Method for producing hematopoietic stem cells derived from differentiated totipotent stem cells, and method for creating a humanized mouse model using the produced hematopoietic stem cells.

Abstract

The present invention relates to a method for producing hematopoietic stem cells derived from totipotent stem cells, and a method for producing a humanized mouse model using the produced hematopoietic stem cells. According to one aspect, the method for producing hematopoietic stem cells can highly efficiently differentiate hematopoietic stem cells from totipotent stem cells without gene insertion, and optimal differentiation conditions have been confirmed by combining low molecular weight compounds and protein growth factors.

Description

Technical Field

[0001] The present invention relates to a method for producing hematopoietic stem cells derived from totipotent stem cells and a method for producing a humanized mouse model using the produced hematopoietic stem cells.

Background Art

[0002] Since research on human diseases has limitations, disease models using animals that are known to be genetically very similar to humans have been usefully utilized. Specifically, the target disease is induced in the model animal, and various therapeutic agents are applied to explore treatment methods. However, when a therapeutic agent that has a therapeutic effect in an animal disease model is applied to humans, it is not possible to know whether it has the same effect. Therefore, the therapeutic agent confirmed in the animal disease model cannot be directly applied to humans and goes through various stages until clinical application.

[0003] To more effectively utilize animal disease models, recently, efforts have been actively advanced to construct humanized animal models having an immune system similar to that of humans. To construct a humanized animal model, particularly a humanized mouse, a method of transplanting hematopoietic stem cells into a mouse lacking immune function has been used. For example, as a result of transplanting human CD34+ cells into SCID (severe combined immune deficiency) mice, it was confirmed that human-derived hematopoietic stem cells were slightly expressed and tissue-reformed in all tissues of the mice.

[0004] Hematopoietic stem cells are derived from umbilical cord blood and mainly exist in the bone marrow. Through proliferation and differentiation, they can produce blood cells such as red blood cells, white blood cells, and platelets, and have characteristics of stem cells such as self-renewal ability, multi-cell division ability, and multi-differentiation potential. In a stable state, the hematopoietic stem cells are about 4X10 per day 11Hematopoietic stem cells can generate individual blood cells. Such hematopoietic stem cells are not only used to create humanized animal models, but their transplantation therapy is clinically active in the treatment of hematological cancers such as acute leukemia, chronic leukemia, aplastic anemia, myelodysplastic syndromes, and multiple myeloma; solid tumors such as breast cancer, kidney cancer, and ovarian cancer; and autoimmune diseases such as refractory systemic lupus erythematosus and refractory rheumatoid arthritis.

[0005] However, because such hematopoietic stem cells exist at a very low rate, isolation is difficult, and the conditions for differentiation and proliferation are complex and intricate. As a result, effective technologies for promoting the differentiation and autologous proliferation of hematopoietic stem cells have yet to be developed.

[0006] As a result, the inventors have developed a method that allows for the highly efficient differentiation of pluripotent stem cells into hematopoietic stem cells using only an optimal combination of small molecule compounds and protein growth factors, without the need for gene insertion, thereby solving the aforementioned problems. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] One embodiment provides a method for producing hematopoietic stem cells, comprising the steps of: (A) primary culturing pluripotent stem cells (PSCs) in a medium containing a glycogen synthase kinase (GSK) inhibitor to obtain mesodermal cells; (B) secondary culturing the mesodermal cells to induce or differentiate them into angioblasts; (C) tertiary culturing the angioblasts to obtain endothelial-to-hematopoietic transition (EHT)-induced angioblasts; and (D) quaternary culturing the EHT-induced angioblasts to induce or differentiate them into hematopoietic stem cells.

[0008] Another embodiment provides a method for creating a humanized animal model, which includes the step of transplanting or introducing hematopoietic stem cells produced by the hematopoietic stem cell production method into an individual other than a human.

[0009] Another embodiment provides a humanized animal model produced by the method for producing the humanized animal model described above. [Means for solving the problem]

[0010] One embodiment provides a method for producing hematopoietic stem cells, comprising the steps of: (A) primary culturing pluripotent stem cells (PSCs) in a medium containing a glycogen synthase kinase (GSK) inhibitor to obtain mesodermal cells; (B) secondary culturing the mesodermal cells to induce or differentiate them into angioblasts; (C) tertiary culturing the angioblasts to obtain EHT (endothelial-to-hematopoietic transition)-induced angioblasts; and (D) quaternary culturing the EHT-induced angioblasts to induce or differentiate them into hematopoietic stem cells.

[0011] In this specification, the term "hematopoietic stem cell (HSC)" refers to the ancestral cells of undifferentiated bone marrow hematopoietic cells that produce red blood cells, white blood cells, and platelets, and is also called hematopoietic stem cell. In healthy individuals, about 1% of the bone marrow blood contains cells (CD34-positive cells) that have the ability to produce all blood cells, and these are called hematopoietic stem cells. As mother cells that produce blood, they are found throughout the body, but are produced in large quantities, especially in the bone marrow. From these cells, red blood cells, white blood cells, and platelets, which are the cells that make up blood, are differentiated and produced. They also have a self-renewal function that allows them to produce exactly the same cells, and they account for about 0.05-0.25% of the total hematopoietic stem cells in the bone marrow. Peripheral blood hematopoietic stem cells originate from the bone marrow and, as hematopoietic stem cells that circulate in the bloodstream, have the properties of self-renewal and differentiation into mature cells.

