continue to increase their DNA content through endomitosis, with a parallel increase in cell size.

[0009] The term "platelet" refers to a cell with a diameter of 1-3 μm that does not have a nucleus but contains RNA. Platelets can express CD41, CD42b, and CD61 on their cell surface. These function primarily in regulating hemostasis by participating in blood clotting, but have also been suggested to play a role in inflammation.

[0010] The term "proplatelet" refers to a megakaryocyte or a cytoplasmic extension that has just been released from a megakaryocyte. Proplatelets are isolated through cytoskeletal rearrangements to form individual platelets.

[0011] As used herein, the term "CHIR99021" refers to a compound having the structural formula of Chemical Formula 1 below.

[0012] [ka]

[0013] As used herein, the term "SB-431542" refers to a compound having the structural formula of Formula 2 below.

[0014] [ka]

[0015] As used herein, the term "UM729" refers to a compound having the structural formula of Chemical Formula 3 below.

[0016] [ka]

[0017] The term "KP-457" as used herein means a compound having the structural formula of Chemical Formula 4 below.

[0018] [ka]

[0019] In the present invention, the term "culture medium" varies depending on the intended use, but may include any common culture medium for animal cell culture that essentially contains inorganic salts, a carbon source, amino acids, bovine serum albumin (BSA), cofactors, etc., and is well known to those skilled in the art. Particularly preferably, the culture medium contains NaCl, KCl, NaHCO3, etc. as inorganic salts, glucose, sodium pyruvate, calcium lactate, etc. as carbon sources, essential and non-essential amino acids including glutamine as amino acids, and other trace elements and buffer solutions, etc. as cofactors.

[0020] The medium may also contain antibiotics. The medium may be any of several known and commercially available media for animal cell culture, including, but not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Endothelial differentiation medium (EDM), Minimal Essential Medium (MEM), Basal Medium Eagle's (BME), RPMI 1640, F-10, F-12, α-Minimal Essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium. In one specific example, StemPro-34 SFM medium may be used.

[0021] In one specific example, the concentration of CHIR99021 in the first medium composition may be 0.5 to 50 uM, 0.5 to 40 uM, 0.5 to 30 uM, 0.5 to 20 uM, 0.5 to 10 uM, 1 to 50 uM, 2 to 50 uM, 3 to 50 uM, 4 to 50 uM, 1 to 40 uM, 2 to 30 uM, 3 to 20 uM, or 4 to 10 uM.

[0022] In one specific example, the concentration of the BMP4 in the first medium composition may be 5 to 500 ng / ml, 5 to 400 ng / ml, 5 to 300 ng / ml, 5 to 200 ng / ml, 5 to 100 ng / ml, 10 to 500 ng / ml, 20 to 500 ng / ml, 30 to 500 ng / ml, 40 to 500 ng / ml, 10 to 400 ng / ml, 20 to 300 ng / ml, 30 to 200 ng / ml, or 40 to 100 ng / ml.

[0023] In one specific example, the concentration of the VEGF in the first medium composition may be 5 to 500 ng / ml, 5 to 400 ng / ml, 5 to 300 ng / ml, 5 to 200 ng / ml, 5 to 100 ng / ml, 10 to 500 ng / ml, 20 to 500 ng / ml, 30 to 500 ng / ml, 40 to 500 ng / ml, 10 to 400 ng / ml, 20 to 300 ng / ml, 30 to 200 ng / ml, or 40 to 100 ng / ml.

[0024] In one specific example, the concentration of the bFGF in the first medium composition can be 1 to 1000 ng / ml, 1 to 900 ng / ml, 1 to 800 ng / ml, 1 to 700 ng / ml, 1 to 600 ng / ml, 1 to 500 ng / ml, 1 to 400 ng / ml, 1 to 300 ng / ml, 1 to 200 ng / ml, 10 to 1000 ng / ml, 20 to 1000 ng / ml, 30 to 1000 ng / ml, 40 to 1000 ng / ml, 10 to 900 ng / ml, 20 to 800 ng / ml, 30 to 700 ng / ml, 40 to 600 ng / ml, 40 to 500 ng / ml, 40 to 400 ng / ml, 40 to 300 ng / ml, 40 to 200 ng / ml, 10 to 200 ng / ml, or 10 to 100 ng / ml.

[0025] In one specific example, the concentration of retinoic acid in the first medium composition may be 0.1 to 10 uM, 0.1 to 8 uM, 0.1 to 6 uM, 0.1 to 4 uM, 0.1 to 2 uM, 0.3 to 10 uM, 0.5 to 10 uM, 0.7 to 10 uM, 0.9 to 10 uM, 0.3 to 8 uM, 0.5 to 6 uM, 0.7 to 4 uM, or 0.9 to 2 uM.

[0026] In one specific example, the concentration of SB-431542 in the first medium composition can be 1 to 100 uM, 1 to 80 uM, 1 to 60 uM, 1 to 40 uM, 1 to 20 uM, 3 to 100 uM, 5 to 100 uM, 7 to 100 uM, 9 to 100 uM, 3 to 80 uM, 5 to 60 uM, 7 to 40 uM, or 9 to 20 uM.

