Method for separating and culturing novel stem cells, isolated novel stem cell, mesenchymal stem cell bank, and method for providing mesenchymal stem cell information

Abstract

The present invention addresses the problem of providing an MSC which is HLA-ABC negative and HLA-G positive.　The problem can be solved by a mesenchymal stem cell isolated from a placenta, said mesenchymal stem cell being HLA-ABC negative and HLA-G positive.

Description

Novel stem cell isolation and culture method, isolated novel stem cells, mesenchymal stem cell bank, and method for providing mesenchymal stem cell information

[0001] The disclosure in this application relates to a novel method for isolating and culturing stem cells, the isolated novel stem cells, a mesenchymal stem cell bank, and a method for providing information on mesenchymal stem cells.

[0002] Mesenchymal stem cells (hereinafter sometimes referred to as "MSCs") are one type of tissue stem cell present in the adult human body. MSCs are present in various locations, such as bone marrow, fat, and the dermis of the skin. The properties of MSCs vary slightly depending on the location. MSCs have the ability to differentiate into tissues (bone, cartilage, blood vessels, muscle, fat, fibroblasts, etc.) classified as mesoderm, which is formed between the ectoderm (epidermis and its derived tissues and nervous system) and the endoderm (digestive tract and its derived tissues) in embryological classification, but can also transdifferentiate into ectoderm (neuronal cells) and endoderm (hepatocytes).

[0003] It has become clear that MSCs have important roles to play in terms of their interferon-γ responsiveness and ability to produce nitric oxide, and that components of proteins (secretome) secreted by MSCs, such as chemokines, have an inhibitory effect on the immunoglobulin (antibody) production of plasma cells. For this reason, MSCs are thought to have immune-regulating capabilities, and even though they are derived from other people, they are said to have a weaker rejection reaction than other cells.

[0004] MSCs are known to be derived from a patient's own adipose tissue, bone marrow, and umbilical cord blood. While using autologous cells is desirable to reduce the risk of rejection, there are issues with the invasiveness of their collection. Bone marrow collection is particularly invasive compared to fat. On the other hand, umbilical cord blood contains many immature, rapidly proliferating cells. Fetal immunity (both innate and adaptive) is immature at birth, and it is said that innate immunity begins to develop around 18 months of age. Umbilical cord blood collected immediately after birth is fetal blood, but because the fetus receives antibodies from the mother through the placenta, antibodies are also present in umbilical cord blood.

[0005] The placenta is a cellular tissue that originally exists as a site of immune tolerance between mother and child, and is composed of specialized somatic cells. Therefore, even though mesenchymal stem cells derived from placental tissue are not autologous, they are expected to be used in regenerative medicine as safe transplant cells and tissues that can avoid immune rejection.

[0006] As an example of mesenchymal stem cells derived from placental tissue, Non-Patent Document 1 describes that mesenchymal stem cells can be obtained from the tissues of the chorionic plate plus amnion (CP), chorionic villi (CV), and decidua (DB) of the placenta in the late stages of pregnancy.

[0007] Fuchi N et al. , “Feasibility of placenta-derived mesenchymal stem cells as a tool for studying pregnancy-related disorders”, Sci. Rep. 2017 Apr 12;7:46220. doi:10.1038 / srep46220. Erratum in: Sci Rep. 2017 Dec 22;7:46922. PMID: 28401946; PMCID: PMC5388876.

[0008] As mentioned above, Non-Patent Document 1 describes that MSCs can be obtained from placental tissue. Incidentally, HLA (Human Leukocyte Antigen) is thought to be one of the factors that cause immune rejection during MSC transplantation. Known types of HLA include HLA-Class I (ABC) and HLA-G.

[0009] HLA-ABC is a transmembrane protein expressed on most nucleated cells and platelets, and is known to be an important immune mechanism involved in distinguishing between self and non-self. In other words, strong expression of HLA-ABC increases the risk of rejection during MSC transplantation. Therefore, it is preferable that MSCs used for transplantation have low HLA-ABC expression.

[0010] HLA-G binds to the NK cell inhibitory receptor KIR2DL4, helping to avoid attacks from NK cells. In other words, high expression of HLA-G is known to promote immune tolerance.

[0011] Therefore, when using MSCs for the purpose of regenerative medicine, it is considered preferable that the expression of HLA-ABC, which is involved in the discrimination between self and non-self, is low and that the expression of HLA-G is high.