[0012] In this specification, the term "pluripotent stem cell (PSC)" refers to a stem cell capable of differentiating into almost all types of cells that constitute the endoderm, mesoderm, and ectoderm. These pluripotent stem cells can proliferate almost permanently or for extended periods in vitro while maintaining an undifferentiated state, exhibit normal chromosome types, and, under appropriate conditions, possess the ability to differentiate into all cells of the three germ layers (ectoderm, mesoderm, and endoderm). Traditionally, embryonic stem cells derived from embryonic tissue of fertilized eggs and embryonic stem cells obtained via blastocysts are representative examples of pluripotent stem cells. However, these embryonic stem cells have the potential to cause immune rejection due to differences in tissue-mediated synthetic antigens between the donor and host. Gene-on-demand pluripotent stem cells have been developed to overcome such problems. One type is replicated germ stem cells, which are obtained in the embryo after the nucleus of a somatic cell is replaced with that of an egg cell. The other is induced pluripotent stem cells (iPS), which are created by reverse differentiating somatic cells through genetic manipulation to produce differentiated totipotent stem cells that are almost identical to germ stem cells. In particular, induced pluripotent stem cells (iPS) make it possible to obtain differentiated totipotent stem cells on demand from a patient's own somatic cells without the bioethical conflicts that existed with germ stem cells and replicated germ stem cells in the past.

[0013] The aforementioned differentiated totipotent stem cells are selected from a group that includes germ stem cells or reverse-differentiated stem cells isolated from early germ, or similar cells thereof, and germ germ cells isolated from primordial germ cells during the fetal stage, and are also human differentiated totipotent stem cells.

[0014] The aforementioned method for producing hematopoietic stem cells is also a method for producing hematopoietic stem cells derived from differentiated totipotent stem cells, specifically a method for inducing / differentiating differentiated totipotent stem cells into hematopoietic stem cells, or a method for enhancing the differentiation of differentiated totipotent stem cells into hematopoietic stem cells. Furthermore, the hematopoietic stem cells produced by the aforementioned method are also CD34-positive (CD34+) cells, specifically CD34-positive and CD45-positive (CD34+CD45+) cells.

[0015] In this specification, the term "differentiation" refers to the process by which unspecified cells develop into specific cells, and in particular, includes the process by which stem cells develop into specific cells. In the present invention, totipotent stem cells are used as cells with differentiation potential, and these totipotent stem cells can differentiate into hematopoietic stem cells via mesoderm (mesoderm-derived cells) and angioblasts.

[0016] In the above method, step (A) is a step of primary culture of differentiated totipotent stem cells, and more specifically, it is a step of inducing or differentiating differentiated totipotent stem cells into mesoderm (mesoderm-derived cells).

[0017] The aforementioned primary culture step involves culturing differentiated pluripotent stem cells in a medium containing a GSK (glycogen synthase kinase) inhibitor, and this medium is also a mesoderm differentiation / induction medium.

[0018] In this specification, the term "GSK (glycogen synthase kinase) inhibitor" refers to a substance that targets GSK1 / 2, which is an upstream molecule of GSK1 / 2 involved in the GSK (glycogen synthase kinase) signaling pathway. The aforementioned GSK inhibitor also includes one or more selected from the group consisting of CHIR99021, 1-azakenpaullone, AZD2858, BIO, ARA014418, Indirubin-3'-monoxime, 5-Iodo-indirubin-3'-monoxime, kenpaullone, SB-415286, SB-216763, Maybridge SEW00923SC, (Z)-5-(2,3-Methylenedioxyphenyl)-imidazolidine-2,4-dione, TWS 119, CHIR98014, SB415286, Tideglusib, LY2090314, and pharmaceutically acceptable salts thereof, specifically CHIR99021 or a pharmaceutically acceptable salt thereof.

[0019] The term "CHIR99021 (6-[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]]-3-pyridinecarbonitrile) (6-[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile)" as used herein is a GSK (glycogen synthase kinase) inhibitor, a substance that targets GSK1 / 2, an upstream molecule of GSK1 / 2 involved in the GSK signaling process, and may be denoted as aminopyrimidine. CHIR99021 is C 22 H 18 It is represented by the chemical formula Cl2N8 and can be expressed by the structural formula shown in Chemical Formula 1 below.

[0020] [ka]

[0021] The aforementioned CHIR99021 is also included in the culture medium at concentrations of 2 to 8 μM, specifically, 2 to 8 μM, 2 to 7.5 μM, 2 to 7 μM, 2 to 6.5 μM, 2 to 6 μM, 2 to 5.5 μM, 2.5 to 8 μM, 2.5 to 7.5 μM, 2.5 to 7 μM, 2.5 to 6.5 μM, 2.5 to 6 μM, 2.5 to 5.5 μM, 3 to 8 μM, 3 to 7.5 μM, 3 to 7 μM, 3 to 6.5 μM, 3 to 6 μM, 3 to It is also included in the culture medium at concentrations of 5.5 μM, 3.5 to 8 μM, 3.5 to 7.5 μM, 3.5 to 7 μM, 3.5 to 6.5 μM, 3.5 to 6 μM, 3.5 to 5.5 μM, 4 to 8 μM, 4 to 7.5 μM, 4 to 7 μM, 4 to 6.5 μM, 4 to 6 μM, 4 to 5.5 μM, 4.5 to 8 μM, 4.5 to 7.5 μM, 4.5 to 7 μM, 4.5 to 6.5 μM, 4.5 to 6 μM, or 4.5 to 5.5 μM.

[0022] In addition, the medium in the primary culture stage does not contain BMP4 and bFGF. Specifically, it may also be a medium in which only a GSK inhibitor is added alone to a basic culture medium.