[0027] In one specific example, the concentration of the PVA in the first medium composition is 0.01 to 10% (w / v), 0.01 to 5% (w / v), 0.01 to 3% (w / v), 0.01 to 2% (w / v), 0.01 to 1% (w / v), 0.01 to 0.8% (w / v), 0.01 to 0.6% (w / v), 0.01 to 0.4% (w / v), 0.01 to 0.2% (w / v), 0.0 It can be 1-0.1% (w / v), 0.03-5% (w / v), 0.05-3% (w / v), 0.09-2% (w / v), 0.1-1% (w / v), 0.05-10% (w / v), 0.1-10% (w / v), 0.5-8% (w / v), 0.7-7% (w / v), 0.8-6% (w / v), 0.9-5% (w / v), or 0.9-2% (w / v).

[0028] In one specific example, the concentration of the SCF in the first medium composition may be 2 to 200 ng / ml, 2 to 150 ng / ml, 2 to 100 ng / ml, 2 to 50 ng / ml, 5 to 200 ng / ml, 10 to 200 ng / ml, 15 to 200 mg / ml, 5 to 150 ng / ml, 10 to 100 ng / ml, or 15 to 50 ng / ml.

[0029] In one specific example, the concentration of IL3 in the first medium composition can be 2 to 200 ng / ml, 2 to 150 ng / ml, 2 to 100 ng / ml, 2 to 50 ng / ml, 5 to 200 ng / ml, 10 to 200 ng / ml, 15 to 200 mg / ml, 5 to 150 ng / ml, 10 to 100 ng / ml, or 15 to 50 ng / ml.

[0030] In one specific example, the concentration of VPA in the first medium composition may be 0.1 to 50 mM, 0.1 to 30 mM, 0.1 to 10 mM, 0.1 to 5 mM, 0.1 to 2 mM, 0.3 to 50 mM, 0.5 to 30 mM, 0.7 to 10 mM, 0.9 to 5 mM, 0.7 to 5 mM, or 0.5 to 3 mM.

[0031] In one specific example, the concentration of the SCF in the second medium composition can be 2 to 200 ng / ml, 2 to 150 ng / ml, 2 to 100 ng / ml, 2 to 50 ng / ml, 5 to 200 ng / ml, 10 to 200 ng / ml, 15 to 200 mg / ml, 5 to 150 ng / ml, 10 to 100 ng / ml, or 15 to 50 ng / ml.

[0032] In one specific example, the concentration of TPO in the second medium composition may be 5 to 500 ng / ml, 5 to 400 ng / ml, 5 to 300 ng / ml, 5 to 200 ng / ml, 5 to 100 ng / ml, 10 to 500 ng / ml, 20 to 500 ng / ml, 30 to 500 ng / ml, 40 to 500 ng / ml, 10 to 400 ng / ml, 20 to 300 ng / ml, 30 to 200 ng / ml, or 40 to 100 ng / ml.

[0033] In one specific example, the concentration of the PVA in the second medium composition can be 0.01 to 1% (w / v), 0.01 to 0.8% (w / v), 0.01 to 0.6% (w / v), 0.01 to 0.4% (w / v), 0.01 to 0.2% (w / v), 0.01 to 0.1% (w / v), 0.03 to 1% (w / v), 0.05 to 1% (w / v), 0.07 to 1% (w / v), 0.09 to 1% (w / v), 0.03 to 0.8% (w / v), 0.05 to 0.6% (w / v), 0.07 to 0.4% (w / v), or 0.09 to 0.2% (w / v).

[0034] In one specific example, the concentration of the bFGF in the second medium composition can be 1 to 100 ng / ml, 1 to 90 ng / ml, 1 to 80 ng / ml, 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 3 to 100 ng / ml, 5 to 100 ng / ml, 7 to 100 ng / ml, 9 to 100 ng / ml, 3 to 90 ng / ml, 5 to 80 ng / ml, 7 to 70 ng / ml, 9 to 60 ng / ml, 15 to 50 ng / ml, 17 to 30 ng / ml, or 1 to 50 ng / ml.

[0035] In one specific example, the concentration of butyzamide in the second medium composition can be 0.01 to 1 uM, 0.01 to 0.8 uM, 0.01 to 0.6 uM, 0.01 to 0.4 uM, 0.01 to 0.2 uM, 0.01 to 0.01 uM, 0.03 to 1 uM, 0.05 to 1 uM, 0.07 to 1 uM, 0.09 to 1 uM, 0.03 to 0.8 uM, 0.05 to 0.6 uM, 0.07 to 0.4 uM, or 0.09 to 0.2 uM.

[0036] In one specific example, the concentration of IL6 in the second medium composition can be 2 to 200 ng / ml, 2 to 150 ng / ml, 2 to 100 ng / ml, 2 to 50 ng / ml, 5 to 200 ng / ml, 10 to 200 ng / ml, 15 to 200 mg / ml, 5 to 150 ng / ml, 10 to 100 ng / ml, or 15 to 50 ng / ml.

[0037] In one specific example, the concentration of UM729 in the second medium composition can be 5 to 500 nM, 5 to 400 nM, 5 to 300 nM, 5 to 200 nM, 5 to 100 nM, 10 to 500 nM, 30 to 500 nM, 50 to 500 nM, 60 to 500 nM, 10 to 400 nM, 30 to 300 nM, 50 to 200 nM, or 60 to 100 nM.

