Preparation and Expansion Culture of Vascular Endothelial Progenitor Cells

Abstract

To provide means for preparing high-purity vascular endothelial progenitor cells in a simple and low-cost manner, and also provide methods for efficient proliferation of vascular endothelial progenitor cells.SOLUTION: High-purity vascular endothelial progenitor cells are prepared by the steps of: differentiating pluripotent stem cells into vascular endothelial progenitor cells; and purifying the vascular endothelial progenitor cells using a difference in adhesion ability between the vascular endothelial progenitor cells constituting the cell population obtained in the previous step and other cells. On the other hand, vascular endothelial progenitor cells are cultured and expanded in the presence of a ROCK inhibitor, a GSK-3β inhibitor, and a TGF-β receptor inhibitor in addition to basic fibroblast growth factor and epidermal growth factor.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to the preparation and expansion culture of vascular endothelial progenitor cells. Specifically, it relates to a method for preparing highly pure vascular endothelial progenitor cells from pluripotent stem cells, a method for efficiently proliferating vascular endothelial progenitor cells, and the like. This application claims priority based on Japanese Patent Application No. 2019-040117 filed on March 6, 2019, and the entire contents of the patent application are incorporated herein by reference.

Background Art

[0002] Human pluripotent stem cells including induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) can proliferate infinitely and have the ability to differentiate into all parts of the human body. Because of this property, human pluripotent stem cells (especially iPS cells) are expected to be used in the construction of drug screening models mimicking human organs and clinical applications in regenerative medicine.

[0003] Vascular endothelial progenitor cells (EPCs) derived from iPS cells are considered for various clinical applications such as blood vessel regeneration, treatment of myocardial infarction by co-transplantation with myocardium, and construction of advanced three-dimensional cell tissues of organs. It is also expected to be used in the construction of pathological models of vascular diseases and in screening systems for new drug development by differentiating into brain capillary endothelial cells. For use for such purposes, a large amount of highly pure vascular endothelial progenitor cells is required.

[0004] Currently, numerous reports have been made that vascular endothelial cells (ECs) and vascular endothelial progenitor cells can be efficiently induced to differentiate from ES cells and iPS cells (see, for example, Non-Patent Documents 1 and 2). However, since the ratio of differentiation into vascular endothelial cells and vascular endothelial progenitor cells is greatly affected by the cell line used and the state before differentiation, in order to obtain high-purity cells, they are often finally purified using antibody beads / magnetic beads or a cell sorter. In addition, there are limited reports on an efficient expansion culture method for iPS cell-derived vascular endothelial progenitor cells. The research group of the present inventors reported a novel method for inducing differentiation of vascular endothelial progenitor cells applying the iPS-sac method in a previous patent application (Patent Document 1). In addition, Patent Document 2 discloses a method for preparing vascular endothelial progenitor cells from embryonic stem cells.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] Although purification methods using antibody beads / magnetic beads or cell sorters can achieve high purification of cells, they cause significant damage to the cells (for example, the cells become exhausted due to the long processing time). In addition, the operation is complicated and the cost is high. Furthermore, the possibility of contamination with foreign components such as components derived from antibodies cannot be excluded. In view of these circumstances, the first problem of the present invention is to provide a means for preparing highly pure vascular endothelial progenitor cells simply and at low cost. The present invention also aims to enable Xeno-freeization (preparation under conditions not containing components derived from foreign species) of the process for preparing vascular endothelial progenitor cells. Furthermore, it is an object of the present invention to provide a method for efficiently proliferating vascular endothelial progenitor cells.

Means for Solving the Problems

[0008] When induced to differentiate into vascular endothelial progenitor cells, iPS cells usually result in a state where vascular endothelial progenitor cells and other unnecessary cells (such as cells with different differentiation directions) are mixed. The inventors of the present invention emphasized simplicity and decided to examine methods and conditions for selectively removing unnecessary cells (in other words, selectively retaining vascular endothelial progenitor cells in the culture system) from various perspectives during the culture process. As a result, it was accidentally found that there is a difference in adhesion ability between vascular endothelial progenitor cells and unnecessary cells. By intervening with a specific detachment treatment, unnecessary cells were selectively removed, and the vascular endothelial progenitor cells were successfully highly purified. That is, the inventors of the present invention established a method for selectively isolating vascular endothelial progenitor cells at the end stage of differentiation by utilizing the difference in adhesion ability between vascular endothelial progenitor cells and other cells. Despite being an extremely simple operation, this method achieved a high purification of vascular endothelial progenitor cells comparable to conventional methods. In addition, by using this method, selection by antibody beads / magnetic beads or cell sorters becomes unnecessary, and damage to cells and contamination with foreign components (such as components derived from antibodies) can be avoided. Notably, the method was applicable to vascular endothelial progenitor cells induced to differentiate under Xeno-free conditions. This fact means that a method capable of making all steps in the process from iPS cell establishment to the generation of vascular endothelial progenitor cells Xeno-free has been successfully established, which is an extremely useful method for practical and clinical applications.

[0009] On the other hand, it was found that when a specific combination of three small-molecule compounds (ROCK inhibitor, GSK3β inhibitor, and TGFβ receptor inhibitor) is used, the proliferation rate of iPS cell-derived vascular endothelial progenitor cells increases dramatically. That is, a "novel combination of small-molecule compounds" that can greatly contribute to the stable supply of vascular endothelial progenitor cells was successfully discovered. This combination is also effective when culturing vascular endothelial progenitor cells under Xeno-free conditions, and its clinical application can be highly anticipated. [1] A method for preparing vascular endothelial progenitor cells, comprising the following steps (1) and (2): (1) A step of differentiating pluripotent stem cells into vascular endothelial progenitor cells; (2) A step of purifying vascular endothelial progenitor cells by utilizing the difference in the adhesion ability between the vascular endothelial progenitor cells and other cells constituting the cell population obtained in step (1). [2] The preparation method according to [1], wherein step (2) consists of the following steps (2-1) and (2-2): (2-1) A step of detaching and removing the other cells from the culture surface by a first detachment treatment using a first cell dissociation solution. (2-2) A detachment treatment using a second cell dissociation solution, which is a second detachment treatment with a higher cell detachment effect than the first detachment treatment, for detaching and recovering the vascular endothelial progenitor cells from the culture surface. [3] The preparation method according to [2], wherein the first detachment treatment and the second detachment treatment satisfy one or more of the following conditions (a) to (d): (a) The second detachment treatment has a longer treatment time than the first detachment treatment. (b) The second detachment treatment has a higher treatment temperature than the first detachment treatment. (c) The second cell dissociation solution used in the second detachment treatment has a higher active ingredient concentration than the first cell dissociation solution used in the first detachment treatment. (d) The second cell dissociation solution used in the second detachment treatment has a stronger action of the active ingredient than the first cell dissociation solution used in the first detachment treatment. [4] The preparation method according to [2] or [3], wherein the first detachment treatment includes contacting the cell population obtained in step (1) with the first cell dissociation solution and subsequent tapping treatment. [5] The preparation method according to any one of [1] to [4], wherein step (1) consists of the following steps (1-1) and (1-2): (1-1) A step of differentiating pluripotent stem cells into mesoderm. (1-2) A step of differentiating the cells obtained in step (1-1) into vascular endothelial progenitor cells. [6] The preparation method according to [5], wherein the culture period in step (1-2) is 2 to 14 days. [7] The preparation method according to [5] or [6], wherein during step (1-2), the culture surface with strong cell adhesion is switched to the culture surface with weak cell adhesion. [8] The preparation method according to [5] or [6], wherein step (1-2) includes the following culture steps (1-2-1) and (1-2-2).: (1-2-1) Culturing in the presence of bone morphogenetic protein 4 and vascular endothelial growth factor, (1-2-2) Culturing in the presence of basic fibroblast growth factor and vascular endothelial growth factor. [9] The preparation method according to [8], wherein the culture period of step (1-2-1) is from 1 day to 7 days, and the culture period of step (1-2-2) is from 1 day to 7 days.

[10] Using a culture surface with strong cell adhesion for the culture in step (1-2-1), The preparation method according to [9], wherein a culture surface with weak cell adhesion is used for the culture in step (1-2-2).

[11] The culture surface with strong cell adhesion is a culture surface coated with a basement membrane component or a fragment thereof on the surface, and the culture surface with weak cell adhesion is a culture surface coated with a material selected from the group consisting of gelatin, poly-D-lysine, and poly-L-lysine on the surface. The preparation method according to [7] or

[10] .

[12] The preparation method according to any one of [1] to

[11] , wherein all steps are performed under conditions not containing components derived from heterologous animals.

[13] The preparation method according to any one of [1] to

[12] , wherein the pluripotent stem cells are induced pluripotent stem cells.

[14] The preparation method according to

[13] , wherein the induced pluripotent stem cells are human induced pluripotent stem cells.