[0012] However, the HLA-ABC and HLA-G described in Non-Patent Document 1 are positive and negative, respectively, which are the exact opposite of the characteristics required for MSCs used in regenerative medicine. Therefore, it would be desirable to obtain placenta-derived MSCs that are negative for HLA-ABC and positive for HLA-G, but currently, MSCs with such characteristics have not been obtained, and no method for isolating and culturing them from placental tissue is known. Furthermore, no cell bank using MSCs with the above characteristics, nor a method for providing information on MSCs stored in a cell bank, is known.

[0013] The present application has been made to solve the above-mentioned problems. As a result of extensive research, the present inventors have newly discovered that when cells collected from placental tissue are cultured in a serum-free medium, HLA-ABC-negative and HLA-G-positive MSCs can be obtained.

[0014] That is, the purpose of the disclosure of this application is to provide a novel method for isolating and culturing stem cells, and the isolated novel stem cells, and more specifically, to provide MSCs isolated from an HLA-ABC-negative and HLA-G-positive placenta, a method for isolating and culturing mesenchymal stem cells from a placenta, a method for purifying and concentrating MSCs having the characteristics disclosed in this application from cells cultured by the isolation and culture method, and a method for isolating MSCs having the characteristics disclosed in this application.Furthermore, the purpose of the disclosure of this application is to provide a cell bank using MSCs having the above-mentioned characteristics, and a method for providing information on MSCs stored in a cell bank.

[0015] The disclosures in this application relate to mesenchymal stem cells isolated from placenta, a method for isolating and culturing mesenchymal stem cells, a method for purifying and concentrating mesenchymal stem cells, a method for isolating mesenchymal stem cells, a mesenchymal stem cell bank, and a method for providing information on mesenchymal stem cells, as shown below.

[0016] (1) Mesenchymal stem cells isolated from a placenta, wherein the mesenchymal stem cells are HLA-ABC negative and HLA-G positive. (2) The mesenchymal stem cells according to (1) above, wherein the mesenchymal stem cells are isolated from placental tissue excluding the amniotic membrane. (3) The mesenchymal stem cells according to (1) above, wherein the mesenchymal stem cells have been cultured using a serum-free medium. (4) The mesenchymal stem cells according to (2) above, wherein the mesenchymal stem cells have been cultured using a serum-free medium. (5) The mesenchymal stem cells according to any one of (1) to (4) above, wherein the placenta is a placenta from after 16 weeks of pregnancy to full term. (6) A method for isolating and culturing HLA-ABC-negative and HLA-G-positive mesenchymal stem cells from a placenta, the method comprising: an enzyme treatment step of shredding placental tissue collected from the placenta and treating it with an enzyme; a first culture step of culturing placental tissue-derived cells obtained by the enzyme treatment in a serum-free medium; and a second culture step of selecting mesenchymal stem cells from the cells cultured in the first culture step and culturing the selected mesenchymal stem cells in a serum-free medium. (7) The method of (6) above, wherein the placenta is placental tissue excluding the amniotic membrane. (8) The method of (6) or (7) above, wherein the placenta is a placenta from after 16 weeks of pregnancy to full term. (9) A method for purifying and concentrating mesenchymal stem cells that are HLA-ABC negative and HLA-G positive, the method comprising a selection step of using an HLA-ABC antibody to select mesenchymal stem cells that are HLA-ABC negative and HLA-G positive from mesenchymal stem cells cultured by the isolation and culture method described in any one of (6) to (8) above, and selecting mesenchymal stem cells that are mesenchymal stem cell marker positive using a mesenchymal stem cell marker antibody. (10) A method for isolating mesenchymal stem cells that are HLA-ABC negative and HLA-G positive, the method comprising an isolation step of isolating the mesenchymal stem cells after carrying out the purification and concentration method described in (9) above.(11) A mesenchymal stem cell bank containing the mesenchymal stem cells according to any one of (1) to (5) above, wherein the mesenchymal stem cell bank contains two or more types of mesenchymal stem cells that differ in at least one of HLA typing, that is, HLA-A locus, HLA-B locus, and HLA-DR locus. (12) The mesenchymal stem cell bank contains at least one HLA-A locus typing selected from the group consisting of A*24:02, A*02:01, A*11:01, A*26:01, A*33:03, and A*31:01, at least one HLA-B locus typing selected from the group consisting of B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01, and B*07:02, and The mesenchymal stem cell bank according to (11) above, which contains mesenchymal stem cells of at least one typing selected from the group consisting of HLA-DR locus typings DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03 and DRB1*01:01. (13) The mesenchymal stem cell bank according to (11) or (12) above, wherein the mesenchymal stem cell bank contains mesenchymal stem cells having at least one haplotype selected from the group consisting of: A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:05.(14) A mesenchymal stem cell information providing method for providing information on mesenchymal stem cells stored in the mesenchymal stem cell bank according to any one of (11) to (13) above, wherein each mesenchymal stem cell constituting the mesenchymal stem cell bank is stored in association with typing information on at least the HLA-A locus, HLA-B locus, and HLA-DR locus among HLA typing, and the mesenchymal stem cell information providing method comprises: an HLA typing information input step for inputting at least typing information on the HLA-A locus, HLA-B locus, and HLA-DR locus of a subject; and based on the input HLA typing information of the subject, among the stored mesenchymal stem cells, mesenchymal stem cell information with matching haplotypes, mesenchymal stem cell information with matching two of the typing information on the HLA-A locus, HLA-B locus, and HLA-DR locus, and and mesenchymal stem cell information that matches one of the typing information for the HLA-A locus, the HLA-B locus, and the HLA-DR locus.