[0038] In one specific example, the concentration of the M-CSF in the second medium composition can be 1 to 100 uM, 1 to 80 uM, 1 to 60 uM, 1 to 40 uM, 3 to 100 uM, 5 to 100 uM, 7 to 100 uM, 9 to 100 uM, 3 to 80 uM, 5 to 60 uM, 7 to 40 uM, or 9 to 20 uM.

[0039] In one specific example, the concentration of the SCF in the three medium compositions can be 2 to 200 ng / ml, 2 to 150 ng / ml, 2 to 100 ng / ml, 2 to 50 ng / ml, 5 to 200 ng / ml, 10 to 200 ng / ml, 15 to 200 mg / ml, 5 to 150 ng / ml, 10 to 100 ng / ml, or 15 to 50 ng / ml.

[0040] In one specific example, the concentration of TPO in the third medium composition may be 5 to 500 ng / ml, 5 to 400 ng / ml, 5 to 300 ng / ml, 5 to 200 ng / ml, 5 to 100 ng / ml, 10 to 500 ng / ml, 20 to 500 ng / ml, 30 to 500 ng / ml, 40 to 500 ng / ml, 10 to 400 ng / ml, 20 to 300 ng / ml, 30 to 200 ng / ml, or 40 to 100 ng / ml.

[0041] In one specific example, the concentration of IL6 in the third medium composition can be 2 to 200 ng / ml, 2 to 150 ng / ml, 2 to 100 ng / ml, 2 to 50 ng / ml, 5 to 200 ng / ml, 10 to 200 ng / ml, 15 to 200 ng / ml, 5 to 150 ng / ml, 10 to 100 ng / ml, or 15 to 50 ng / ml.

[0042] In one specific example, the concentration of fasudil in the third medium composition can be 1 to 100 uM, 1 to 80 uM, 1 to 60 uM, 1 to 40 uM, 1 to 20 uM, 3 to 100 uM, 5 to 100 uM, 7 to 100 uM, 9 to 100 uM, 3 to 80 uM, 5 to 60 uM, 7 to 40 uM, or 9 to 20 uM.

[0043] In one specific example, the concentration of butyzamide in the third medium composition can be 0.01 to 1 uM, 0.01 to 0.8 uM, 0.01 to 0.6 uM, 0.01 to 0.4 uM, 0.01 to 0.2 uM, 0.01 to 0.1 uM, 0.02 to 1 uM, 0.03 to 1 uM, 0.04 to 1 uM, 0.05 to 1 uM, 0.02 to 0.8 uM, 0.03 to 0.6 uM, 0.04 to 0.4 uM, or 0.05 to 0.2 uM.

[0044] In one specific example, the concentration of the bFGF in the third medium composition can be 1 to 100 ng / ml, 1 to 90 ng / ml, 1 to 80 ng / ml, 1 to 70 ng / ml, 1 to 60 ng / ml, 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 3 to 100 ng / ml, 5 to 100 ng / ml, 7 to 100 ng / ml, 9 to 100 ng / ml, 3 to 90 ng / ml, 5 to 80 ng / ml, 7 to 70 ng / ml, 9 to 60 ng / ml, 15 to 50 ng / ml, 17 to 30 ng / ml, or 1 to 50 ng / ml.

[0045] As used herein, "positive" or "+" can refer to a cell marker (i.e., a greater amount or concentration of the marker) compared to other reference cells. A cell can be positive for a marker if the marker is present inside or on the cell and can be used to distinguish the cell from one or more other cell types. It can also mean that the cell has enough of the marker to produce a signal, e.g., a signal from a cell measurement device, that is greater than background. For example, if cells can be detectably labeled with an antibody specific for CD56 and the signal from this antibody is detectably greater than a control (e.g., background), the cells can be described as "positive for CD56" or "CD56+." The term "negative" or "-" can mean that the marker cannot be detected using an antibody specific for a particular cell surface marker compared to background. For example, if cells cannot be detectably labeled with an antibody specific for CD3, the cells can be described as "negative for CD3" or "CD3-."

[0046] In one specific example, the platelet progenitor cells differentiated using the medium composition of the present invention for inducing differentiation of stem cells into platelet progenitor cells may have 50%, 60%, 70%, 80%, or 90% or more of the cells in the total cell population positive for CD41a and CD42b.

[0047] In one embodiment, the stem cells can be pluripotent stem cells.

[0048] In one embodiment, the stem cells can be human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0049] As used herein, the term "stem cell" refers to a cell that has the ability to self-renew and differentiate into two or more cells, and can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.

[0050] In the present invention, "human pluripotent stem cells (hPSCs)" refers to stem cells with pluripotent or totipotent self-renewal capabilities, capable of differentiating into cells of all tissues of an individual, and includes embryonic stem cells and induced pluripotent stem cells. Embryonic stem cells are derived from the inner cell mass extracted from a blastocyst embryo, which is formed just before a fertilized egg implants in the mother's uterus, and cultured in vitro. These cells possess the pluripotent or totipotent self-renewal capabilities, capable of differentiating into cells of all tissues of an individual. In a broader sense, they also include embryoid bodies derived from embryonic stem cells. "Induced pluripotent stem cells" refer to cells induced to have pluripotent differentiation capabilities through an artificial reverse differentiation process from differentiated cells, and are also known as reverse differentiation stem cells. Artificial reverse differentiation processes can be carried out using viral or non-viral vectors such as retroviruses and lentiviruses, non-viral vectors such as proteins and cell extracts, or reverse differentiation processes using stem cell extracts, compounds, etc. Induced pluripotent stem cells have almost the same properties as embryonic stem cells, specifically, they exhibit similar cell morphology, have similar gene and protein expression patterns, are pluripotent in vitro and in vivo, form teratomas, and when inserted into mouse blastocysts, form chimera mice and are capable of germline transmission of genes.