[15] Vascular endothelial progenitor cells obtained by the preparation method according to any one of [1] to

[14] .

[16] The vascular endothelial progenitor cells according to

[15] , having a purity of 90% or more.

[17] A cell preparation containing the vascular endothelial progenitor cells according to

[15] or

[16] .

[18] Use of the vascular endothelial progenitor cells according to

[15] or

[16] for constructing blood vessels in vitro or constructing a human blood-brain barrier model.

[19] An expansion culture method of vascular endothelial progenitor cells, including the step of culturing the vascular endothelial progenitor cells in the presence of a ROCK inhibitor, a GSK-3β inhibitor, and a TGF-β receptor inhibitor in addition to basic fibroblast growth factor and epidermal growth factor.

[20] The ROCK inhibitor is Y-27632, the GSK-3β inhibitor is CHIR 99021, and the TGF-β receptor inhibitor is A 83-01, and the expansion culture method described in

[19] .

[21] The expansion culture method according to

[19] or

[20] , wherein the vascular endothelial progenitor cells are cells obtained by inducing differentiation of pluripotent stem cells.

[22] The expansion culture method according to

[21] , wherein the pluripotent stem cells are induced pluripotent stem cells.

[23] The expansion culture method according to

[22] , wherein the induced pluripotent stem cells are human induced pluripotent stem cells.

[24] The expansion culture method according to

[19] or

[20] , wherein the vascular endothelial progenitor cells are cells prepared by the preparation method according to any one of [1] to

[14] .

[25] The expansion culture method according to

[19] or

[20] , wherein the vascular endothelial progenitor cells are vascular endothelial progenitor cells previously collected from a living body or cells maintained or proliferated in vitro thereof.

[26] The expansion culture method according to any one of

[19] to

[25] , wherein the step is performed under conditions not containing components derived from a heterologous animal.

Brief Description of Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] 1. Method for preparing vascular endothelial progenitor cells The first aspect of the present invention relates to a method for preparing vascular endothelial progenitor cells (EPCs) from pluripotent stem cells (hereinafter also referred to as "the preparation method of the present invention"). According to the present invention, vascular endothelial progenitor cells having the ability to differentiate into vascular endothelial cells (ECs) can be obtained. If the vascular endothelial progenitor cells are induced to differentiate under appropriate conditions, or if the vascular endothelial progenitor cells are placed in an environment suitable for differentiation induction (typically, transplantation / administration to a site where vascular endothelial cells are present in the living body or in the vicinity thereof), vascular endothelial cells will be generated. Vascular endothelial progenitor cells are used in regenerative therapies and angiogenesis experiments for severe ischemic diseases such as coronary artery disease and lower limb ischemic diseases. The preparation method of the present invention enables the simple and inexpensive preparation of such highly useful vascular endothelial progenitor cells.

[0012] The "pluripotent stem cells" refer to cells that have both the ability to differentiate into all the cells constituting the living body (pluripotency) and the ability to produce daughter cells having the same differentiation ability as themselves through cell division (self-renewal ability). Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing the presence or absence of teratoma formation containing cells of each of the three germ layers (ectoderm, mesoderm, endoderm).

[0013] Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), etc., but are not limited thereto as long as they are cells having both pluripotency and self-renewal ability. Preferably, ES cells or iPS cells are used. More preferably, iPS cells are used. The pluripotent stem cells are preferably cells of mammals (for example, primates such as humans and chimpanzees, rodents such as mice and rats), and particularly preferably human cells. Therefore, in the most preferred embodiment of the present invention, human iPS cells are used as the pluripotent stem cells.

[0014] ES cells can be established, for example, by culturing pre-implantation early embryos, inner cell masses constituting the early embryos, single blastomeres, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, J. A. et al., Science, 282, 1145-1147 (1998)). As the early embryo, an early embryo produced by nuclear transfer of the nucleus of a somatic cell may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Akira Iritani et al. (Protein, Nucleic Acid, Enzyme, 44, 892 (1999)), Baguisi et al. (Nature Biotechnology, 17, 456 (1999)), Wakayama et al. (Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000), Tachibana et al. (Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013) in press)). As the early embryo, parthenogenetic embryos may be used (Kim et al. (Science, 315, 482-486 (2007)), Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al. (Cloning Stem Cells, 9, 432-449 (2007)), Revazova et al. (Cloning Stem Cells, 10, 11-24 (2008)).In addition to the above-mentioned papers, for the preparation of ES cells, see Strelchenko N., et al. Reprod Biomed Online. 9: 623-629, 2004; Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al Stem Cells 24: 2669-2676, 2006; Wassarman, P.M. et al. Methods in Enzymology, Vol.365, 2003, etc. It should be noted that fusion ES cells obtained by cell fusion of ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

[0015] Some ES cells are available from preservation institutions or are commercially available. For example, human ES cells can be obtained from the Institute for Frontier Medical Sciences, Kyoto University (such as KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0016] EG cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, SCF, etc. (Matsui et al., Cell, 70, 841-847 (1992), Shamblott et al., Proc. Natl. Acad. Sci. USA, 95 (23), 13726-13731 (1998), Turnpenny et al., Stem Cells, 21(5), 598-609, (2003)).

[0017] "Induced pluripotent stem cells (iPS cells)" are cells that are produced by reprogramming somatic cells, such as by introducing reprogramming factors, and have pluripotency (the ability to differentiate into multiple cell types) and proliferative ability. Induced pluripotent stem cells exhibit properties similar to those of ES cells. The somatic cells used for the production of iPS cells are not particularly limited and may be differentiated somatic cells or undifferentiated stem cells. Also, their origin is not particularly limited, but preferably somatic cells of mammals (e.g., primates such as humans and chimpanzees, rodents such as mice and rats), particularly preferably human somatic cells, are used. iPS cells can be produced by various methods reported so far. It is also naturally assumed that iPS cell production methods to be developed in the future will be applied.

[0018] The most basic method for producing iPS cells is a method of introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, which are transcription factors, into cells using a virus (Takahashi K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007). Regarding human iPS cells, there are reports of establishment by introducing four factors, Oct4, Sox2, Lin28, and Nonog (Yu J, et al: Science 318(5858), 1917-1920, 2007). Establishment of iPS cells by introducing three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), two factors, Oct3 / 4 and Klf4 (Kim J B, et al: Nature 454 (7204), 646-650, 2008), or only Oct3 / 4 (Kim J B, et al: Cell 136 (3), 411-419, 2009) has also been reported. In addition, a method of introducing a protein, which is the expression product of a gene, into cells (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al: Cell Stem Cell 4, 472-476, 2009) has also been reported. On the other hand, there are also reports that it is possible to improve the production efficiency and reduce the factors to be introduced by using an inhibitor BIX-01294 against histone methyltransferase G9a, a histone deacetylase inhibitor valproic acid (VPA), or BayK8644, etc. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J, et al: PLoS. Biol. 6 (10), e 253, 2008).Regarding gene transfer methods, studies are also underway. In addition to retroviruses, lentiviruses (Yu J, et al: Science 318(5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903), 945-949, 2008), plasmids (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been used to develop techniques for gene transfer.

[0019] Cells in which transformation into iPS cells, that is, reprogramming (reinitialization) has occurred can be selected using as an indicator the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript. The selected cells are recovered as iPS cells.

[0020] iPS cells can also be obtained, for example, from the National University Corporation Kyoto University or the RIKEN BioResource Center, National Institute of Advanced Industrial Science and Technology.

[0021] In this specification, "differentiate" means to act so as to differentiate along a specific cell lineage, that is, to induce differentiation. In the preparation method of the present invention, after inducing differentiation of pluripotent stem cells into vascular endothelial progenitor cells, the vascular endothelial progenitor cells are purified. Specifically, the following steps (1) and (2) are performed in the preparation method of the present invention. (1) Step of differentiating pluripotent stem cells into vascular endothelial progenitor cells (2) Step of purifying vascular endothelial progenitor cells by utilizing the difference in the adhesion ability between vascular endothelial progenitor cells and other cells constituting the cell population obtained in step (1)

[0022] <Step (1) Induction of differentiation into vascular endothelial progenitor cells> In this step, pluripotent stem cells are cultured and differentiated into vascular endothelial progenitor cells. In other words, pluripotent stem cells are cultured under conditions that induce differentiation into vascular endothelial progenitor cells. As long as the pluripotent stem cells differentiate into vascular endothelial progenitor cells, the culture conditions are not particularly limited. Typically, a two-step differentiation induction described below is performed so that the pluripotent stem cells differentiate into vascular endothelial progenitor cells via mesoderm, that is, a step of differentiating pluripotent stem cells into mesoderm (step (1-1)) and a step of differentiating the obtained cells into vascular endothelial progenitor cells (step (1-2)).