[0023] The primary culture stage may also be a stage of culturing totipotent stem cells for 24 to 72 hours. Specifically, it may be culturing for 24 to 72 hours, 24 to 66 hours, 24 to 60 hours, 24 to 54 hours, 30 to 72 hours, 30 to 66 hours, 30 to 60 hours, 30 to 54 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 36 to 54 hours, 42 to 72 hours, 42 to 66 hours, 42 to 60 hours, or 42 to 54 hours.

[0024] In the method, the step (B) is a step of secondary-culturing the cells that have been primary-cultured. Specifically, it is also a step of inducing or differentiating mesoderm (mesoderm-derived cells) differentiated from totipotent stem cells into angioblasts.

[0025] The term "hemangioblast" in this specification is a pluripotent precursor cell that can differentiate into hematopoietic stem cells or endothelial cells. In a mouse embryo, on the 7th day of the embryo, when blood islands appear in the yolk sac, hematopoiesis begins, and hematopoietic cells and vascular structures are formed from the blood islands. Hemangioblasts are precursor cells that form blood islands.

[0026] The aforementioned secondary culture step may involve culturing mesoderm (mesoderm-derived cells) differentiated from primary cultured cells or differentiated totipotent stem cells in a medium containing one or more selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor). Specifically, it may involve culturing in a medium containing BMP4, VEGF, and bFGF. The aforementioned medium is also an angioblast differentiation / induction medium.

[0027] The aforementioned BMP4 is also included in the culture medium at concentrations of 20 to 80 ng / ml, specifically 20 to 80 ng / ml, 20 to 75 ng / ml, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 80 ng / ml, 25 to 75 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 80 ng / ml, 30 to 75 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, 30 to 60 ng / ml, 3 It is also included in the culture medium at concentrations of 0 to 55 ng / ml, 35 to 80 ng / ml, 35 to 75 ng / ml, 35 to 70 ng / ml, 35 to 65 ng / ml, 35 to 60 ng / ml, 35 to 55 ng / ml, 40 to 80 ng / ml, 40 to 75 ng / ml, 40 to 70 ng / ml, 40 to 65 ng / ml, 40 to 60 ng / ml, 40 to 55 ng / ml, 45 to 80 ng / ml, 45 to 75 ng / ml, 45 to 70 ng / ml, 45 to 65 ng / ml, 45 to 60 ng / ml, or 45 to 55 ng / ml.

[0028] The bFGF mentioned above is also included in the culture medium at a concentration of 80 to 120 ng / ml, specifically at concentrations of 80 to 120 ng / ml, 80 to 115 ng / ml, 80 to 110 ng / ml, 80 to 105 ng / ml, 85 to 120 ng / ml, 85 to 115 ng / ml, 85 to 110 ng / ml, 85 to 105 ng / ml, 90 to 120 ng / ml, 90 to 115 ng / ml, 90 to 110 ng / ml, 90 to 105 ng / ml, 95 to 120 ng / ml, 95 to 115 ng / ml, 95 to 110 ng / ml, or 95 to 105 ng / ml.

[0029] The aforementioned VEGF is also included in the culture medium at concentrations of 20 to 80 ng / ml, specifically 20 to 80 ng / ml, 20 to 75 ng / ml, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 80 ng / ml, 25 to 75 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 80 ng / ml, 30 to 75 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, 30 to 60 ng / ml, 3 It is also included in the culture medium at concentrations of 0 to 55 ng / ml, 35 to 80 ng / ml, 35 to 75 ng / ml, 35 to 70 ng / ml, 35 to 65 ng / ml, 35 to 60 ng / ml, 35 to 55 ng / ml, 40 to 80 ng / ml, 40 to 75 ng / ml, 40 to 70 ng / ml, 40 to 65 ng / ml, 40 to 60 ng / ml, 40 to 55 ng / ml, 45 to 80 ng / ml, 45 to 75 ng / ml, 45 to 70 ng / ml, 45 to 65 ng / ml, 45 to 60 ng / ml, or 45 to 55 ng / ml.

[0030] The aforementioned secondary culture stage may involve culturing for 24 to 72 hours, specifically for 24 to 72 hours, 24 to 66 hours, 24 to 60 hours, 24 to 54 hours, 30 to 72 hours, 30 to 66 hours, 30 to 60 hours, 30 to 54 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 36 to 54 hours, 42 to 72 hours, 42 to 66 hours, 42 to 60 hours, or 42 to 54 hours.

[0031] In the above method, step (C) is a step in which the secondarily cultured cells are subjected to tertiary culture, and specifically, it is a step in which EHT (endothelial-to-hematopoietic transition) is induced in hemangioblasts differentiated from mesoderm (mesoderm-derived cells) to obtain EHT-induced hemangioblasts.

[0032] In this specification, the term "EHT (endothelial-to-hematopoietic transition)" refers to the process of transitioning from endothelial characteristics to hematopoietic characteristics, specifically, to inducing an increase in hematopoietic maturity from cells exhibiting endothelial traits.

[0033] The aforementioned tertiary culture step also involves culturing the secondary cultured cells or angioblasts differentiated from mesoderm in a medium containing a TGF-β inhibitor, and this medium is also an EHT induction medium.