[0051] Another aspect provides a method for inducing differentiation of stem cells into platelet progenitor cells, comprising the steps of (i) inducing differentiation of stem cells into hematopoietic progenitor cells, (ii) inducing differentiation of hematopoietic progenitor cells into megakaryocyte progenitor cells, and (iii) inducing maturation of megakaryocyte progenitor cells into platelet progenitor cells. The same content as above also applies to the method.

[0052] The step (i) will now be described in detail.

[0053] The step (i) can include a step of inducing differentiation of stem cells into hematopoietic stem cells or hematopoietic progenitor cells in the presence of a first medium composition.

[0054] In one embodiment, the step (i) can include inducing mesoderm in stem cells, inducing vascular endoderm, including endothelial to hematopoietic conversion, and / or inducing differentiation into hematopoietic stem cells or hematopoietic progenitor cells.

[0055] In one specific example, the step (i) may be carried out for 1 to 20 days, 2 to 19 days, 3 to 18 days, 4 to 17 days, or 5 to 16 days.

[0056] During the induction of differentiation in step (i), the first medium composition may be replaced with a first medium composition having a different composition.

[0057] In one embodiment, the step of inducing mesoderm may be performed in the presence of a medium containing CHIR.

[0058] In one embodiment, the step of inducing vascular endoderm may be carried out in the presence of a medium containing BMP4, VEGF and / or bFGF.

[0059] In one embodiment, the step involving the endothelial to hematopoietic conversion may be performed in the presence of a medium containing VEGF, bFGF, SB-431542 and / or retinoic acid.

[0060] In one embodiment, the step of inducing differentiation into hematopoietic stem cells or hematopoietic progenitor cells may be carried out in the presence of PVA, bFGF, SCF, IL3 and / or VPA.

[0061] The step (ii) will now be described in detail.

[0062] The step (ii) can include inducing differentiation of hematopoietic progenitor cells or hematopoietic stem cells into megakaryocyte progenitor cells in the presence of a second medium composition.

[0063] In one specific example, the step (ii) may be carried out for 1 to 20 days, 5 to 18 days, or 7 to 16 days.

[0064] During the induction of differentiation in step (ii), the first medium composition may be replaced with a second medium composition having a different composition.

[0065] In one specific example, the step of inducing differentiation of the hematopoietic progenitor cells or hematopoietic stem cells into megakaryocyte progenitor cells may be performed in the presence of a medium containing SCF, TPO, PVA, bFGF, butyzamide, IL6, and / or UM729.

[0066] The step (iii) will now be described in detail.

[0067] The step (iii) can include inducing maturation of megakaryocyte progenitor cells into platelet progenitor cells in the presence of a third medium composition.

[0068] In one specific example, the step (iii) may be carried out for 5 to 14 days or 7 to 12 days.

[0069] During the induction of differentiation in step (iii), the third medium composition may be replaced with a third medium composition having a different composition.

[0070] In one specific example, the step of inducing the maturation of megakaryocyte progenitor cells into platelet progenitor cells may be performed in the presence of a medium containing SCF, TPO, IL6, fasudil, bFGF, and / or butyzamide.

[0071] In one embodiment, the method includes the steps of (i) inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells in the presence of a first medium composition containing one or more selected from the group consisting of CHIR99021, Bone Morphogenetic Protein 4 (BMP4), Vascular Endothelial Growth Factor (VEGF), Basic Fibroblast Growth Factor (bFGF), Retinoic acid, SB-431542, Polyvinyl Alcohol (PVA), Stem Cell Factor (SCF), Interleukin 3 (IL3), Valproic acid (VPA), and combinations thereof; and (ii) inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells in the presence of a first medium composition containing one or more selected from the group consisting of Stem Cell Factor (SCF), Thrombopoietin (TPO), Polyvinyl Alcohol (PVA), Basic Fibroblast Growth Factor (bFGF), Butyzamide, Interleukin 6 (IL6), UM729, Macrophage Colony-Stimulating Factor (M-CSF), and combinations thereof. and (iii) a step of inducing the differentiation of megakaryocyte precursor cells into platelet precursor cells in the presence of a third medium composition containing one or more selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), interleukin 6 (IL6), fasudil, butyzamide, basic fibroblast growth factor (bFGF), and combinations thereof.

[0072] In one embodiment, the present invention provides platelets derived from platelet progenitor cells differentiated by the method for inducing differentiation of stem cells into platelet progenitor cells.

[0073] The platelets derived from the platelet precursor cells can include a process of culturing the platelet precursor cells obtained by the method for inducing differentiation of stem cells into platelet precursor cells, and recovering platelets from the culture.