[0023] Step (1-1) Differentiation into mesoderm In this step, pluripotent stem cells are cultured and differentiated into mesoderm. In other words, pluripotent stem cells are cultured under conditions that induce differentiation into mesoderm. As long as the pluripotent stem cells differentiate into mesoderm, the culture conditions are not particularly limited. For example, according to past reports (for example, Sriram G. et al, Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions. Stem Cell Research & Therapy 2015;6:261 is referred to), after culturing in the presence of a GSK-3β inhibitor, it is cultured in the presence of basic fibroblast growth factor (bFGF).

[0024] "In the presence of a GSK-3β inhibitor" is synonymous with the condition where a GSK-3β inhibitor is added to the medium. Therefore, to perform culturing in the presence of a GSK-3β inhibitor, a medium to which a GSK-3β inhibitor has been added may be used. Examples of GSK-3β inhibitors include CHIR 99021, SB216763, CHIR 98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, 1-Azakenpaullone. An example of the added concentration of a GSK-3β inhibitor (in the case of CHIR 99021) is 1 μM to 100 μM, preferably 3 μM to 30 μM. The culturing period (culture period) in the presence of a GSK-3β inhibitor is, for example, 1 day to 4 days, preferably 1 day to 2 days. In addition, regarding the added concentration when using a compound different from the exemplified compound, i.e., CHIR99021, a person skilled in the art can set it according to the above concentration range in consideration of the characteristics of the compound to be used and the difference in characteristics (especially the difference in activity) between the exemplified compound. Also, whether the set concentration range is appropriate can be confirmed by preliminary experiments according to the following examples.

[0025] "In the presence of basic fibroblast growth factor (bFGF)" is synonymous with the condition where bFGF is added to the medium. Therefore, to perform culturing in the presence of bFGF, a medium to which bFGF has been added may be used. An example of the added concentration of bFGF is 5 ng / mL to 500 ng / mL, preferably 10 ng / mL to 200 ng / mL. Preferably, human bFGF (for example, human recombinant bFGF) is used as bFGF. Incidentally, bFGF is also called fibroblast growth factor 2 (FGF2).

[0026] Step (1-2) Differentiation into vascular endothelial progenitor cells In this step, the cells obtained in step (1-1) are cultured and differentiated into vascular endothelial progenitor cells. In other words, they are cultured under conditions that induce differentiation into vascular endothelial progenitor cells. As long as differentiation induction into vascular endothelial progenitor cells is possible, the culture conditions are not particularly limited. For example, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), bFGF, etc. are used as differentiation-inducing factors to induce differentiation into vascular endothelial progenitor cells.

[0027] In the next step, namely step (2) (purification of vascular endothelial progenitor cells), in order to improve the efficiency and operability of the detachment treatment, etc., it is preferable to switch from a culture surface with strong cell adhesiveness to a culture surface with weak cell adhesiveness during the process of step (1-2). In other words, at the end of step (1-2), the cells should be cultured on a culture surface with weak cell adhesiveness. According to this mode, in step (2), unnecessary cells, that is, cells other than vascular endothelial progenitor cells, can be easily detached.

[0028] For example, a culture surface coated with one or more types of basement membrane components (such as laminin (such as laminin 511), collagen (such as type IV collagen) 4, entactin, vitronectin, fibronectin) or fragments thereof (such as vitronectin-N, E8 fragment of laminin (such as laminin 511)) etc. on the surface of a culture container (such as a culture dish, culture flask, microplate for cell culture) can be adopted as the "culture surface with strong cell adhesiveness". Thus, the "culture surface with strong cell adhesiveness" can be constituted by using substances that show high adhesive force to cells. The "culture surface with weak cell adhesiveness" is one with weak cell adhesiveness (that is, relatively weak cell adhesiveness) in comparison with the "culture surface with strong cell adhesiveness", and it is also a culture surface with cell adhesiveness similar to the "culture surface with strong cell adhesiveness". The "culture surface with weak cell adhesiveness" can be constituted, for example, by coating one or more types of gelatin, poly-D-lysine, poly-L-lysine, etc. on the surface of the culture container. Incidentally, the strength or weakness of cell adhesiveness may be set according to the difference in the concentration of the surface coating agent (the density of the cell adhesion substance present on the culture surface).

[0029] The period of step (1-2) (culture period) is, for example, 2 to 14 days, preferably 4 to 10 days.

[0030] In a preferred embodiment, step (1-2) is a two-stage culture, namely, culture in the presence of BMP4 and VEGF (step (1-2-1)) and culture in the presence of bFGF and VEGF (step (1-2-2)) performed after the said culture. According to this embodiment, while inducing efficient differentiation into vascular endothelial progenitor cells, a state with a high cell density can be formed before the next step (step (2)). Keeping the cell density high is preferable in that it can increase the difference in the adhesion ability between vascular endothelial progenitor cells and other cells, which is advantageous for the selective detachment of cells other than vascular endothelial progenitor cells.

[0031] "In the presence of BMP4 and VEGF" in step (1-2-1) is synonymous with the condition where BMP4 and VEGF are added to the culture medium. Therefore, in order to perform the culture in the presence of BMP4 and VEGF, a culture medium to which BMP4 and VEGF are added may be used. BMP4 is a differentiation-inducing factor, and by its use, differentiation into vascular endothelial progenitor cells occurs. The added concentration of BMP4 is, for example, 5 mg / mL to 200 ng / mL, preferably 10 mg / mL to 100 mg / mL. On the other hand, the added concentration of VEGF, which is an angiogenesis factor, is, for example, 10 ng / mL to 200 ng / mL, preferably 20 ng / mL to 100 ng / mL. Preferably, human BMP4 (e.g., human recombinant BMP4) and human VEGF (e.g., human recombinant VEGF) are used.

[0032] The period of step (1-2-1) (culture period) is, for example, 1 to 7 days, preferably 2 to 5 days. If the culture period is too short, the expected effect (i.e., induction of differentiation into vascular endothelial progenitor cells) cannot be sufficiently obtained. On the other hand, if the culture period is too long, induction of differentiation into different cells is caused.

[0033] In step (1-2-2), the phrase "in the presence of bFGF and VEGF" is synonymous with the condition where bFGF and VEGF are added to the culture medium. Therefore, to perform the culture in the presence of bFGF and VEGF, a culture medium supplemented with bFGF and VEGF can be used. The addition concentration of bFGF is, for example, 1 ng / mL to 5200 ng / mL, preferably 2 ng / mL to 100 ng / mL. The addition concentration of VEGF is, for example, 10 ng / mL to 200 ng / mL, preferably 20 ng / mL to 100 ng / mL. Similar to the above-described steps, preferably human bFGF (e.g., human recombinant bFGF) and human VEGF (e.g., human recombinant VEGF) are used.

[0034] The period of step (1-2-2) (culture period) is, for example, 1 day to 7 days, preferably 2 days to 5 days. If the culture period is too short, the expected effects (i.e., inducing differentiation into vascular endothelial progenitor cells while proliferating the cells) cannot be sufficiently obtained. On the other hand, if the culture period is too long, it causes induction of differentiation into different cells.

[0035] In a preferred embodiment, the above-described switching of the culture surface, i.e., the switching from the "culture surface with strong cell adhesiveness" to the "culture surface with weak cell adhesiveness", is synchronized with the switching from step (1-2-1) to step (1-2-2). In other words, a "culture surface with strong cell adhesiveness" is used for the culture in step (1-2-1), and a "culture surface with weak cell adhesiveness" is used for the culture in step (1-2-2). According to this embodiment, the operation can be simplified, and cell damage can be minimized.

[0036] In each step (step (1), step (1-1), step (1-2), step (1-2-1), step (1-2-2)) constituting the present invention, subculture may be performed in the middle. For example, when the cells become confluent or sub-confluent, a part of the cells is collected and transferred to another culture vessel, and the culture is continued. It is preferable to set a low cell density to promote differentiation. For example, 1×10 4 cells / cm 2 ~1×10 6 cells / cm 2Cells may be seeded at an appropriate cell density. For cell recovery, a cell dissociation solution or the like may be used. As the cell dissociation solution, for example, proteolytic enzymes such as trypsin-EDTA, various collagenases (e.g., collagenase IV), metalloproteases, dispase, etc. can be used alone or in appropriate combinations. Also, TrypLE TM (Invitrogen), Acutase TM (Innova Cell Technologies), Accumax TM (Innova Cell Technologies), etc., commercially available cell dissociation solutions may also be used.

[0037] When recovering cells associated with medium exchange, subculture, etc., in order to suppress cell death, it is advisable to pre-treat the cells with an ROCK inhibitor such as Y-27632 (Rho-associated coiled-coil forming kinase / Rho-binding kinase).