[0034] In this specification, the term "TGF-β inhibitor" refers to a molecule that inhibits the interaction between TGF-β and the TGF-β receptor (TGF-βR), thereby inhibiting the TGF-β pathway. The TGF-β inhibitor may also include one or more molecules selected from the group consisting of SB-431542 (SB4), galunisertib, LY2109761, SB525334, SP505124, GW788388, LY364947, RepSox, SD-208, vactosertib, LY3200882, PF-06952229, and pharmaceutically acceptable salts thereof, and more specifically, SB-431542 (SB4) or a pharmaceutically acceptable salt thereof.

[0035] In this specification, the term "SB-431542 (SB4)" refers to a TGF-β inhibitor, C 22 H 16 It is represented by the chemical formula N4O3 and can be expressed by the structural formula shown in Chemical Formula 2 below.

[0036] [ka]

[0037] The aforementioned SB-431542 is also included in the culture medium at concentrations of 7 to 13 μM, specifically, 7 to 13 μM, 7 to 12.5 μM, 7 to 12 μM, 7 to 11.5 μM, 7 to 11 μM, 7 to 10.5 μM, 7.5 to 13 μM, 7.5 to 12.5 μM, 7.5 to 12 μM, 7.5 to 11.5 μM, 7.5 to 11 μM, 7.5 to 10.5 μM, 8 to 13 μM, 8 to 12.5 μM, 8 to 12 μM, 8 to 11.5 μM, 8 to 11 μM, 8 to It is also included in the culture medium at concentrations of 10.5 μM, 8.5 to 13 μM, 8.5 to 12.5 μM, 8.5 to 12 μM, 8.5 to 11.5 μM, 8.5 to 11 μM, 8.5 to 10.5 μM, 9 to 13 μM, 9 to 12.5 μM, 9 to 12 μM, 9 to 11.5 μM, 9 to 11 μM, 9 to 10.5 μM, 9.5 to 13 μM, 9.5 to 12.5 μM, 9.5 to 12 μM, 9.5 to 11.5 μM, 9.5 to 11 μM, or 9.5 to 10.5 μM.

[0038] The culture medium used in the tertiary culture stage may also contain retinoic acid (RA) or a pharmaceutically acceptable salt thereof.

[0039] The retinoic acid may also be present in the culture medium at a concentration of 0.5 to 1.5 μM, specifically at concentrations of 0.5 to 1.5 μM, 0.5 to 1.3 μM, 0.5 to 1.1 μM, 0.7 to 1.5 μM, 0.7 to 1.3 μM, 0.7 to 1.1 μM, 0.9 to 1.5 μM, 0.9 to 1.3 μM, or 0.9 to 1.1 μM.

[0040] The culture medium used in the tertiary culture stage may also include one or more substances selected from the group composed of VEGF and bFGF, specifically, it may include VEGF and bFGF.

[0041] The VEGF and bFGF are each included in the culture medium at concentrations of 20 to 80 ng / ml, specifically 20 to 80 ng / ml, 20 to 75 ng / ml, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 80 ng / ml, 25 to 75 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 80 ng / ml, 30 to 75 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, and 30 to 60 ng / ml. It is also included in the culture medium at concentrations of g / ml, 30 to 55 ng / ml, 35 to 80 ng / ml, 35 to 75 ng / ml, 35 to 70 ng / ml, 35 to 65 ng / ml, 35 to 60 ng / ml, 35 to 55 ng / ml, 40 to 80 ng / ml, 40 to 75 ng / ml, 40 to 70 ng / ml, 40 to 65 ng / ml, 40 to 60 ng / ml, 40 to 55 ng / ml, 45 to 80 ng / ml, 45 to 75 ng / ml, 45 to 70 ng / ml, 45 to 65 ng / ml, 45 to 60 ng / ml, or 45 to 55 ng / ml.

[0042] The aforementioned tertiary culture stage may involve culturing for 12 to 44 hours, specifically for 12 to 44 hours, 12 to 40 hours, 12 to 36 hours, 12 to 32 hours, 12 to 28 hours, 16 to 44 hours, 16 to 40 hours, 16 to 36 hours, 16 to 32 hours, 16 to 28 hours, 20 to 44 hours, 20 to 40 hours, 20 to 36 hours, 20 to 32 hours, or 20 to 28 hours.

[0043] In the above method, step (D) is a step in which the tertiary cultured cells are subjected to quaternary culture, and is also a step in which hemangioblasts, specifically EHT-induced hemangioblasts, are differentiated / induced into hematopoietic stem cells. The differentiated hematopoietic stem cells are also CD34+ hematopoietic stem cells.

[0044] The aforementioned fourth-stage culture step also involves culturing the tertiary-cultured cells, or EHT-induced angioblasts, in a medium containing PVA (polyvinyl alcohol) or a pharmaceutically acceptable salt thereof. This medium is also a hematopoietic stem cell differentiation / induction medium.

[0045] The aforementioned PVA is also included in the culture medium at a concentration of 0.01% to 0.5% (w / v), specifically, 0.01 to 0.5% (w / v), 0.01 to 0.4% (w / v), 0.01 to 0.3% (w / v), 0.01 to 0.2% (w / v), 0.01 to 0.15% (w / v), 0.03 to 0.5% (w / v), 0.03 to 0.4% (w / v), 0.03 to 0.3% (w / v), 0.03 to 0.2% (w / v), 0.03 to 0.15% (w / v), 0.05 to 0.5% (w / v), 0.05 to 0.4% ( It is also included in the culture medium at concentrations of w / v, 0.05 to 0.3% (w / v), 0.05 to 0.2% (w / v), 0.05 to 0.15% (w / v), 0.07 to 0.5% (w / v), 0.07 to 0.4% (w / v), 0.07 to 0.3% (w / v), 0.07 to 0.2% (w / v), 0.07 to 0.15% (w / v), 0.09 to 0.5% (w / v), 0.09 to 0.4% (w / v), 0.09 to 0.3% (w / v), 0.09 to 0.2% (w / v), or 0.09 to 0.15% (w / v).