[0017] By using the method for isolating and culturing mesenchymal stem cells disclosed in the present application, mesenchymal stem cells that are HLA-ABC negative and HLA-G positive can be obtained.

[0018] FIG. 1 is a schematic diagram of a placenta. FIG. 2A is a photograph, substituted for a drawing, of a dish on day 5 after the start of cell culture in the isolation culture of Example 1. The scale bar is 200 μm. FIG. 2B is a photograph, substituted for a drawing, showing a phase-contrast image and an immunostained photograph of cells on day 14 of culture in the isolation culture of Example 1. FIG. 3 is a photograph, substituted for a drawing, showing a phase-contrast image and an immunostained photograph of MSCs after the second culture step in Example 1. The scale bar is 200 μm. FIG. 4 is a photograph, substituted for a drawing, showing a phase-contrast image and an immunostained photograph using a different antibody from that in FIG. 3. The scale bar is 200 μm. FIG. 5 is a graph showing the results of FCM analysis of cells on day 14 of culture after the second culture step in Example 1. FIG. 6 is a graph showing the results of FCM analysis of cells on day 14 of culture after the second culture step in Example 1 (the labeled antibody is different from that in FIG. 5). Figure 7 is a graph showing the results of FCM analysis after carrying out the purification and concentration method of Example 2. Figure 8 is a photograph, substituted for a drawing, showing a phase contrast image photograph and an immunostained photograph of cells isolated and cultured in Comparative Example 1. The scale bars are all 200 μm. Figure 9 is a graph showing the results of FCM analysis of cells isolated and cultured in Comparative Example 1. Figure 10 is a graph showing the results of FCM analysis of cells isolated and cultured in Comparative Example 2. Figure 11 is a photograph, substituted for a drawing, showing a phase contrast image photograph and an immunostained photograph of MSCs cultured for five passages in Example 3. The scale bars are all 100 μm. Figure 12 is a graph showing the results of FCM analysis of MSCs cultured for five passages in Example 3.

[0019] The mesenchymal stem cells isolated from the placenta (hereinafter, mesenchymal stem cells isolated from the placenta may also be referred to simply as "MSCs"), the method for isolating and culturing mesenchymal stem cells (hereinafter, may also be referred to simply as the "isolation and culturing method"), the method for purifying and concentrating mesenchymal stem cells (hereinafter, may also be referred to simply as the "purification and concentration method"), and the method for isolating mesenchymal stem cells (hereinafter, may also be referred to simply as the "isolation method") disclosed in the present application are described in detail below.

[0020] Furthermore, in this specification, (1) a numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits, (2) numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors that are generally acceptable in the technical field, and (3) when it is written "approximately ____-shaped," it is interpreted as including not only the exact ____-shaped, but also a shape that is understood to be roughly ____-shaped.

[0021] (Embodiments of MSCs) MSCs according to the embodiments are cells isolated from the placenta, are HLA-ABC negative and HLA-G positive, and express mesenchymal stem cell markers (hereinafter referred to as "MSC markers").