[0074] The platelet recovery can be obtained by inducing the recovery in the presence of a medium containing bFGF, butyzamide, fasudil and / or KP-457.

[0075] In one embodiment, a platelet product or blood product comprising said platelets is provided.

[0076] The production of a platelet preparation according to the present invention can include the steps of culturing megakaryocytes by the method according to the present invention to produce platelets, recovering a platelet-rich fraction from the culture, and removing blood cell components other than platelets from the platelet fraction. The step of removing blood cell components can be performed by removing blood cell components other than platelets, including megakaryocytes, using a leukocyte removal filter or the like. More specific methods for producing platelet preparations are described, for example, in International Publication No. 2011 / 034073.

[0077] The production of a blood product according to the present invention can include the steps of producing a platelet product by the method according to the present invention and mixing the platelet product with other components, such as red blood cells.

[0078] Other components that contribute to stabilizing the cells can be added to the platelet and blood products.

[0079] Another embodiment is a kit for inducing differentiation of stem cells into platelet precursor cells, comprising: (i) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of CHIR99021, Bone Morphogenetic Protein 4 (BMP4), Vascular Endothelial Growth Factor (VEGF), Basic Fibroblast Growth Factor (bFGF), Retinoic acid, SB-431542, Polyvinyl Alcohol (PVA), Stem Cell Factor (SCF), Interleukin 3 (IL3), Valproic acid (VPA), and combinations thereof; and (ii) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of SCF (Stem Cell Factor), Thrombopoietin (TPO), Polyvinyl Alcohol (PVA), Basic Fibroblast Growth Factor (bFGF), Butyzamide, Interleukin 6 (IL6), and combinations thereof. (iii) a second medium composition for inducing differentiation of hematopoietic progenitor cells or hematopoietic stem cells into megakaryocyte progenitor cells, the second medium composition containing one or more selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), interleukin 6 (IL6), fasudil, butyzamide, basic fibroblast growth factor (bFGF), and combinations thereof; and (iv) a third medium composition for maturing megakaryocyte progenitor cells into platelet progenitor cells, the third medium composition containing one or more selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), interleukin 6 (IL6), fasudil, butyzamide, basic fibroblast growth factor (bFGF), and combinations thereof. The same parts as those described above also apply to the kit. [Effects of the Invention]

[0080] According to one embodiment of the medium composition for inducing differentiation of stem cells into platelet precursor cells, the efficiency of differentiation into platelet precursor cells can be increased, and differentiation into platelet precursor cells can be achieved without genetic manipulation. [Brief explanation of the drawings]

[0081] [Figure 1] FIG. 1 is a schematic diagram showing the process of differentiating stem cells into platelet precursor cells. [Figure 2] FIG. 1 is a schematic diagram showing the process of differentiating stem cells into platelet precursor cells. [Figure 3] This was the result of inducing differentiation of stem cells into platelet precursor cells and then confirming CD markers specific to platelet precursor cells. [Figure 4] This was the result of inducing differentiation of stem cells into platelet precursor cells and then confirming CD markers specific to platelet precursor cells. [Figure 5] 1 shows the results of comparing the differentiation efficiency into platelet precursor cells by checking CD markers between platelet precursor cells differentiated by a conventional differentiation induction method and platelet precursor cells of Example 1 according to the present invention. [Figure 6] 1 shows the results of confirming the morphology of platelet precursor cells differentiated according to the present invention. [Figure 7] 1 shows the results of confirming the morphology of platelet precursor cells differentiated according to the present invention. [Figure 8] This shows the results of confirming whether or not platelets are released from platelet precursor cells differentiated according to the present invention. [Figure 9] This shows the results of confirming whether or not platelets are released from platelet precursor cells differentiated according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0082] The present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0083] The terms or words used in the specification and claims of the present invention should not be construed as being limited to their ordinary or dictionary meanings, but should be construed as meanings and concepts that are consistent with the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concepts of terms in order to best describe the invention.

[0084] Throughout the present specification, when a part "comprises" a certain element, this does not mean that it may further include other elements, but does not exclude other elements, unless otherwise specified.

[0085] Throughout the present specification, "A and / or B" means A or B, or A and B.

[0086] Example 1

[0087] A schematic diagram of the process of differentiating stem cells into platelet precursor cells (megakaryocytes) according to Example 1 is shown in FIG.

[0088] 1.1. Differentiation into hematopoietic progenitor cells

[0089] Human pluripotent stem cells (hPSCs) were maintained in stemMACS-iPS brew Media for 2 days. The basal differentiation medium was StemMACS-SFM (+ StemMACS sup) + 200µg / ml human transferrin + 2mM L-glutamine + 0.5mM L-ascobic acid + 0.45mM MTG (1-thioglycerol) + 1% penicillin / streptomy.

[0090] The cells were treated with CHIR99021 alone at 5 μM for two days. They were then treated with differentiation medium supplemented with BMP4 50 ng / ml, VEGF 50 ng / ml, and bFGF 100 ng / ml for two days. The following day, they were cultured in medium supplemented with VEGF 50 ng / ml, bFGF 50 ng / ml, SB-431542 10 μM, and retinoic acid 1 μM for one day.