[0046] The culture medium used in the fourth stage of culture may also contain one or more selected components from the group composed of SCF (stem cell factor) and bFGF, specifically, it may contain SCF and bFGF.

[0047] The aforementioned SCF is also included in the culture medium at concentrations of 20 to 80 ng / ml, specifically 20 to 80 ng / ml, 20 to 75 ng / ml, 20 to 70 ng / ml, 20 to 65 ng / ml, 20 to 60 ng / ml, 20 to 55 ng / ml, 25 to 80 ng / ml, 25 to 75 ng / ml, 25 to 70 ng / ml, 25 to 65 ng / ml, 25 to 60 ng / ml, 25 to 55 ng / ml, 30 to 80 ng / ml, 30 to 75 ng / ml, 30 to 70 ng / ml, 30 to 65 ng / ml, 30 to 60 ng / ml, 3 It is also included in the culture medium at concentrations of 0 to 55 ng / ml, 35 to 80 ng / ml, 35 to 75 ng / ml, 35 to 70 ng / ml, 35 to 65 ng / ml, 35 to 60 ng / ml, 35 to 55 ng / ml, 40 to 80 ng / ml, 40 to 75 ng / ml, 40 to 70 ng / ml, 40 to 65 ng / ml, 40 to 60 ng / ml, 40 to 55 ng / ml, 45 to 80 ng / ml, 45 to 75 ng / ml, 45 to 70 ng / ml, 45 to 65 ng / ml, 45 to 60 ng / ml, or 45 to 55 ng / ml.

[0048] The aforementioned bFGF is also included in the culture medium at concentrations of 7 to 13 ng / ml, specifically, 7 to 13 ng / ml, 7 to 12.5 ng / ml, 7 to 12 ng / ml, 7 to 11.5 ng / ml, 7 to 11 ng / ml, 7 to 10.5 ng / ml, 7.5 to 13 ng / ml, 7.5 to 12.5 ng / ml, 7.5 to 12 ng / ml, 7.5 to 11.5 ng / ml, 7.5 to 11 ng / ml, 7.5 to 10.5 ng / ml, 8 to 13 ng / ml, 8 to 12.5 ng / ml, 8 to 12 ng / ml, 8 to 11.5 ng / ml, 8 to 11 ng / ml, and 8 to 10.5 It is also included in the culture medium at concentrations of ng / ml, 8.5 to 13 ng / ml, 8.5 to 12.5 ng / ml, 8.5 to 12 ng / ml, 8.5 to 11.5 ng / ml, 8.5 to 11 ng / ml, 8.5 to 10.5 ng / ml, 9 to 13 ng / ml, 9 to 12.5 ng / ml, 9 to 12 ng / ml, 9 to 11.5 ng / ml, 9 to 11 ng / ml, 9 to 10.5 ng / ml, 9.5 to 13 ng / ml, 9.5 to 12.5 ng / ml, 9.5 to 12 ng / ml, 9.5 to 11.5 ng / ml, 9.5 to 11 ng / ml, or 9.5 to 10.5 ng / ml.

[0049] The culture medium used in the fourth stage of cultivation does not contain one or more selected elements from the group composed of VEGF, IL-3, and IL-6; specifically, it does not contain VEGF, IL-3, or IL-6.

[0050] The aforementioned fourth-stage culture step involves culturing for 168 to 360 hours, specifically 168 to 360 hours, 168 to 348 hours, 168 to 336 hours, 168 to 324 hours, 168 to 312 hours, 168 to 300 hours, 168 to 288 hours, 192 to 360 hours, 192 to 348 hours, 192 to 336 hours, 192 to 324 hours, 192 to 312 hours, and 192 to 300 hours. These are also cultures that last for 192 to 288 hours, 216 to 360 hours, 216 to 348 hours, 216 to 336 hours, 216 to 324 hours, 216 to 312 hours, 216 to 300 hours, 216 to 288 hours, 240 to 360 hours, 240 to 348 hours, 240 to 336 hours, 240 to 324 hours, 240 to 312 hours, 240 to 300 hours, or 240 to 288 hours.

[0051] The aforementioned method for producing hematopoietic stem cells also includes an additional step of culturing the hematopoietic stem cells differentiated from differentiated pluripotent stem cells to maintain their function.

[0052] The aforementioned step of culturing hematopoietic stem cells is intended to maintain the biological and physiological characteristics of differentiated hematopoietic stem cells, specifically by suppressing aging and differentiation of differentiated hematopoietic stem cells and maintaining their CD34+ potential.

[0053] The aforementioned culture for maintenance may utilize the culture medium from the fourth stage of culture, and can be cultured for 1 to 20 days.

[0054] The above method also includes the additional step of recovering the quaternarily cultured cells, specifically hematopoietic stem cells differentiated from differentiated totipotent stem cells.

[0055] The basic culture medium used in the hematopoietic stem cell manufacturing method described above may be a general culture medium known in the industry to be suitable for stem cell culture and differentiation. Examples include, but are not limited to, DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), Stempro 34-SFM, and Stempro 34.