[0022] FIG. 1 shows an outline of the placenta. The placenta is composed of, from the fetal side, the amnion, the chorionic hair growth portion (chorion), and the decidua basalis (decidua). The decidua is a tissue formed by transformation of the endometrium, and therefore contains many maternal cells. The MSCs according to the embodiment are relatively abundant on the chorionic side of the decidua and in the chorion, but are also present throughout the chorion. Therefore, the MSCs according to the embodiment are isolated from placental tissue excluding the amnion. Among placental tissues excluding the amnion, the chorion on the decidua side and the decidua are preferred. Furthermore, the placenta is said to be fully formed by 15 to 16 weeks of pregnancy. Therefore, the placenta used in the present application includes placentas from 16 weeks of pregnancy or later, and from the viewpoint of ease of acquisition, placentas from 28 weeks of pregnancy or later to full term are included.

[0023] As described above, HLA-ABC is known to be an important immune mechanism involved in the discrimination between self and non-self. Therefore, strong expression of HLA-ABC increases the risk of rejection during MSC transplantation. Therefore, low HLA-ABC expression is preferred for the MSCs disclosed in the present application. As used herein, "positive" means that a higher signal is detected by an analytical method such as flow cytometry, which utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. Furthermore, "negative" means that a signal equivalent to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected. When the term "negative" is used, the expression level is not zero as long as it is within the meaning of "negative" described above. In other words, some expression is not a problem, but zero expression is preferred.

[0024] As mentioned above, it is known that high expression of HLA-G promotes immune tolerance. Therefore, when using MSCs for the purpose of regenerative medicine, it is considered preferable that the expression of HLA-ABC, which is involved in the discrimination between self and non-self, is low and the expression of HLA-G is high. Therefore, the MSCs disclosed in the present application are preferably HLA-ABC negative and HLA-G positive.

[0025] Furthermore, the MSCs disclosed in the present application express MSC markers, including, but not limited to, CD29, CD71, CD90, CD105, etc. Cells expressing mesenchymal stem cell markers can also be referred to as MSCs.

[0026] Diseases that can be expected to be treated when MSCs are transplanted for therapeutic purposes include, but are not limited to, IgA nephropathy, arthritis, myocardial infarction, diabetic foot ulcers, acute graft-versus-host disease, brain injury, spinal cord injury and associated neurological symptoms and functional disorders, complex anal fistula in Crohn's disease patients, acute respiratory distress syndrome due to pneumonia, hypophosphatasia, knee osteoarthritis, non-infectious pulmonary complications after hematopoietic stem cell transplantation, meniscus injury, Duchenne muscular dystrophy, etc. Transplantation of MSCs for therapeutic purposes is not limited to humans. In this specification, the term "MSC" refers to human MSCs as well as MSCs obtained from mammals such as pets such as dogs, cats, and mice; and livestock such as cows, horses, goats, and sheep.

[0027] The MSCs disclosed in this application have the following advantages: (1) While MSCs are known to have a low risk of immune rejection, the MSCs disclosed in this application have low (negative) expression of HLA-ABC, which is involved in the differentiation of self from non-self, and high (positive) expression of HLA-G, which promotes immune tolerance. Therefore, they are particularly suitable for use in regenerative medicine, such as transplantation of MSCs from other donors (allotransplantation). (2) As shown in the examples below, the isolated MSCs disclosed in this application maintain their low (negative) expression of HLA-ABC and high (positive) expression of HLA-G, even after repeated subculture. Therefore, MSCs with consistent properties can be stably provided. (3) The isolated MSCs can be cryopreserved. Therefore, by storing the isolated MSCs, MSCs for transplantation can be easily prepared by thawing and expanding them when needed. (4) By performing HLA-ABC typing tests on isolated MSCs, it is possible to construct a cell bank of MSCs with low HLA-ABC expression. (5) As shown in Example 5 and Comparative Example 3 described below, the MSCs disclosed in the present application have the ability to secrete hepatocyte growth factor (HGF) extracellularly when cultured in serum-free medium. MSCs have a wide range of therapeutic effects, including tissue repair and immunomodulatory activity, many of which are contributed by cytokines and growth factors secreted by MSCs (paracrine effect). HGF is one of the most potent tissue regeneration-promoting factors, and has diverse biological activities, including anti-fibrosis, anti-inflammatory, angiogenesis promotion, and cell proliferation promotion. The MSCs disclosed in the present application exhibit the unexpected effect of being HLA-ABC negative and HLA-G positive, and capable of secreting HGF.