[0038] Other culture conditions (such as culture temperature) in each step constituting the present invention may be the conditions generally adopted in the culture of animal cells. That is, for example, it may be cultured in an environment of 37°C and 5% CO2. Also, as the basal medium, for example, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), MEMα medium, serum-free medium for vascular endothelial cells (Human Endothelial SFM (Gibco), Medium 200 (Gibco), etc.), Ham's F12 medium (HamF12) (SIGMA, Gibco, etc.), Dulbecco's Modified Eagle Medium (D-MEM) (Nacalai Tesque, Inc., Sigma, Gibco, etc.), Glasgow's Minimal Essential Medium (Gibco, etc.), RPMI1640 medium, MCDB107 medium (Functional Peptide Research Institute), etc. can be used. A medium obtained by mixing two or more basal media, for example, a mixed medium of D-MEM and Ham's F12 medium, may also be used.

[0039] Examples of other components that can be added to the medium include serum (such as fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (such as Knockout serum replacement (KSR), etc.), antibiotics (such as penicillin, streptomycin, etc.), supplements (such as ITS-G supplement), L-glutamine, magnesium L-ascorbic acid phosphate salt n-hydrate, non-essential amino acids (NEAA), 2-mercaptoethanol, Chemically Defined Lipid Concentrate (Gibco).

[0040] In a preferred embodiment, step (1) is carried out under Xeno-free conditions. When step (1) is composed of steps (1-1) and (1-2), these two steps will be carried out under Xeno-free conditions. Similarly, when step (1-2) is composed of steps (1-2-1) and (1-2-2), these two steps will also be carried out under Xeno-free conditions. When culturing under Xeno-free conditions, usually, a serum-free medium is employed, and preferably, KnockOut TM SR XenoFree Medium (Gibco), ITS-G, hormones (such as human recombinant hydrocortisone, etc.), Chemically Defined Lipid Concentrate, etc. are added.

[0041] Differentiation into vascular endothelial progenitor cells can be determined or evaluated, for example, using the expression of vascular endothelial progenitor cell markers as an indicator. Examples of vascular endothelial progenitor cell markers include PECAM1 (CD31), CD34, CDH5 (VE-Cadherin), FLK1 (VEGFR-2). Among them, CD34 is specific to vascular endothelial progenitor cells and is a particularly useful vascular endothelial progenitor cell marker.

[0042] On the one hand, when inducing differentiation into vascular endothelial cells, if the cells show the functions of vascular endothelial cells, it can be confirmed that they have differentiated into vascular endothelial progenitor cells. For the evaluation of the functions of vascular endothelial cells, the Dil-Ac LDL uptake test and the tube formation test can be used.

[0043] <Step (2) Purification of Vascular Endothelial Progenitor Cells> A cell population containing vascular endothelial progenitor cells is obtained in step (1). In step (2) following step (1), the vascular endothelial progenitor cells are purified. "Purifying vascular endothelial progenitor cells" means increasing the ratio (i.e., the abundance or content rate) of vascular endothelial progenitor cells constituting the cell population. Therefore, if the ratio of vascular endothelial progenitor cells is higher after the treatment compared to before the treatment, the treatment corresponds to purification. The degree of purification (purity) is not particularly limited. The purity can vary depending on the state of the cells used, the culture conditions, etc., but by going through step (2), a purity of, for example, 80% or more, preferably 90% or more, and more preferably 95% or more can be achieved. The purity can be calculated using the following formula. For the measurement of the number of cells, a hemocytometer or the like can be used. Purity (%) = (Number of vascular endothelial progenitor cells) / (Total number of cells constituting the cell population) × 100

[0044] Step (2) is one of the greatest features of the present invention. Based on the finding that the adhesion force to the culture surface differs between vascular endothelial progenitor cells and other cells, the difference in the adhesion ability between vascular endothelial progenitor cells and other cells constituting the cell population obtained in step (1) is utilized to purify the vascular endothelial progenitor cells. As shown in the examples described later, vascular endothelial progenitor cells show a higher adhesion ability than other cells. Therefore, typically, the vascular endothelial progenitor cells are purified by preferentially or selectively detaching and removing other cells with low adhesion ability (so that the vascular endothelial progenitor cells are selectively maintained in the culture system). In this case, for example, the following steps (2-1) and (2-2) can be performed. (2-1) Step of detaching and removing the other cells from the culture surface by the first detachment treatment using the first cell dissociation solution (2-2) A detachment process using the second cell detachment solution, wherein the vascular endothelial progenitor cells are detached from the culture surface and recovered by a second detachment process having a higher cell detachment effect than the first detachment process

[0045] Step (2-1) is a process of selectively removing unnecessary cells, i.e., cells other than vascular endothelial progenitor cells. After this process, the remaining cells are recovered by step (2-2) to obtain a cell population containing vascular endothelial progenitor cells with high purity.

[0046] In the first detachment process of step (2-1), typically, after weakening the adhesive force between the cells and the culture surface by bringing the cell population obtained in step (1) into contact with the first detachment solution (a state in which the cells are easily peeled off is formed), an impact is applied by tapping, pipetting, etc., to dissociate the unnecessary cells to be removed, i.e., cells other than vascular endothelial progenitor cells, from the culture surface. The dissociated cells are removed, and the next step, i.e., step (2-2), is performed. "Tapping" refers to applying an intermittent and rapid impact to the contents by bouncing the wall surface of the culture vessel, etc., with a finger, rod, etc., or by gripping the upper part of the culture vessel and lightly using a snap to strike the culture vessel against the wall surface, etc.

[0047] In step (2-2) following step (2-1), the remaining cells, i.e., vascular endothelial progenitor cells, are detached from the culture surface and recovered by the same operation using the second cell detachment solution. Note that the contact of the cell detachment solution with the cell population is usually not instantaneous, and the contact state is maintained for a certain period of time.

[0048] In order to achieve high purification of vascular endothelial progenitor cells by steps (2-1) and (2-2), for example, the conditions of the first detachment process in step (2-1) and the second detachment process in step (2-2) are set so as to satisfy one or more of the following conditions (a) to (d). Note that as the active ingredients of the first cell detachment solution and the second cell detachment solution, proteolytic enzymes such as trypsin-EDTA, various collagenases (e.g., collagenase IV), metalloprotease, and dispase can be used alone or in appropriate combinations. Also, TrypLE TM(Invitrogen), Acutase TM (Innova Cell Technologies), Accumax TM (Innova Cell Technologies), etc., you may also use commercially available cell dissociation solutions. (a) The processing time of the second detachment process is longer than that of the first detachment process (b) The processing temperature of the second detachment process is higher than that of the first detachment process (c) The concentration of the active ingredient in the second cell dissociation solution used for the second detachment process is higher than that in the first cell dissociation solution used for the first detachment process (d) The action of the active ingredient in the second cell dissociation solution used for the second detachment process is stronger than that in the first cell dissociation solution used for the first detachment process

[0049] Regarding conditions (a) to (c), usually, the first cell dissociation solution and the second cell dissociation solution with the same active ingredient are prepared, and the first detachment process and the second detachment process are performed. Therefore, it is easy to prepare the cell dissociation solution. In this regard, in conditions (a) and (b), a cell dissociation solution with the same composition including the active ingredient can be used, which is particularly advantageous. Also, condition (a) only requires setting a difference in processing time between the first detachment process and the second detachment process, and is simpler than condition (b) that sets a difference in processing temperature, and can be said to be the optimal condition.

[0050] On the other hand, conditions (c) and (d) are conditions for using a second cell dissociation solution with a higher detachment effect than the first cell dissociation solution. When these conditions are adopted, two types of cell dissociation solutions with different active ingredient concentrations (in the case of condition (c)) or different active ingredients themselves (in the case of condition (d)) need to be prepared.

[0051] The optimal conditions for the first detachment process and the second detachment process may vary depending on the type and state of the cells used, the active ingredient of the cell dissociation solution, or other factors, but can be set by preliminary experiments while referring to the disclosure of this specification. Hereinafter, examples of the active ingredient and concentration of the cell dissociation solution, as well as the processing time and processing temperature of the detachment process, are given. Active ingredients of cell dissociation solution: EDTA, trypsin-EDTA, various collagenases, metalloproteases, dispase Concentration of active ingredients in cell dissociation solution: EDTA: 0.5 mM - 500 mM, trypsin-EDTA: 0.005 - 0.5%, collagenase IV: 0.005 - 0.5%, dispase: 100 - 2000 PU / mL Treatment time of detachment treatment: 5 seconds - 1 hour Treatment temperature of detachment treatment: 10°C - 38°C

[0052] In the case of condition (a), for example, the treatment time of the second detachment treatment is set to 1.5 to 10 times the treatment time of the first detachment treatment. In the case of condition (b), for example, the first detachment treatment is performed at 20°C - 30°C, and the second detachment treatment is performed at 30°C - 38°C. In the case of condition (c), for example, the concentration of the active ingredient in the second cell dissociation solution is set to 1.5 to 10 times the concentration of the active ingredient in the first cell detachment solution. Regarding condition (d), for example, the active ingredient in the first cell dissociation solution is collagenase or EDTA (two or more of these may be combined), and the active ingredient in the second cell detachment solution is trypsin-EDTA or dispase (two or more of these may be combined).