[0056] The basic medium may be supplemented with additives. Generally, it may also contain neutral buffers in an isotonic solution (e.g., phosphates and / or high-concentration bicarbonates) and protein nutrients (e.g., serum (e.g., FBS), serum substitutes, albumin, or essential and non-essential amino acids (e.g., glutamine)). Furthermore, it may also contain lipids (fatty acids, cholesterol, serum HDL or LDL extracts) and other components found in most preservation mediums of this type (e.g., insulin or transferrin, nucleosides or nucleotides, pyruvates, sugar sources in any ionized form or salt (e.g., glucose), selenium, glucocorticoids (e.g., hydrocortisone), and / or reducing agents (e.g., β-mercaptoethanol). The medium may also contain antibiotics such as penicillin, streptomycin, gentamicin, or a mixture of two or more of these.

[0057] The above-mentioned method for producing hematopoietic stem cells involves differentiating pluripotent stem cells into hematopoietic stem cells, and comprises: 1) culturing pluripotent stem cells in a medium containing 4 to 6 μM CHIR99021 for approximately 2 days to differentiate / induce them into mesoderm (mesoderm-derived cells / mesoderm-derived stem cells); 2) culturing the differentiated mesoderm in a medium containing 40 to 60 ng / ml BMP4, 90 to 100 ng / ml bFGF, and 40 to 60 ng / ml VEGF for approximately 2 days to differentiate / induce them into angioblasts; 3) culturing the angioblasts in a medium containing 40 to 60 ng / ml bFGF, 40 to 60 ng / ml VEGF, 9 to 11 μM MSB-431542, and 0.8 to 1.2 μM retinoic acid for approximately 1 day to induce EHT; and 4) dividing the EHT-induced angioblasts into 0.05% to 0.15% This method also includes a step of culturing for approximately 11 days in a medium containing PVA, 40 to 60 ng / ml SCF, and 9 to 11 ng / ml bFGF to differentiate / induce hematopoietic stem cells. Furthermore, it involves using Stempro 34-SFM (+Stempro supplement) medium containing 200 μg / ml human transferrin, 2 mM L-glutamine, 0.5 mM L-ascorbic acid, 0.45 mM MTG (1-thioglycerol), and 1% penicillin / streptomycin as the basic culture medium.

[0058] Another embodiment provides a method for creating a humanized animal model, which includes the step of transplanting or introducing hematopoietic stem cells produced by the hematopoietic stem cell production method into an individual other than a human. Any content that overlaps with the above description also applies to the above method.

[0059] The aforementioned humanized animal models include primates; mammals such as mice, hamsters, guinea pigs, rats, dogs, cats, horses, and cattle, and specifically, mice.

[0060] The step of transplanting or introducing hematopoietic stem cells involves introducing hematopoietic stem cells derived from differentiated totipotent stem cells produced by the method described above into the tail vein of a mouse, specifically 1-2 x 10 5 It also involves introducing individual cells.

[0061] The individuals other than the aforementioned humans are also individuals in which the functions of T cells, B cells, NK cells, etc. are completely lacking and immune deficiency has been induced; specifically, they are NSG (NOD scid gamma) mice that have been treated with radioactivity.

[0062] Another embodiment provides a humanized animal model produced by the method for producing the humanized animal model described above. Any content that overlaps with the above description also applies to the animal model.

[0063] The aforementioned humanized animal models include primates; mammals such as mice, hamsters, guinea pigs, rats, dogs, cats, horses, and cattle; specifically, mice. [Effects of the Invention]

[0064] According to one embodiment, the method, without gene insertion, allows for the identification of optimal differentiation conditions through a combination of small molecule compounds and protein growth factors, and enables highly efficient differentiation of pluripotent stem cells into hematopoietic stem cells. [Brief explanation of the drawing]

[0065] [Figure 1] This figure schematically illustrates the novel hematopoietic stem cell differentiation protocol derived from pluripotent stem cells according to the present invention. [Figure 2] This figure specifically illustrates the hematopoietic stem cell differentiation protocol derived from pluripotent stem cells according to the present invention. [Figure 3] This figure shows the results of comparative experiments to establish the primary conditions for hematopoietic stem cell induction. [Figure 4] This figure shows the results of comparative experiments to establish the conditions for the mesoderm induction stage. [Figure 5]This figure shows the results of comparative experiments to establish the conditions for the EHT induction stage. [Figure 6] This figure shows the results of comparative experiments to establish secondary conditions for hematopoietic stem cell induction. [Figure 7] This figure illustrates the process of creating a humanized mouse model using hematopoietic stem cells produced by the method of the present invention. [Figure 8] This figure schematically illustrates the method for producing hematopoietic stem cells according to the present invention, and the method for creating a humanized mouse model using the same. [Modes for carrying out the invention]

[0066] The invention will be further described below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by them.

[0067] Example 1: Novel human pluripotent stem cell differentiation protocol The present invention relates to a method for differentiating human pluripotent stem cells to produce human hematopoietic stem cells, and a method for producing humanized mice using the hematopoietic stem cells produced by the said method. The said method consists of 1) a mesoderm induction / differentiation stage, 2) a hemangioblast induction / differentiation stage, 3) an endothelial-to-hematopoietic transition (EHT) induction stage, and 4) a hematopoietic stem cell (HSC) induction / differentiation stage, and essential small molecule compounds and / or growth factors may be applied at each stage (Figure 1).