[0028] (Embodiment of Separation and Culture Method) Next, an isolation and culture method according to an embodiment will be described. The isolation and culture method according to an embodiment is a culture method for obtaining the above-mentioned MSCs from a placenta. The isolation and culture method includes an enzyme treatment step, a first culture step, and a second culture step.

[0029] In the enzyme treatment step, placental tissue collected from the placenta may be cut into pieces, for example, 5 mm or less. The enzymes used in the enzyme treatment step are not particularly limited as long as they are enzymes commonly used in the field of cell culture. Examples include, but are not limited to, collagenase, trypsin, papain, dispase, etc. As mentioned above, placental tissues include placental tissues excluding the amnion, with the chorion and decidua on the decidual side being preferred. Placenta includes placentas from 16 weeks of pregnancy to full term.

[0030] The first culture step is a step of culturing placental tissue-derived cells obtained by enzyme treatment in a serum-free medium. As shown in the Examples and Comparative Examples described below, in conventional methods such as those described in Non-Patent Document 1, placental tissue-derived cells obtained by enzyme treatment are cultured in a serum-containing medium. In this case, the MSCs are HLA-ABC positive and HLA-G negative, which are the exact opposite of the MSC characteristics required for regenerative medicine. On the other hand, the isolation and culture method disclosed in the present application is an invention that newly discovered that by culturing cells in a serum-free medium, the characteristics of low HLA-ABC expression (negative) and high HLA-G expression (positive) can be obtained.

[0031] The medium is not particularly limited as long as it does not contain serum. As the medium, a medium commonly used for cell culture can be used as long as it does not contain serum. Examples of the medium include, but are not limited to, DMEM liquid medium, DMEM / F12 liquid medium, and Neurobasal medium.

[0032] In the second culture step, first, MSCs are selected from the cells cultured in the first culture step. Placental tissue contains many cells other than MSCs. Therefore, in the second culture step, the MSCs disclosed in the present application are selected by first excluding cells based on cell shape, HLA-ABC positive cells, etc. Then, similar to the first culture step, the selected MSCs can be cultured using a serum-free medium. Note that the medium used in the second culture step may have the same medium components as those in the first culture step, or may be different, as long as it is serum-free.

[0033] The isolation and culture method according to the embodiment provides the following advantages. (1) MSCs that are HLA-ABC negative, HLA-G positive, and express MSC markers can be isolated and cultured from placental tissue using a simple method that uses a serum-free medium. (2) The MSCs isolated in (1) above can be cultured and expanded by culturing them in a serum-free medium. Therefore, by expanding and culturing MSCs with HLA-ABC typing matched from the above-mentioned MSC bank, they can also be used as MSCs for allogeneic transplantation.

[0034] (Embodiment of Purification and Concentration Method) Next, a purification and concentration method according to an embodiment will be described. The purification and concentration method is performed on MSCs cultured by the isolation and culture method according to the embodiment described above. By selecting MSCs during the second culture step, many of the cells cultured in the second culture step are considered to be MSCs, but it is also possible that cells other than MSCs may be contaminated. It is also possible that MSCs may contain cells that do not have the characteristics disclosed in the present application. By performing the purification and concentration method, MSCs having the characteristics disclosed in the present application can be concentrated. The purification and concentration method is not particularly limited as long as it can select MSCs having the characteristics disclosed in the present application from cells contained in a cell culture medium. It is not particularly limited, but it is sufficient to select HLA-ABC-negative cells using an HLA-ABC antibody and MSC marker-positive cells using an MSC marker antibody. As shown in the examples below, when the isolation and culture method disclosed in the present application is performed, the cultured MSCs are HLA-G-positive. Therefore, no special selection process is required for HLA-G.

[0035] Furthermore, after carrying out the purification and concentration method, an isolation method may be optionally carried out to isolate MSCs having desired characteristics from the concentrated MSC culture medium. By isolating and preserving MSCs having desired characteristics, they can be used as parent lines for regenerative medicine, etc. Furthermore, the isolated MSCs may be subcultured as needed. When subcultured, a serum-free medium may be used, as in the first and second culture steps.