[0053] TrypLE TM (Invitrogen), Acutase TM (Innova Cell Technologies), Accumax TM If commercially available cell dissociation solutions such as TrypLE (Invitrogen), Acutase (Innova Cell Technologies), Accumax (Innova Cell Technologies), etc. are used, the first and second detachment treatments can be performed more simply. In this case, considering factors such as reduction in the type / number of cell dissociation solutions used and simplicity of operation, preferably any one of conditions (a) - (c), more preferably condition (a) or (c), and even more preferably condition (a) is adopted.

[0054] The purified cells (high-purity vascular endothelial progenitor cells) are used for various purposes. Also, they are subjected to culture for maintenance and proliferation as needed. Or they are stored until use (culturing also falls under the category of use here).

[0055] For culturing for maintenance and proliferation, various culturing methods (for example, reference may be made to reports such as James D. et al, Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nature Biotechnology. 2010;28(2):161-6. and Mien T.X. et al. Differentiation of Human Embryonic stem cells to endothelial progenitor cells on laminins in defined and xeno-free systems. Stem cell reports. 2016;7:802-16., etc.) can be used as long as they are suitable for culturing vascular endothelial progenitor cells. In a preferred embodiment, the cells after purification are cultured by the expansion culturing method of the present invention described below.

[0056] The storage method when storing the cells after purification may follow conventional methods. For example, use TC protector (DS Pharma Biomedical), Cell Banker (ZenoArk), Stem Cell Banker (ZenoArk), Cell Reserver One (Nacalai), etc. and store them by cryopreservation.

[0057] 2. Uses of Vascular Endothelial Progenitor Cells The second aspect of the present invention relates to the use of vascular endothelial progenitor cells obtained by the preparation method of the present invention (for convenience of explanation, hereinafter referred to as "vascular endothelial progenitor cells of the present invention"). As a first use, a cell preparation containing the vascular endothelial progenitor cells of the present invention is provided. As described above, vascular endothelial progenitor cells are expected to be applied to the treatment of coronary artery disease, lower limb ischemia diseases (such as Buerger's disease and atherosclerotic obliterans), etc. In fact, clinical trials using vascular endothelial progenitor cells have also been conducted and good results have been reported. The cell preparation of the present invention can be used for the treatment (regenerative medicine) of various diseases for which administration / transplantation of vascular endothelial progenitor cells is considered effective.

[0058] The cell preparation of the present invention can be prepared, for example, by suspending the vascular endothelial progenitor cells of the present invention in physiological saline, a buffer solution (e.g., phosphate buffer solution), etc. In order to be able to administer a therapeutically effective amount of cells, for example, 1×10 5 cells to 1×10 10 cells may be contained as the amount for a single administration. The cell content can be appropriately adjusted in consideration of the purpose of use, the target disease, the gender, age, body weight of the recipient, the condition of the affected part, the condition of the cells, etc.

[0059] Dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained in the cell preparation of the present invention for the purpose of protecting cells, antibiotics, etc. for the purpose of preventing the contamination of bacteria, and various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of inducing cell activation, proliferation, or differentiation. Furthermore, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained in the cell preparation of the present invention.

[0060] Instead of using it as a cell preparation for regenerative medicine, the vascular endothelial progenitor cells of the present invention can also be used to construct materials used in regenerative medicine, specifically, blood vessels for transplantation in vitro. Regarding the method of constructing blood vessels using vascular endothelial progenitor cells, it is detailed, for example, in Vascular Regeneration Therapy (supervised by Hiroo Imura, Diagnostic and Therapeutic Society).

[0061] The vascular endothelial progenitor cells of the present invention can also be used for constructing a human blood-brain barrier model. For example, by seeding vascular endothelial progenitor cells in the insert part of a transwell, seeding pericytes on the back side thereof, and seeding astrocytes on the bottom surface of the well, a structure similar to the blood-brain barrier in vivo can be mimicked. (Nakagawa S, Maria A. D, Nakao S, Honda M, Hayashi M, Nakaoke R, Kataoka Y, Niwa M. Cellular and Molecular Neurobiology. 27:687-694, 2007).

[0062] 3. Expansion culture of vascular endothelial progenitor cells The third aspect of the present invention relates to a method for expanding the culture of vascular endothelial progenitor cells. "Expansion culture" means increasing the number of cells while maintaining the properties of the cells. When vascular endothelial progenitor cells are subjected to expansion culture, the vascular endothelial progenitor cells can be proliferated while maintaining their properties (particularly the ability to differentiate into vascular endothelial cells). According to the expansion culture method of the present invention, a high proliferation rate can be obtained, enabling efficient expansion culture. Therefore, the present invention is useful as a means for preparing a large amount of vascular endothelial progenitor cells useful for clinical applications, research applications, etc., and contributes to the stable supply of vascular endothelial progenitor cells.

[0063] In the expansion culture method of the present invention, a characteristic step of "culturing vascular endothelial progenitor cells in the presence of a ROCK inhibitor, a GSK-3β inhibitor, and a TGF-β receptor inhibitor in addition to basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)" is performed. "In the presence of a ROCK inhibitor, a GSK-3β inhibitor, and a TGF-β receptor inhibitor in addition to basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)" is synonymous with the condition where bFGF, EGF, a ROCK inhibitor, a GSK-3β inhibitor, and a TGF-β receptor inhibitor are added to the culture medium. The first two (bFGF, EGF) are factors generally used when culturing vascular endothelial progenitor cells and vascular endothelial cells. Examples of the addition concentration of bFGF are 5 ng / mL to 200 ng / mL, preferably 10 ng / mL to 50 ng / mL, and examples of the addition concentration of EGF are 5 ng / mL to 500 ng / mL, preferably 10 ng / mL to 200 ng / mL. On the other hand, the latter three (ROCK inhibitor, GSK-3β inhibitor, and TGF-β receptor inhibitor) are characteristic of the present invention, that is, the point of using a combination of these three compounds forms the basis of the present invention.

[0064] Although not bound by theory, it can be expected that the ROCK inhibitor has the effect of suppressing cell death and enhancing cell proliferation ability. In addition, the GSK-3β inhibitor has a wnt pathway activation effect and can be expected to enhance cell proliferation ability via the wnt / β-catenin signal. The TGF-β receptor inhibitor can be expected to enhance cell proliferation ability by suppressing the TGFβ signal known as a cell growth inhibitor. By using these three compounds in combination and their synergistic effect, a dramatic improvement in the proliferation ability of vascular endothelial progenitor cells can be achieved.

[0065] For example, Y-27632 can be used as a ROCK inhibitor. On the other hand, examples of GSK-3β inhibitors include CHIR 99021, SB216763, CHIR 98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, 1-Azakenpaullone. Examples of TGF-β receptor inhibitors are A 83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, RepSox.

[0066] Examples of the addition concentration of the ROCK inhibitor (in the case of Y-27632) are 1 μM to 50 μM, preferably 3 μM to 30 μM. Examples of the addition concentration of the GSK-3β inhibitor (in the case of CHIR 99021) are 0.1 μM to 100 μM, preferably 1 μM to 30 μM. Examples of the addition concentration of the TGF-β receptor inhibitor (in the case of A 83-01) are 0.05 μM to 20 μM, preferably 0.1 μM to 10 μM.

[0067] In addition, regarding the addition concentration when using a compound different from the exemplified compounds, namely Y-27632, CHIR99021, and A 83-01, those skilled in the art can set it according to the above concentration range in consideration of the differences in the characteristics of the compound to be used and the exemplified compounds (especially the differences in activity). Also, whether the set concentration range is appropriate can be confirmed by preliminary experiments according to the following examples.

[0068] Other culture conditions may follow conventional methods (for example, reports such as Ikuno T. et al, Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP. PLOS ONE. 2017;12:3 and Li S. et al, Inhibition of cell growth and induction of inflammation by endosulfan in HUVEC-C cells. Environmental Toxicology. 2016;12:1785-1795 can be referred to), that is, a general medium can be used (refer to the description of the medium in the first aspect), and for example, it can be cultured under the environment of 37°C and 5% CO2.

[0069] Examples of other components that can be added to the medium include serum (such as fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (such as Knockout serum replacement (KSR)), antibiotics (such as penicillin, streptomycin, etc.), supplements (such as ITS-G supplement), L-glutamine, magnesium L-ascorbic acid phosphate salt n-hydrate, non-essential amino acids (NEAA), 2-mercaptoethanol, and Chemically Defined Lipid Concentrate.