[0091] 1.2. Differentiation into megakaryocyte progenitor cells

[0092] After differentiation into hematopoietic progenitor cells, the cells were cultured in a culture medium supplemented with 0.1% PVA, 25 ng / ml SCF, 50 ng / ml TPO, and 10 ng / ml bFGF from day 5 to day 12, with the medium being changed every day.

[0093] 1.3. Maturation into platelet precursor cells (megakaryocytes)

[0094] After differentiation into megakaryocyte progenitor cells, the cells were cultured from day 12 to day 21 with the addition of SCF 25 ng / ml, TPO 50 ng / ml, IL6 25 ng / ml, and Fasudil 10 uM.

[0095] Example 2.

[0096] A schematic diagram of the process of differentiating stem cells into platelet precursor cells (megakaryocytes) according to Example 2 is shown in FIG.

[0097] 2.1. Differentiation into hematopoietic stem cells

[0098] Human pluripotent stem cells (hPSCs) were maintained for 2 days in stemMACS-iPS brew Media. The basal differentiation medium was StemMACS-SFM (+ StemMACS sup) + 200µg / ml human transferrin + 2mM L-glutamine + 0.5mM L-ascobic acid + 0.45mM MTG (1-thioglycerol) + 1% penicillin / streptomycin.

[0099] CHIR99021, known to induce intermediate endoderm, was treated alone at concentrations of 5µM-8µM for two days. To induce vascular endoderm, the cells were then treated for two days in differentiation medium supplemented with 50ng / ml BMP4, 50ng / ml VEGF, and 100ng / ml bFGF. The following day, the cells were cultured in medium supplemented with 50ng / ml VEGF, 50ng / ml bFGF, 10µM SB-431542, and 1µM retinoic acid for one day to induce endothelial-to-hematopoietic transition (EHT).

[0100] After differentiation, the cells were cultured in a medium supplemented with 1% PVA, 10 ng / ml bFGF, and 25 ng / ml SCF for 4 days (days 5 to 9), and then in a medium supplemented with 1% PVA, 10 ng / ml bFGF, and 10 ng / ml SCF for 2 days (days 9 to 11). From days 11 to 16, the cells were further cultured in a medium supplemented with 1% PVA, 10 ng / ml bFGF, 10 ng / ml SCF, 20 ng / ml IL3, and 1 mM VPA to differentiate into hematopoietic stem cells.

[0101] 2.2. Differentiation into megakaryocyte progenitor cells

[0102] Hematopoietic stem cells differentiated according to Example 2.1 above were differentiated into megakaryocyte progenitor cells using a basal differentiation medium containing IMDM, 5% FBS, 1% ITS-X, 4 mM L-Glutamine, 0.5 mM L-Ascrobic Acid, 0.45 mM MTG, 1% PVA, 50 μg / ml gentamycin, and 25 U heparin.

[0103] After differentiation into hematopoietic stem cells, the cells were cultured for 7 days in a culture medium supplemented with 20 ng / ml bFGF, 0.1 μM butyzamide, 25 ng / ml IL6, and 70 nM UM729, and then for 9 days from day 7 to day 16 after differentiation, the cells were cultured in a culture medium supplemented with 20 ng / ml bFGF, 0.05 μM butyzamide, and 10-30 ng / ml M-CSF.

[0104] 2.3. Maturation into platelet precursor cells (megakaryocytes)

[0105] After differentiation into megakaryocyte progenitor cells, from day 16 to day 23, the cells were cultured in a culture medium supplemented with 20 ng / ml bFGF, 0.05 uM butyzamide, and 10 uM Fasudil to mature megakaryocytes.

[0106] Comparative Example 1

[0107] 1.1. Differentiation into hematopoietic progenitor cells

[0108] Human pluripotent stem cells (hPSCs) were cultured for 6 days in STEMSpan-ACF media + 50ng / ml BMP4 + 50ng / ml VEGF + 50ng / ml bFGF.

[0109] 1.2. Differentiation into megakaryocyte progenitor cells

[0110] From day 6 to day 12 after differentiation into hematopoietic progenitor cells, differentiation was induced using STEMdiff APEL media + 25 ng / ml TPO + 25 ng / ml SCF + 25 ng / ml FLT3L + 10 ng / ml IL3 + 10 ng / ml IL6 + 5 U / ml heparin.

[0111] 1.3. Platelet precursor cell (megakaryocyte) maturation and platelet production

[0112] From day 12 to day 20 after differentiation into megakaryocytic progenitor cells, the cells were cultured with STEMSpan-ACF 25ng / ml TPO + 25ng / ml SCF + 10ng / ml IL6 + 10ng / ml IL9 + 5U / ml heparin.

[0113] Comparative Example 2

[0114] 2.1. Differentiation into hematopoietic progenitor cells

[0115] Human pluripotent stem cells (hPSCs) were maintained for 2 days in stemMACS-iPS brew Media. The basal differentiation medium was StemMACS-SFM (+ StemMACS sup) + 200µg / ml human transferrin + 2mM L-glutamine + 0.5mM L-ascorbic acid + 0.45mM MTG (1-thioglycerol) + 1% penicillin / streptomycin.