[0036] The purification and enrichment method disclosed in the present application has the following advantages. (1) In actual clinical practice, it is expected that isolated and preserved MSCs will be cultured and used. By implementing the purification and enrichment method, the probability of cells other than the MSCs disclosed in the present application being present in the culture medium is reduced, thereby improving the convenience of isolating and preserving MSCs with desired characteristics. (2) During primary isolation and culture from placental tissue, significantly elongated, thin, spindle-shaped, strongly HLA-ABC-positive MSCs derived from the mother are present. By implementing the purification and enrichment method using HLA-ABC antibodies early in the culture process, strongly HLA-ABC-positive MSCs derived from the mother can be removed. Because strongly HLA-ABC-positive MSCs derived from the mother have rapid cell proliferation, removing them at an early stage facilitates the purification and enrichment of the MSCs disclosed in the present application. (3) By removing leukocyte cells contained in placental tissue during the purification and enrichment process, the risk of unexpected cell differentiation or degeneration of the enriched MSCs due to cytokines secreted from the leukocyte cells can be avoided.

[0037] The MSCs, isolation and culture methods, and purification and concentration methods according to the above-described embodiments are merely examples of each embodiment and are not limited to the described examples. Other components may be added or removed within the scope of the technical concept disclosed in the present application. For example, in the above-described example, MSCs having the characteristics disclosed in the present application are isolated after performing the purification and concentration method. However, MSCs having the characteristics disclosed in the present application may also be isolated from cells after performing the second culture step.

[0038] (Embodiment of Mesenchymal Stem Cell Bank) Next, a mesenchymal stem cell (MSC) bank (hereinafter sometimes simply referred to as "bank") according to an embodiment will be described. The bank according to the embodiment includes two or more types of MSCs that are the MSCs according to the above-described embodiment, but that differ in at least one of the HLA typings (sometimes referred to as "HLA type"), namely, the HLA-A locus, the HLA-B locus, and the HLA-DR locus, in other words, different haplotypes, which are combinations of the HLA-A locus, the HLA-B locus, and the HLA-DR locus.

[0039] As described above, the MSCs according to the embodiment are negative for HLA-ABC, which is involved in distinguishing between self and non-self, and therefore are unlikely to cause immune rejection. However, although the MSCs according to the embodiment are negative for HLA-ABC, they do have the genes that express HLA-A antigens, HLA-B antigens, and HLA-C antigens. Therefore, from the perspective of minimizing the risk of immune rejection as much as possible, it is preferable to provide patients with MSCs of the same HLA typing.

[0040] The HLA type possessed by mammalian cells is determined by the combination of two inherited types, one inherited from each parent. A combination of the same HLA types is an HLA homozygote, and a combination of different HLA types is an HLA heterozygote. When MSCs (or induced biological tissues) are transplanted into a patient, immune rejection is unlikely to occur if one of the HLA types of the transplanted MSCs matches one of the patient's HLA types. Therefore, HLA homozygotes are preferred for MSCs stocked in a bank, as this increases the range of patients that can be covered by a single HLA type. Note that even HLA heterozygote MSCs are unlikely to cause immune rejection if they completely match the patient's HLA type (combination of two types). Therefore, the MSCs contained in the bank may be HLA heterozygotes.

[0041] There are six known types of human HLA, each with different functions and structures: HLA-A, HLA-B, and HLA-C, known as class I molecules, and HLA-DR, HLA-DQ, and HLA-DP, known as class II molecules. To minimize immune rejection, it is desirable to match three of the six types: HLA-A, HLA-B, and HLA-DR.

[0042] Incidentally, it is known that there are numerous genetic variations in each of the HLA-A locus, the HLA-B locus, and the HLA-DR locus. It is also known that the tendency of genetic variations differs depending on race. For example, in the allele groups of Japanese people, it is known that (1) at the HLA-A locus, genotypes such as HLA-A*02, HLA-A*24, and HLA-A*01 are relatively common, (2) at the HLA-B locus, genotypes such as HLA-B*40, HLA-B*46, HLA-B*15, and HLA-B*51 are relatively common, and (3) at the HLA-DR locus, genotypes such as DRB1*04, DRB1*01, and DRB1*15 are relatively common.

[0043] The MSCs contained in the bank are desirably stored in association with at least information on the genotype of the HLA-A locus, information on the genotype of the HLA-B locus, information on the genotype of the HLA-DR locus, and a haplotype, which is a combination of the HLA-A locus, B locus, and DR locus. If at least one of the genotypes (typing) of the HLA-A locus, HLA-B locus, and HLA-DR locus is different, they can be said to be types of MSCs with different HLA types (different haplotypes).