[0070] In a preferred embodiment, the above steps are carried out under Xeno-free conditions. When culturing under Xeno-free conditions, usually, a serum-free medium is employed, and preferably, KnockOut TM SR XenoFree Medium, hormones (such as human recombinant hydrocortisone), ITS-G, Chemically Defined Lipid Concentrate, etc. are added.

[0071] Subculture as needed. Usually, subculture is performed when the cells reach a confluent or sub-confluent state to promote further growth and maintenance of the cells. The operations for subculture are the same as those in the first aspect, and the detailed description thereof is omitted.

[0072] The vascular endothelial progenitor cells to be subjected to the expansion culture method of the present invention are not particularly limited. In addition to vascular endothelial progenitor cells obtained by inducing differentiation of pluripotent stem cells, vascular endothelial progenitor cells previously collected from a living body or cells obtained by maintaining or proliferating them in vitro (cells immediately after collection, cells stored after collection, cells maintained in culture after collection, cells cultured and proliferated after collection, etc.) can be subjected to the expansion culture method of the present invention.

[0073] The differentiation induction method when using vascular endothelial progenitor cells obtained by inducing differentiation of pluripotent stem cells is not particularly limited. Therefore, not only the previously reported differentiation induction methods (for example, see the reports such as Lian X. et al, Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. Stem cell reports. 2014;3(5):804-16. and Mien T.X. et al. Differentiation of Human Embryonic stem cells to endothelial progenitor cells on laminins in defined and xeno-free systems. Stem cell reports, 2016;7:802-16.), but also vascular endothelial progenitor cells prepared by differentiation induction methods to be developed in the future can be subjected to the expansion culture method of the present invention. The "pluripotent stem cells" are as defined in the description of the preparation method of the present invention above, and embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), etc. correspond to pluripotent stem cells. As the pluripotent stem cells, preferably ES cells or iPS cells are used, and more preferably iPS cells (particularly preferably human iPS cells) are used.

[0074] As a preferred embodiment, the expansion culture method of the present invention is applied to the vascular endothelial progenitor cells obtained by the preparation method of the present invention. That is, highly purified pluripotent stem cell-derived vascular endothelial progenitor cells prepared through a characteristic purification process are expanded in culture to obtain highly purified vascular endothelial progenitor cells suitable for various uses (particularly medical uses).

[0075] When using vascular endothelial progenitor cells pre-collected from a living body, the living body is typically a human. For example, vascular endothelial progenitor cells derived from patients with diseases where a therapeutic effect is expected by transplantation / transfer of vascular endothelial progenitor cells, such as coronary artery disease and lower limb ischemia diseases (such as Buerger's disease and atherosclerotic obliterans), can be subjected to the expansion culture method of the present invention.

[0076] The cells after expansion culture, similar to the cells obtained by the preparation method of the present invention, can be used for various purposes (refer to the column of item 2 above). If not used immediately, they can be stored. The description of the cell storage method in the first aspect is incorporated herein by reference.

Examples

[0077] Aiming to develop a method for purifying vascular endothelial progenitor cells induced to differentiate from pluripotent stem cells, the following experiments were conducted. Also, studies were carried out on the expansion culture of vascular endothelial progenitor cells.

[0078] 1. Method (1) Coating of the culture surface Gelatin: A 0.1% gelatin solution was allowed to stand at 37°C for 1 hour or at 4°C overnight. Vitronectin-N (VTN-N): Diluted with D-PBS (-) to a concentration of 1 μg / cm 2 and allowed to stand at 37°C for 1 to 2 hours. Fibronectin: Diluted with D-PBS (-) to a concentration of 1 μg / cm 2 and allowed to stand at 37°C for 1 hour or at 4°C overnight.

[0079] (2) Culture of human body-derived cells Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from ScienCell and cultured using ECM medium (ScienCell). Passage was performed once every three days, and medium change was performed on the second day after passage. In addition, fibronectin-coated culture dishes were used for culturing. The cell seeding density was 5,000 cells / cm 2 2.

[0080] (3) Culture of human iPS cells Experiments were performed using the 610B1 strain (established at Kyoto University), which was established by introducing five factors (Oct3 / 4, Sox2, Klf4, L-Myc, Lin28) into human umbilical cord blood using an episomal vector (pCXLE). The 610B1 strain was cultured at 37°C in a CO2 incubator under 5% CO2 / 95% air conditions using Essential 8 flex medium (Gibco). For the detachment solution during passage of the 610B1 strain, 0.5 M EDTA (pH 8.0) or TrypLE TM Select (Gibco) was used. When TrypLE TM Select was used, 10 μM Y-27632 was added immediately after passage and the medium was changed the next day. In addition, culturing was performed on culture dishes coated with VTN-N.

[0081] (4) Differentiation of human iPS cells into vascular endothelial progenitor cells The cells were seeded at 2×10 4 cells / cm 2Cells were seeded on VTN-N coated culture dishes at a density of TM and cultured in Essential 8 Flex medium for 24 hours. Then, they were cultured for 1 day in a medium supplemented with 5 μM CHIR99021 in modified DMEM / F12 (450 mmol / L monothioglycerol, 50 μg / mL L-ascorbic acid phosphate magnesium salt n-hydrate, 1 / 10 ITS, 2 mM Gluta-MAX, 0.1% chemically defined concentrate, 1% penicillin-streptomycin). Subsequently, they were cultured for 1 day in a medium supplemented with 50 ng / mL bFGF in modified DMEM / F12. Then, they were cultured for 3 days in a medium supplemented with 50 ng / mL VEGF and 25 ng / mL BMP4 in modified DMEM / F12. During this period, the medium was changed daily. On the 5th day, cells treated with 10 μM Y-27632 for 1 hour were 4 dissociated with TrypL 2 E Select (Gibco) and passaged into new gelatin-coated culture dishes at 3.5×10

[0082] (5) Purification of EPCs Cells on the 8th day of differentiation treated with 10 μM Y-27632 for 1 hour were TrypLE TMTreat with Select (Gibco) for 45 - 60 seconds to remove some of the cells other than EPCs (Extra cells). Tap the culture dish several times (tapping) to completely remove the Extra cells. At this point, if the cells at the edge of the culture dish are difficult to remove, aspirate the cells at the edge of the culture dish in a circular motion. Then wash with PBS three or more times and then TrypLE again TM After treating with Select for 6 - 12 minutes to detach EPCs, collect them in a centrifuge tube, and after centrifugation (1000 rpm, 5 min), reseed them in a new culture dish.

[0083] (6) Culture of differentiated cells The purified cells were cultured in modified Human endothelial SFM medium supplemented with 10 ng / mL of EGF and 20 ng / mL of bFGF, with DMSO (DMSO group) or 10 μM of Y - 27632, 0.5 μM of A 83 - 01, and 3 μM of CHIR99021 (YAC group). For culture under xeno - free conditions, a medium obtained by adding DMSO or YAC to modified medium 200 supplemented with 2.5 μM of hydrocortisone, 10 ng / mL of EGF, and 20 ng / mL of bFGF was used. Medium exchange was performed on the 1st and 3rd days after seeding. Sub - culture was performed at intervals of 3 - 5 days. At the time of sub - culture, after washing once with PBS, TrypLE was used at 37°C TM Treat with Select for 5 minutes or more to detach the cells. Then, collect the cells in a 15 mL tube or 50 mL tube with medium and centrifuge at 1,000 rpm for 5 minutes. Then, resuspend with medium and reseed at 1.5×10 4 cells / cm 2 onto a culture dish coated with VTN - N. The cell count was performed by trypan blue staining and automatically measured with Countess II (Invitrogen).

[0084] (7) Cell Titer Glo 2.0 assay The Cell Titer Glo 2.0 assay was performed according to the enclosed manual. Luminescence was measured using Synergy HTX Gen5 with the Gain set to auto. For the measurement, cells on day 11 of differentiation were seeded at 0.5×10 3 cells / cm 2 in a 48-well plate and cultured for 3 days.

[0085] (8) Angiogenesis assay (tube formation assay) Endothelial cells selectively obtained on day 8 of differentiation were seeded at 7.5×10 4 cells (under normal conditions) or 1×10 5 cells (under Xeno-free conditions) in a 24-well plate coated with 300 μL of Matrigel. As the medium, modified Human Endothelial SFM supplemented with 50 ng / mL of VEGF, 10 ng / mL of EGF, and 20 ng / mL of bFGF (under normal conditions) or modified medium 200 supplemented with 2.5 μM of Hydrocortisone, 50 ng / mL of VEGF, 10 ng / mL of EGF, and 20 ng / mL of bFGF (under Xeno-free conditions) was used. After 20 hours, the cells were reacted with Calcein-AM (a live cell observation reagent) at a final concentration of 2 μM for 30 minutes, and the cell morphology was observed under a microscope. Cells after day 8 were seeded at 0.5×10 5 to 1×10 5 cells under the same conditions as above. For the 20-hour culture, the medium used for maintenance culture until just before (containing growth factors and small molecule compounds) with 50 ng / mL of VEGF newly added was used under each condition.