[0116] The cells were treated with CHIR99021 alone at 5 μM for two days. They were then treated with differentiation medium supplemented with BMP4 50 ng / ml, VEGF 50 ng / ml, and bFGF 100 ng / ml for two days. The following day, they were cultured in medium supplemented with VEGF 50 ng / ml, bFGF 50 ng / ml, SB-431542 10 μM, and retinoic acid 1 μM for one day.

[0117] 2.2. Differentiation into megakaryocyte progenitor cells

[0118] After differentiation into hematopoietic progenitor cells, the cells were cultured in a culture medium supplemented with 0.1% PVA, 25 ng / ml SCF, 50 ng / ml TPO, and 10 ng / ml bFGF from day 5 to day 12, with the medium being changed every day.

[0119] 2.3. Maturation into platelet precursor cells (megakaryocytes)

[0120] From day 12 to day 21 after differentiation into megakaryocyte progenitor cells, the cells were cultured with 25 ng / ml SCF, 50 ng / ml TPO, 25 ng / ml IL6, and 1 uM SR1.

[0121] Experimental Example 1: Identification of specific markers for platelet precursor cells

[0122] To confirm that the cells obtained in Examples 1 and 2 were differentiated into platelet progenitor cells, CD markers specific to platelet progenitor cells were examined using FACS (Fluorescence-activated cell sorting, BD FACSCalibur™). Specifically, cells were harvested at each differentiation stage and analyzed for CD markers. The results are shown in Figures 3 and 4.

[0123] As shown in Figure 3, the megakaryocyte markers CD41a and CD42b, which were barely expressed on day 12 of differentiation, increased to 26.7% on day 14, 47.6% on day 19, and 80.5% on day 21, confirming that differentiation was occurring with increasing efficiency.

[0124] As shown in Figure 4, the megakaryocyte markers CD41a and CD42b were 19.4% on day 7 of differentiation, 25.5% on day 16, 79.3% on day 23, and 90.2% on day 30, confirming that differentiation proceeded with increasing efficiency.

[0125] The above results demonstrate that the methods of Examples 1 and 2 enable efficient differentiation of human pluripotent stem cells into platelet precursor cells.

[0126] Furthermore, to examine the differentiation efficiency, the platelet precursor cells obtained in the comparative example were examined for CD markers in the same manner and compared. The results are shown in Figure 5.

[0127] As shown in Figure 5, when differentiation was performed using the method of Comparative Example 1, the number of CD41a / CD42b double positive cells was 10.7%, and when differentiation was performed using the method of Comparative Example 2 with the addition of SR1, the number was 32.9%. When differentiation was performed using the method of Example 1, it was found that the addition of fasudil showed a high differentiation efficiency (80.5%).

[0128] Experimental Example 2: Morphological confirmation of differentiated platelet precursor cells

[0129] To examine the morphological characteristics of the cells obtained in Examples 1 and 2, the differentiated platelet precursor cells were observed under a microscope and stained with Giemsa staining. The results are shown in Figures 6 and 7.

[0130] As shown in Figures 6 and 7, the differentiated platelet precursor cells were found to exhibit a multinuclear structure.

[0131] Experimental Example 3: Confirmation of platelet release

[0132] To examine whether the cells obtained in Example 1 release platelets, the cells were transferred to another gelatin-coated plate on day 23 after differentiation and cultured in the medium of Example 1.3 to observe the platelet formation process. The results are shown in Figure 8.

[0133] Furthermore, to examine whether the cells obtained in Example 2 release platelets, the cells were cultured for 7 days from day 23 after differentiation in a culture medium supplemented with 20 ng / ml bFGF, 0.05 μM butyzamide, 10 μM Fasudil, and 15 μM KP-457, and the platelet formation process was observed. The results are shown in Figure 9.

[0134] As shown in FIGS. 8 and 9, on the 28th day of differentiation (7 days after transfer to another plate), it was confirmed that the platelet precursor cells adhered to the culture dish and released platelets.

[0135] From the above results, it was found that platelets could be obtained by culturing the cells obtained in Examples 1 and 2.

Claims

1. As a medium composition for inducing differentiation of stem cells into platelet progenitor cells, (i) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of CHIR99021, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, SB-431542, PVA (Poly Vinyl Alcohol), SCF (Stem Cell Factor), IL3 (Interleukin 3), VPA (Valproic acid), and combinations thereof; (ii) a second medium composition for inducing differentiation of hematopoietic progenitor cells or hematopoietic stem cells into megakaryocyte progenitor cells, the second medium composition comprising one or more selected from the group consisting of SCF (Stem Cell Factor), TPO (Thrombopoietin), PVA (Poly Vinyl Alcohol), bFGF (Basic Fibroblast Growth Factor), butyzamide, IL6 (Interleukin 6), UM729, M-CSF (Macrophage Colony-Stimulating Factor), and combinations thereof; (iii) a third medium composition for maturation of megakaryocyte progenitor cells into platelet progenitor cells, comprising one or more selected from the group consisting of SCF (Stem Cell Factor), TPO (Thrombopoietin), IL6 (Interleukin 6), fasudil, butyzamide, bFGF (Basic Fibroblast Growth Factor), and combinations thereof; A medium composition comprising:

2. The medium composition according to claim 1, wherein the first medium composition has a concentration of CHIR99021 of 0.5 to 50 uM, a concentration of BMP4 of 50 to 500 ng / ml, a concentration of VEGF of 5 to 500 ng / ml, a concentration of bFGF of 1 to 1000 ng / ml, a concentration of retinoic acid of 0.1 to 10 uM, and a concentration of SB-431542 of 1 to 100 uM.