[0044] In order to provide MSCs that are compatible with the HLA types of many patients, the bank contains at least two or more types of MSCs with different HLA types. The more types of MSCs with different HLA types contained in the bank, the wider the range of patients to which they can be provided, so the types of MSCs contained in the bank may be 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more.

[0045] Furthermore, it is desirable for the bank to contain MSCs with HLA types that match those prevalent in humans, as this will broaden the range of compatible patients. For example, the website of the Japan Society for Histocompatibility Research (https: / / drive.google.com / file / d / 1tn5Sgd2fkFf1bO0lnSBjmY4wiipZMgWD / view) lists the HLA types frequently occurring in Japanese people at the subtype level. According to the website, the most common subtypes are as follows: HLA-A: A*24:02, A*02:01, A*11:01, A*26:01, A*33:03, A*31:01. Among these, A*24:02 is the most common. HLA-B: B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01, B*07:02. Among these, B*52:01 is most frequent. HLA-DR: DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03, DRB1*01:01. Among these, DRB1*04:05 is most frequent.

[0046] Therefore, the bank preferably includes MSCs with the following typings: - For typing of the HLA-A locus, it is preferable to include at least one selected from the group consisting of A*24:02, A*02:01, A*11:01, A*26:01, A*33:03, and A*31:01, and it is more preferable to include A*24:02. - For typing of the HLA-B locus, it is preferable to include at least one selected from the group consisting of B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01, and B*07:02, and it is more preferable to include B*52:01. For typing of the HLA-DR locus, it is preferable that at least one selected from the group consisting of DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03, and DRB1*01:01 is included, and it is more preferable that DRB1*04:05 is included.

[0047] In addition, the Kyoto University MSC Research Foundation website (https: / / www.cira-foundation.or.jp / j / provision-of-ips-cells / homozygous / ) states that the most common haplotypes of HLA-A, B, and DR are A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:05, in that order. Therefore, when identifying the MSCs contained in the bank by HLA haplotype, it is preferable that at least one selected from the group consisting of A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:05 is included, and it is more preferable that A*24:02-B*52:01-DRB1*15:02 is included.

[0048] When MSCs are heterozygous, at least one of the HLA types may include the above-mentioned HLA-A locus typing, HLA-B locus typing, HLA-DRB1 typing, and HLA haplotype. Note that the above-mentioned typing is an example for Japanese people. When creating a bank for a race other than Japanese, typing frequencies for that race may be investigated, and MSCs of frequently occurring types may be included.

[0049] (Embodiment of Method for Providing Mesenchymal Stem Cell Information) Next, a method for providing mesenchymal stem cell (MSC) information according to an embodiment will be described. The method for providing MSC information according to an embodiment is a method for providing MSC information contained in the MSC bank according to the embodiment described above. As described above, each MSC constituting the MSC bank is stored in association with typing information for at least the HLA-A locus, HLA-B locus, and HLA-DR locus of HLA typing, and optionally with a haplotype, which is a combination of the HLA-A locus, B locus, and DR locus. In addition to the above-described HLA-A locus, B locus, DR locus, and haplotype information, MSCs may also be stored in association with typing information for the HLA-C locus, DQ locus, and DP locus, as well as information such as cell type, race, and passage number. Hereinafter, information associated with MSCs may be collectively referred to as "MSC information."

[0050] The MSC information providing method includes at least an HLA typing information input step of inputting HLA typing information of a subject, and an MSC information providing step.

[0051] In the HLA typing information input step, it is sufficient that typing information for at least the HLA-A locus, HLA-B locus, and HLA-DR locus of the subject is input. Note that, in the case where the subject is heterozygous, typing information for both alleles of the HLA-A locus, HLA-B locus, and HLA-DR locus may be input. In the MSC information providing step, if MSCs with a matching haplotype are included among the stored MSCs based on the input HLA typing information of the subject, MSC information with the matching haplotype may be provided. In the case where the haplotypes do not match, MSC information with two pieces of matching information among the typing information for the HLA-A locus, HLA-B locus, and HLA-DR locus, or MSC information with one piece of matching information may be provided. If the typing of the subject's HLA-A locus, HLA-B locus, and HLA-DR locus does not match at all with the stored MSCs, information may be provided to the effect that there are no stored MSCs that match at least one typing of the subject.