[0086] (9) Immunostaining method Cells on a 96-well plate were fixed in 4% paraformaldehyde at room temperature for 15 minutes, washed twice with PBS containing glycine, and then permeabilized with PBS containing 0.1% Triton X-100 at room temperature for 25 minutes. After blocking with 5% donkey serum at room temperature for 20 minutes, the primary antibody was reacted at room temperature for 2 hours. After washing three times with PBS, the secondary antibody was reacted at a 200-fold dilution at room temperature for 60 minutes. At this time, DAPI, a staining reagent, was also reacted simultaneously (final concentration 1 μg / mL). Then, it was washed three times with PBS and analyzed using an Opera High Content Imaging System (PerkinElmer). The ratio of CD31- and CD144-positive cells was automatically calculated by the Opera analysis system as the number of cells with a certain level of brightness or higher / total number of cells. The average number of positive cells in 6 fields of view in 1 well was calculated, and this was done for 3 wells (n = 3).

[0087] (10) Acetylated LDL uptake assay On day 10 of differentiation, cells were reacted with acetylated LDL labeled with Dil at a final concentration of 10 μg / mL for 5 hours, then reacted with Hoechist 33342 for 30 minutes, washed four times with the medium, and analyzed using an Opera High Content Imaging System. The ratio of acetylated LDL-positive cells was automatically calculated by the Opera analysis system as the number of cells with a certain level of brightness or higher / total number of cells. The average number of positive cells in 6 fields of view in 1 well was calculated, and this was done for 3 wells (n = 3).

[0088] (11) Detection of β-galactosidase β-galactosidase was detected using the Cellular Senescence Detection Kit - SPiDER-βGal (Dojindo) according to the enclosed manual. Cells were seeded in a 96-well plate and cultured for 3 days. After washing once with the medium, the cells were reacted for 1 hour in the medium (50 μl) in which Barifomycin A1 working solution was diluted 1000-fold. Then, the medium (50 μl) supplemented with SPiDER β-Gal working solution (1 / 1000 volume) and Hoechist 33342 (final concentration 10 μg / mL) was added to each well and cultured for 30 minutes. After that, the cells were washed twice with the medium and analyzed using an Opera High-Content Imaging System. The ratio of β-galactosidase-positive cells was automatically calculated by the analysis system of the Opera as the number of cells with luminance above a certain level / the total number of cells. The average number of positive cells in 6 fields of view in one well was calculated, and this was done for 3 wells (n = 3).

[0089] (12) RNA extraction Agencourt (registered trademark) RNAdvance TM Extraction was performed according to the enclosed manual of the Tissue Kit.

[0090] (13) Reverse transcription reaction Complementary DNA (cDNA) was synthesized using ReverTra Ace (registered trademark) qPCR RT Master Mix according to the enclosed manual.

[0091] (14) RT-qPCR method RT-qPCR was performed using the KAPA SYBR Fast qPCR Kit with cDNA as the template, and the reaction was carried out according to the enclosed manual. The results were corrected using HPRT as the endogenous control

[0092] The characteristics of the marker genes used in this study are shown below. PECAM1 (CD31): A representative vascular endothelial cell marker CD34: A marker for hematopoietic cells and vascular endothelial progenitor cells CDH5 (VE-Cadherin): An adhesion molecule belonging to the cadherin family and expressed in vascular endothelial cells vWF: A representative vascular endothelial cell marker FLK1: A representative mesoderm-vascular endothelial cell marker OCT4: A representative undifferentiated marker BRACHYURY: A representative mesoderm marker

[0093] (15) Measurement of purity by flow cytometry A 15 mL tube containing the medium in which purified EPCs (on the 14th day of differentiation) were suspended was centrifuged at 100×g for 5 minutes, and the medium was aspirated. Then, it was fixed with 1 mL of 4% paraformaldehyde for 10 minutes. Centrifuged at 100×g for 5 minutes, the 4% paraformaldehyde was aspirated, and permeabilization treatment was performed at room temperature for 10 minutes using chilled methanol. The fixed cells were centrifuged at 100×g for 5 minutes, resuspended in 5 mL of Flow Cytometry Staining Buffer, and centrifuged at 100×g for 5 minutes. After aspirating the Flow Cytometry Staining Buffer, a solution prepared by adding FITC-PECAM1 (10-fold dilution) and PE-CD34 (10-fold dilution) to 50 μL of Flow Cytometry Staining Buffer was added, transferred to a 1.5 mL tube, and rotated at 4°C for 1 hour. Then, it was centrifuged at 100×g at 4°C for 5 minutes, washed once with Flow Cytometry Staining Buffer, suspended in 500 μL of Flow Cytometry Staining Buffer, and analyzed using CytoFLEX (Beckman Coulter, Inc.). In addition, the following antibodies were used. PE-CD34 antibody (Beckman Coulter, Inc. IM1459U) FITC-PECAM1 (Beckman Coulter, Inc. IM1431U)

[0094] (16) Freezing and thawing of cells Freezing of differentiated cells was performed using TC Protector, CELLBANKER, STEM-CELLBANKER (containing DMSO) or Cell Reservoir One (containing DMSO). After resuspension with each cryopreservative solution, cells were frozen in a -80°C deep freezer for 1 hour. During thawing, cells were semi-thawed in a 37°C water bath. Then, 1 mL was taken from 10 mL of medium in a pre-warmed 15 mL tube or 50 mL tube and poured into the semi-thawed cell suspension. After complete thawing, the suspension was returned to the tube and centrifuged at 100×g for 5 minutes. Then, the medium was aspirated and the cells were resuspended in fresh medium for subsequent experiments. When seeding thawed cells, 4 cells / cm 2 were seeded at a density of 2×10

[0095] 2. Results and Discussion (1) Induction of differentiation into EPCs First, based on previous reports (Sriram G. et al, Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions, Stem Cell Research & Therapy. 2015;6:261), cells were treated with 5 μM CHIR99021 until day 1 and 50 ng / ml bFGF until day 2 to induce differentiation into mesoderm. From day 2 to day 5 of differentiation, cells were directed towards EPCs with 25 ng / mL BMP4 and 50 ng / mL VEGF (Figure 1A). Modified DMEM / F12 was used as the medium.

[0096] Thereafter, from day 5 to day 8 of differentiation, the cells were induced to further differentiate into mature EPCs in modified human endothelial SFM supplemented with 10 ng / mL of bFGF and 50 ng / mL of VEGF. When the gene expression levels of cells before differentiation (iPSCs), on day 2, day 5, and day 8 of differentiation were analyzed by RT-qPCR, it was found that OCT4, an undifferentiated marker, decreased over time, and BRACHYURY, a mesoderm marker, peaked on day 2 of differentiation. In addition, CD34, a vascular endothelial progenitor cell marker, peaked on day 5, and PECAM1 (CD31), a mature vascular endothelial cell marker, increased significantly on day 5 and day 8. Thus, it was confirmed that the induction of differentiation into EPCs proceeded according to this protocol (Figure 1B).

[0097] Two populations with clearly different morphologies were observed on day 8 of differentiation. One was spindle-shaped cells, which are typical features of endothelial cells, and the other type of cells (Extra cells) had a clearly different morphology from them (Figure 1C). We developed a method of first detaching only the Extra cells and then detaching and subculturing the EPCs by taking advantage of the difference in adhesion ability between the two cell populations (Figure 1C, Figure 2A).

[0098] (2) Purification of vascular endothelial progenitor cells Treat with TrypLE TM Select at 37°C for 45 - 60 seconds. When the extra cells were detached, the culture dish was tapped several times to completely detach the extra cells. Then, it was washed with PBS three or more times, and the adherent spindle-shaped cells were detached again with TrypLE TM Select and seeded into a new culture dish (Figure 2A).

[0099] Subsequently, the protein expression of cells on the 8th and 10th days of differentiation (Non Purified EPCs: NP-EPCs) and purified cells (Purified EPCs: P-EPCs) was examined by immunostaining. The localization of CD31 and VE-Cadherin on the cell membrane was confirmed. Furthermore, the expression of CD34, a marker for vascular endothelial progenitor cells, was also confirmed. In addition, there were cells (Extra cells) in NP-EPCs that did not express CD31, VE-Cadherin, or CD34. However, since all the cells after selection expressed vascular endothelial (progenitor) cell markers, it was confirmed that P-EPCs had a higher purity compared to NP-EPCs (Figure 2C). Subsequently, to actually confirm the degree of purification, the number of positive cells for CD31, VE-Cadherin, and CD34 was calculated using the expression of CD31, CD144, and CD34 as indicators. As a result, P-EPCs showed significantly higher values compared to NP-EPCs. The purity of P-EPCs was estimated to be 90% or higher. On the other hand, as a result of confirming the purity of purified cells (P-EPCs) on the 14th day of differentiation by flow cytometry, the purity was 98.45%. That is, cells of extremely high purity were obtained (Figure 7).