[0068] The specific experiment for producing the aforementioned human hematopoietic stem cells was carried out using the following method. First, isolated human pluripotent stem cells (hPSCs) were maintained in stemMACS-iPS brew medium for 2 days. The basic differentiation culture medium consisted of the following composition: Stempro 34-SFM (+ stempro sup) + 200 μg / ml human transferrin + 2 mM L-glutamine + 0.5 mM L-ascorbic acid + 0.45 mM MTG (1-thioglycerol) + 1% penicillin / streptomycin. To induce mesoderm from the isolated hPSCs, CHIR99021 (5 μM), a representative GSK inhibitor, was used alone for approximately 2 days. Next, to induce hemangioblasts, cells were treated with 50 ng / ml BMP4, 50 ng / ml VEGF, and 100 ng / ml bFGF for 2 days. Then, 50 ng / ml VEGF, 50 ng / ml bFGF, SB-431542 (10 μM) as a representative example of a TGF-β inhibitor, and 1 μM retinoic acid (RA) were added, and the cells were cultured for about 1 day. Finally, the cells were cultured for about 11 days with the addition of 0.1% (w / v) PVA, 50 ng / ml SCF (stem cell factor), and 10 ng / ml bFGF (Figure 2).

[0069] In most existing hematopoietic stem cell differentiation technologies, differentiation of hPSCs is initiated by forming a community using EB (embryonic body), then the community is dismantled into single cells, and CD34+ HSCs are extracted. Gene insertion has been attempted to improve differentiation efficiency. However, the HSC differentiation method of the present invention has the excellent effect of efficiently differentiating HSCs and easily obtaining them using only an optimal combination of low-differentiation compounds and protein growth factors, without the complex processes and gene insertions mentioned above.

[0070] In the following, experiments were conducted to establish optimal conditions for developing a novel hematopoietic stem cell differentiation method according to the present invention, by comparing it with existing publicly available differentiation protocols.

[0071] Example 2: Establishment of primary conditions for hematopoietic stem cell induction To establish the primary conditions for hematopoietic stem cell induction, the following experiments were conducted.

[0072] Specifically, VEGF (vascular endothelial growth factor) is widely known to play a role in inducing mesoderm and inducing and maintaining hemangioblasts (precursors of hematopoietic stem cells), and has been primarily used during the hematopoietic stem cell induction period. Therefore, in order to confirm what effect the exclusion of VEGF during this hematopoietic stem cell induction stage has on the final hematopoietic stem cell generation efficiency, we examined the hematopoietic stem cell differentiation results with and without VEGF (10 ng / ml) under the same conditions (50 ng / ml SCF, 10 ng / ml bFGF, 20 ng / ml IL-3, and 10 ng / ml IL-6) (Figure 3A).

[0073] To confirm the differentiation results and to determine the ratio of CD34+CD45+ cells, FACS was performed. Specifically, to identify cells expressing both CD34 and CD45, anti-CD34 antibodies and anti-CD45 antibodies, each linked to the fluorescent substances PE and PerCP / Cy5.5 respectively, were diluted in a 1:25 ratio in staining buffer (PBS containing 1% FBS). The cells were then stained with the antibody cocktail at 4°C for 35 minutes. After washing off residual antibody with the staining buffer, another 200 μl of staining buffer was added, and the cells were analyzed using a fluid cell analyzer.

[0074] As a result, we confirmed that the ratio of CD34+CD45+ cells in the hematopoietic stem cell induction stage was significantly superior when VEGF was removed compared to when VEGF was included (Figure 3B). Based on these results, it can be seen that removing VEGF in the hematopoietic stem cell induction stage has a significantly superior hematopoietic stem cell differentiation effect, contrary to what is currently known.

[0075] Example 3: Establishment of conditions for mesoderm induction To establish the conditions for the mesoderm (mesoderm-derived cells / mesoderm-derived stem cells) induction stage, the following experiments were conducted.

[0076] Specifically, existing methods involved treating cells with BMP4 (bone morphogenetic protein 4), VEGF, and CHIR99021 to induce mesoderm. However, it was determined that these conditions did not correspond to the sequence of steps required to differentiate stem cells into hematopoietic stem cells. Therefore, we examined the hematopoietic stem cell differentiation efficiency when only CHIR99021, a representative GSK inhibitor, was treated during the mesoderm induction stage, excluding the previously used BMP4 (5 ng / ml) and VEGF (50 ng / ml) (Figure 4A).

[0077] As a result, it was confirmed that the ratio of CD34+CD45+ cells when CHIR99021 alone was treated during the mesoderm induction stage was significantly superior compared to when BMP4, VEGF, and CHIR99021 were treated (Figure 4B). Based on these results, it can be seen that not treating with BMP4 and VEGF during the mesoderm induction stage has a significantly superior hematopoietic stem cell differentiation effect.

[0078] Example 4: Establishment of conditions for the EHT induction step To establish the conditions for the induction stage of EHT (endothelial-to-hematopoietic transition), the following experiment was conducted.

[0079] Specifically, in order to induce CD34+HSCs, hemangioblasts must be induced after mesoderm induction. These hemangioblasts can then be differentiated into CD34+HSCs by inducing endothelial-to-hematopoietic transition (EHT), but it is important to establish appropriate culture conditions to maximize the efficiency of EHT.

[0080] To this end, in addition to VEGF and bFGF (basic fibroblast growth factor), we investigated the hematopoietic stem cell differentiation efficiency when SB-431542 (SB4), a representative example of a TGF-β inhibitor, was treated for various durations (Figure 5A). As a result, we confirmed that when SB4 was treated for 24 hours or less, the ratio of CD34+CD45+ cells was significantly superior, and that when SB4 was treated for a longer period, the hematopoietic stem cell differentiation efficiency decreased significantly (Figure 5B). Furthermore, we confirmed that when SB4 and retinoic acid (RA) were treated simultaneously, the hematopoietic stem cell differentiation efficiency increased significantly compared to when SB4 was treated alone (Figures 5C and 5D).