3. The medium composition according to claim 1, wherein the first medium composition has a concentration of CHIR99021 of 0.5 to 50 uM, a concentration of BMP4 of 50 to 500 ng / ml, a concentration of VEGF of 5 to 500 ng / ml, a concentration of bFGF of 1 to 1000 ng / ml, a concentration of retinoic acid of 0.1 to 10 uM, a concentration of SB-431542 of 1 to 100 uM, a concentration of PVA of 0.01 to 10% (w / v), a concentration of SCF of 2 to 200 ng / ml, a concentration of IL3 of 2 to 200 ng / ml, and a concentration of VPA of 0.1 to 50 mM.

4. 2. The medium composition according to claim 1, wherein the second medium composition has an SCF concentration of 2 to 200 ng / ml, a TPO concentration of 5 to 500 ng / ml, a PVA concentration of 0.01 to 1% (w / v), and a bFGF concentration of 1 to 100 ng / ml.

5. The medium composition according to claim 1, wherein the second medium composition contains bFGF at a concentration of 1 to 100 ng / ml, butyzamide at a concentration of 0.01 to 1 uM, IL6 at a concentration of 2 to 200 ng / ml, UM729 at a concentration of 5 to 500 nM, and M-CSF at a concentration of 1 to 100 uM.

6. 2. The medium composition according to claim 1, wherein the third medium composition contains SCF at a concentration of 2 to 200 ng / ml, TPO at a concentration of 5 to 500 ng / ml, IL6 at a concentration of 2 to 200 ng / ml, and fasudil at a concentration of 1 to 100 uM.

7. 2. The medium composition according to claim 1, wherein the concentration of bFGF in the third medium composition is 1 to 100 ng / ml, the concentration of butyzamide is 0.01 to 1 uM, and the concentration of fasudil is 1 to 100 uM.

8. The medium composition according to claim 1, wherein 50% or more of the differentiated platelet precursor cells in the entire cell population are positive for CD41a and CD42b.

9. The medium composition according to claim 1, wherein the stem cells are human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

10. (i) inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells in the presence of a first medium composition containing one or more selected from the group consisting of CHIR99021, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, SB-431542, PVA (Poly Vinyl Alcohol), SCF (Stem Cell Factor), IL3 (Interleukin 3), VPA (Valproic acid), and combinations thereof; (ii) inducing differentiation of hematopoietic progenitor cells or hematopoietic stem cells into megakaryocytic progenitor cells in the presence of a second medium composition containing one or more selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), polyvinyl alcohol (PVA), basic fibroblast growth factor (bFGF), butyzamide, interleukin 6 (IL6), UM729, macrophage colony-stimulating factor (M-CSF), and combinations thereof; (iii) inducing maturation of megakaryocyte progenitor cells into platelet progenitor cells in the presence of a third medium composition containing one or more selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), interleukin 6 (IL6), fasudil, butyzamide, basic fibroblast growth factor (bFGF), and combinations thereof; A method for inducing differentiation of stem cells into platelet progenitor cells, comprising:

11. The method according to claim 10, wherein the step (i) comprises culturing stem cells in a medium composition containing BMP4, VEGF, and bFGF for 1 to 20 days.

12. The method according to claim 10, wherein the step (ii) comprises culturing the hematopoietic progenitor cells or hematopoietic stem cells in a medium composition containing bFGF or butyzamide for 1 to 20 days.

13. The method of claim 10, wherein the step (iii) comprises culturing the megakaryocyte progenitor cells in a medium composition containing fasudil for 5 to 14 days.

14. Platelets derived from platelet precursor cells differentiated by the method of claim 10.

15. A platelet preparation or blood preparation comprising the platelets of claim 14.

16. As a kit for inducing differentiation of stem cells into platelet progenitor cells, (i) a first medium composition for inducing differentiation of stem cells into hematopoietic progenitor cells or hematopoietic stem cells, the first medium composition comprising one or more selected from the group consisting of CHIR99021, BMP4 (Bone Morphogenetic Protein 4), VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), retinoic acid, SB-431542, PVA (Poly Vinyl Alcohol), SCF (Stem Cell Factor), IL3 (Interleukin 3), VPA (Valproic acid), and combinations thereof; (ii) a second medium composition for inducing differentiation of hematopoietic progenitor cells or hematopoietic stem cells into megakaryocyte progenitor cells, the second medium composition comprising one or more selected from the group consisting of SCF (Stem Cell Factor), TPO (Thrombopoietin), PVA (Poly Vinyl Alcohol), bFGF (Basic Fibroblast Growth Factor), butyzamide, IL6 (Interleukin 6), UM729, M-CSF (Macrophage Colony-Stimulating Factor), and combinations thereof; (iii) a third medium composition for maturation of megakaryocyte progenitor cells into platelet progenitor cells, comprising one or more selected from the group consisting of SCF (Stem Cell Factor), TPO (Thrombopoietin), IL6 (Interleukin 6), fasudil, butyzamide, bFGF (Basic Fibroblast Growth Factor), and combinations thereof; Kit including:

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