[0100] Subsequently, to confirm whether P-EPCs have the function of vascular endothelial cells, an acetylated LDL uptake test was conducted. As a result, most of the cells retained the ability to take up acetylated LDL (Figure 2E). Subsequently, a tube formation assay using Matrigel was performed, and vascular-like structures were observed (Figure 2E). From the above, it was confirmed that P-EPCs have the function of vascular endothelial cells.

[0101] On the other hand, when protein expression was compared between the cases where EPCs differentiated from other iPS cell lines (606A1 and 648A1) were purified (P) and not purified (NP) using the same method, it was confirmed that highly pure cells were obtained by purification (Figure 8A), which verified the high versatility. Also, EPCs derived from the 606A1 cell line and those from the 648A1 cell line retained the ability to take up acetylated LDL (Figure 8B), and in the tube formation assay, they showed the ability to form vascular-like structures (Figure 8C), demonstrating the function as vascular endothelial cells.

[0102] (3) Generation of highly pure EPCs under xeno-free conditions Subsequently, since vascular endothelial progenitor cells are expected to be applied to regenerative medicine, induction of differentiation was attempted under conditions free of components derived from heterologous sources (xeno-free). Specifically, the medium used for culturing from day 5 to day 8 was changed from modified human endothelial SFM to modified Medium 200 with hydrocortisone added, and the coating agent was changed from gelatin to VTN-N (Figure 3A). Under these conditions, XF-EPCs that underwent purification on day 8 of differentiation had almost the same expression levels of vascular endothelial cell-related gene groups as those in the normal protocol (Figure 3B). Furthermore, the expression of CD31, CD144, and CD34 was confirmed, and it was found that the purity was also as high as that in the normal protocol (Figure 3C). Also, they had the ability to take up acetylated LDL (Figure 3D), which is one of the functions of vascular endothelial cells, and the ability to form tubes (Figure 3E). From the above, it was shown that differentiation into EPCs is possible even under xeno-free conditions, and the purification method using the difference in cell-cell adhesion ability is applicable.

[0103] (4) Establishment of an expansion culture method for iPS cell-derived EPCs It was found that there is room for improvement in the proliferation ability of the prepared iPS cell-derived EPCs. Therefore, in order to increase the proliferation ability of EPCs, we focused on low-molecular-weight compounds that are inexpensive and easy to handle, and explored an effective method for improving the EPC proliferation ability. We focused on a combination of three low-molecular-weight compounds (Y-27632, a ROCK inhibitor; CHIR99021, a GSK3β inhibitor; A83-01, a TGFβ inhibitor) that confer undifferentiated properties and increase the proliferation ability of mouse hepatocytes. Y-27632 has the function of increasing the proliferation ability of iPS cell-derived ECs, and there is a report that SB43152, a similar compound to A-8301 among TGFβ inhibitors, increases the proliferation ability of ES cell-derived ECs. Furthermore, there is a report that CHIR99021 increases the proliferation ability of HUVECs. Therefore, we examined all combinations of single compound groups, two-compound groups, and three-compound groups (YAC group). As a result, the EPCs in the YAC group showed the highest proliferation ability (Figure 4A). Also, when the DMSO group and the YAC group were passaged, it was confirmed that the YAC group could be passaged while maintaining the proliferation ability for a longer time, and moreover, the phenotype as a vascular endothelial progenitor cell was maintained (Figures 4B and 4C). Also, it was confirmed that there was less cell senescence in the YAC group compared to the DMSO group (Figure 4D). Furthermore, the YAC group (on the 39th day of differentiation) highly expressed the vascular endothelial progenitor cell marker CD34 compared to HUVEC (Figure 9).

[0104] (5) Effect of the combination of Y-27632, A83-01, and CHIR99021 under xeno-free conditions When purified XF-EPCs on the 8th day of differentiation were cultured for 3 days in the absence (DMSO group) or presence (YAC group) of YAC, the number of cells in the YAC group was clearly increased compared to the DMSO group (Figure 5). That is, the cell proliferation effect of YAC was obtained even under xeno-free conditions.

[0105] (6) Examination of the cell proliferation rate when Y-27632, A83-01, and CHIR99021 are removed from the medium When YAC was removed from the medium, the enhanced proliferative ability decreased again (Figure 6). Thus, it was confirmed that EPCs stimulated by YAC were not cells that had acquired the autonomous proliferative ability, which is a characteristic seen in cancer cells. Therefore, it is considered that EPCs proliferated by YAC can be used for regenerative medicine and the like.

[0106] (7) Effects of freeze-thaw on EPCs Cells on day 11 of differentiation were subjected to freeze-thaw treatment, and the effects of freeze-thaw on the cells were examined (Figure 10A). Cells subjected to freeze-thaw treatment (frozen cells) and cells not subjected to freezing treatment (non-frozen cells) showed equivalent survival rates (Figure 10B), and no significant difference was observed in the expression of each marker between the two (Figure 10C). Also, from the results of immunostaining (Figures 11A, B), acetylated LDL uptake test (Figure 11C), and tube formation assay results (Figure 11D), it was shown that frozen cells and non-frozen cells had equivalent functions as vascular endothelial cells.

[0107] 3. Conclusions In this study, by utilizing the difference in the adhesion ability between human iPS cell-derived EPCs and other cells at the late stage of differentiation, high-purity EPCs could be simply and non-damagingly isolated without using a cell sorter or magnetic beads. Furthermore, by combining and adding multiple low-molecular-weight compounds, the proliferative ability of EPCs was successfully increased dramatically. Also, the obtained EPCs maintained their functions even after freeze-thaw treatment. This fact indicates that cells can be cryopreserved while maintaining their characteristics, which has extremely great practical significance. This result is expected to greatly contribute to the stable supply of human iPS cell-derived vascular endothelial progenitor cells for various applications such as regenerative medicine.

Industrial Applicability

[0108] The preparation method of the present invention enables the preparation of highly pure vascular endothelial progenitor cells despite the simple operation. The highly pure vascular endothelial progenitor cells are useful for constructing drug screening models and clinical applications (such as the treatment of coronary artery disease and lower limb ischemia diseases, and the in vitro construction of blood vessels for regenerative medicine, etc.). The present invention is expected to be applied to and used for these purposes. On the other hand, the expansion culture method of the present invention enables the preparation of a large amount of vascular endothelial progenitor cells, and for example, contributes to the stable supply of vascular endothelial progenitor cells. Both the preparation method and the expansion culture method of the present invention are suitable for Xeno-free, and particularly great contributions to the clinical applications (such as regenerative medicine) of vascular endothelial progenitor cells are expected.

[0109] This invention is not limited to the descriptions of the embodiments and examples of the above invention. Various modifications are also included in this invention within the scope that can be easily conceived by those skilled in the art without departing from the description of the claims. The contents of the papers, published patent gazettes, and patent gazettes explicitly stated in this specification shall be cited by incorporating all of their contents.

Claims

1. A method for expanding vascular endothelial progenitor cells, comprising a step of culturing vascular endothelial progenitor cells in the presence of a ROCK inhibitor, a GSK-3β inhibitor and a TGF-β receptor inhibitor in addition to basic fibroblast growth factor and epidermal growth factor.

2. The expansion culture method according to claim 1, wherein the ROCK inhibitor is Y-27632, the GSK-3β inhibitor is CHIR 99021, and the TGF-β receptor inhibitor is A 83-01.

3. The expansion culture method according to claim 1 or 2, wherein the vascular endothelial precursor cells are cells obtained by inducing differentiation of pluripotent stem cells.

4. The expansion culture method according to claim 3 , wherein the pluripotent stem cells are induced pluripotent stem cells.

5. The expansion culture method according to claim 4, wherein the induced pluripotent stem cells are human induced pluripotent stem cells.

6. The expansion culture method according to claim 1 or 2, wherein the vascular endothelial precursor cells are cells prepared by a preparation method comprising the following steps (1) and (2): (1) differentiating pluripotent stem cells into vascular endothelial progenitor cells; (2) A step of purifying vascular endothelial precursor cells by utilizing the difference in adhesive ability between the vascular endothelial precursor cells and other cells that constitute the cell population obtained in step (1).

7. The expansion culture method according to claim 1 or 2, wherein the vascular endothelial precursor cells are vascular endothelial precursor cells previously collected from a living body or cells obtained by maintaining or growing the vascular endothelial precursor cells in vitro.

8. The method for expansion according to any one of claims 1 to 7, wherein the process is carried out under conditions free of components derived from xenogeneic animals.

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