[0081] Based on the results described above, it was found that after angioblast induction, the EHT induction stage shows a remarkably superior hematopoietic stem cell differentiation effect when SB4 is treated for about 24 hours, and the combination of SB4 and retinoic acid shows an even superior hematopoietic stem cell differentiation effect.

[0082] Example 5: Establishment of secondary conditions for hematopoietic stem cell induction To establish secondary conditions for hematopoietic stem cell induction, the following experiments were conducted.

[0083] Specifically, existing methods primarily use IL-3 and IL-6 in the culture process to maintain the CD34+ potential of hematopoietic stem cells differentiated from human pluripotent stem cells. However, this can promote aging and differentiation of the differentiated hematopoietic stem cells, slightly reducing the efficiency of CD34+ potential maintenance. Therefore, we investigated the efficiency of hematopoietic stem cell differentiation maintenance when treating with PVA (polyvinyl alcohol) instead of the existing IL-3 and IL-6 treatments during the induction and maintenance stages of hematopoietic stem cells (Figure 6A).

[0084] As a result, we confirmed that when PVA was treated during the hematopoietic stem cell induction stage, excluding IL-3 and IL-6, not only was the hematopoietic stem cell differentiation efficiency increased, but the maintenance efficiency of CD34+ hematopoietic stem cells after differentiation was also remarkably superior (Figures 6B and 6C). Based on these results, it can be seen that, contrary to what is currently known, treating with PVA during the hematopoietic stem cell induction stage, excluding IL-3 and IL-6, has a remarkably superior effect on hematopoietic stem cell differentiation and maintenance.

[0085] Example 6: Creation of humanized mice Based on the experimental results of Examples 2 to 5 described above, a novel hematopoietic stem cell differentiation protocol was established in Example 1, and humanized mice were created using hematopoietic stem cells produced by the above method.

[0086] Specifically, hematopoietic stem cells 1-2 x 10⁻¹⁰ derived from human differentiated totipotent stem cells produced by the method of Example 1 above. 5 Individual cells were injected into the tail vein of radioactively treated immunodeficient NSG mice. Twelve weeks after injection, the presence of human mitochondrial genes was checked in the peripheral blood of the mice, and 22 weeks after injection, the presence or absence of human CD45-positive cells was checked in the peripheral blood of the mice (Figure 7A).

[0087] To detect human mitochondrial DNA in the peripheral blood of the aforementioned mice, a PCR reaction was performed. Specifically, 2x PCRBIO HS Taq Mix red was used as the polymerase, and the annealing temperature was set to 58°C for 35 cycles. Subsequently, the PCR reaction product was loaded onto a 1% agarose gel containing EtBr at 100V for 20 minutes, and the PCR band was confirmed. The primer sequences used for human mitochondria detection are as follows. Forward: 5'- CAACACTAAAGGACGAACCTGA-3'(Sequence ID 1) Reverse: 5'- TCGTAAGGGGTGGATTTTTC-3'(Sequence ID 2)

[0088] As a result, human mitochondrial genes were detected in the peripheral blood of mice 12 weeks after injection (Figure 7B), and it was confirmed that more than 25% of the peripheral blood of mice contained human CD45-positive cells 22 weeks after injection (Figure 7C).

[0089] Based on the results described above, it can be seen that hematopoietic stem cells produced by the hematopoietic stem cell differentiation protocol method of Example 1 can also be usefully used to create humanized animal models.

[0090] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that the invention can be easily modified into other specific forms without altering the technical idea or essential features. Accordingly, the embodiments described above should be understood in all respects as illustrative and not limiting.

Claims

1. (A) A step in which differentiated pluripotent stem cells (PSCs) are primary cultured in a medium containing a GSK (glycogen synthase kinase) inhibitor but not containing BMP4 (bone morphogenetic protein 4) and VEGF (vascular endothelial growth factor) to obtain mesodermal cells, (B) The step of secondary culture of the mesodermal cells and inducing or differentiating them into angioblasts, (C) The step of culturing the angioblasts in a third culture to obtain angioblasts that have undergone EHT (endothelial-to-hematopoietic transition), (D) The step of quaternary culturing the EHT-induced angioblasts in a medium that does not contain VEGF, and inducing or differentiating them into hematopoietic stem cells, A method for producing hematopoietic stem cells, including [the specified element].

2. The method according to claim 1, wherein the GSK (glycogen synthase kinase) inhibitor is CHIR99021.

3. The method according to claim 1, wherein the secondary culture is performed in a culture medium containing one or more selected from the group consisting of BMP4 (bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor).

4. The method according to claim 1, wherein the tertiary culture is cultured in a medium containing a TGF-β inhibitor.

5. The method according to claim 4, wherein the TGF-β inhibitor is SB-431542.

6. The method according to claim 4, wherein the culture medium is further comprising retinoic acid (RA) or a pharmaceutically acceptable salt thereof.

7. The method according to claim 4, wherein the culture medium further comprises one or more selected from the group consisting of VEGF and bFGF.

8. The method according to claim 1, wherein the quaternary culture is cultured in a medium containing PVA (polyvinyl alcohol) or a pharmaceutically acceptable salt thereof.

9. The method according to claim 8, wherein the culture medium further comprises one or more selected from the group consisting of SCF and bFGF.

10. A method for producing a humanized animal model, comprising the step of transplanting or introducing hematopoietic stem cells produced by the method of claim 1 into an individual other than a human.

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