[0052] As described above, MSCs according to the embodiment are cells that are unlikely to cause immune rejection. After clinical trials, etc., have been conducted to determine whether a given haplotype is mismatched, as long as the typing information for the HLA-A locus, HLA-B locus, and HLA-DR locus matches, immune rejection is unlikely to occur. A discrimination step may be performed in which predetermined reference values ​​are determined and MSCs stored in a bank are identified as suitable for the subject based on the subject's HLA typing information. The discrimination step may identify the MSCs most suitable for the subject, or may identify multiple types of cells suitable for the subject. After the discrimination step is performed, a provision step of the identified cells may be performed. Of course, if the haplotypes are completely matched, MSCs matching the subject's haplotype may be provided without performing the discrimination step.

[0053] The method for providing MSC information is not particularly limited as long as each of the above-mentioned steps is carried out. For example, the MSC information may be stored in a computer, and an HLA typing information input step, an MSC information provision step, and optionally an additional discrimination step may be carried out.

[0054] The MSC bank and MSC information providing method according to the embodiments have the following advantages: (1) MSCs that are unlikely to cause immune rejection reactions can be provided to patients. (2) As described above, the MSCs according to the embodiments are cells that are unlikely to cause immune rejection reactions. Taking these characteristics and HLA typing into consideration, they can also be used in drug discovery research, etc. (3) Because the MSCs according to the embodiments are produced from placenta-derived cells, at least one of the HLA types will be the same for at least the mother or child of the placenta donor. Therefore, by associating donor information with the MSCs, they can be used preferably when future treatment using MSCs is required for the donor's mother or child.

[0055] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the technical scope of the disclosure in the present application.

[0056] [Isolation and Culture Method] Example 1 MSCs were isolated and cultured from placental tissue using the following procedure. First Culture Step (1) Placental tissue (decidua partially including the chorion) was excised from a placenta at 38 weeks of gestation and cut into pieces of 5 mm or less. (2) Enzyme treatment was performed using collagenase (Sigma-Aldrich). (3) The enzyme-treated placental tissue was washed using phosphate-buffered saline (PBS). (4) Fibrin and other particles that were not dispersed by the enzyme treatment were removed using a cell strainer with a mesh size of 100 μm. (5) Cells were seeded in serum-free medium in a cell culture dish and cultured in a 5% carbon dioxide incubator at 37°C. The medium used was a mixture of equal volumes of serum-free DMEM / F12 medium (Cat No. 11320033) and Neurobasal medium (Cat No. 21103049). The medium was replaced every 2 to 3 days.

[0057] Figure 2A is a photograph of a dish on day 5 after the start of cell culture. Thin, spindle-shaped cells, indicated by the arrowheads in Figure 2A, were observed. As will be described later, these cells were significantly elongated, thin, spindle-shaped, HLA-ABC-positive MSCs.

[0058] [Immunostaining of cells on day 14 of culture (1)] The above-mentioned <First culture step> was subsequently carried out. Note that day 14 of culture refers to the number of days after enzyme treatment. Thin, spindle-shaped cells were stained using the following procedure: (1) The culture medium was aspirated and the cells were washed once with PBS. (2) The cells were fixed with 4% paraformaldehyde solution for 10 minutes at room temperature. (3) The fixative was removed and the cells were washed once with PBS. (4) The cells were reacted with a nonspecific reaction blocking reagent (Protein Block Liquid, Agilent) for 5 minutes at room temperature. (5) The CD105 mouse antibody (Thermo Fisher Scientific) was diluted 100-fold in PBS containing 1% bovine serum albumin (BSA) and reacted at 37°C for 60 minutes. (6) The cells were washed three times with PBS. (7) Anti-mouse Alexa Fluor 594-labeled secondary antibody (Themo Fisher Scientific) was diluted 500-fold in PBS containing 1% BSA, and HLA-ABC-FITC-labeled antibody (Beckman Coulter) was diluted 10-fold and reacted at 37°C for 60 minutes. (8) The sections were washed three times with PBS. (9) The sections were mounted in VECTASHIELD Mounting Medium (Vector Laboratories), a fluorescent mounting medium containing nuclear staining solution (4',6-diamidino-2-phenylindole; DAPI). (10) The sections were observed and photographed using a fluorescent inverted microscope (IX-71, Olympus).

[0059] The photographs taken are shown in Figure 2B. In Figure 2B, "PH" represents a phase-contrast image, "CD105 + DAPI" represents a fluorescent image of CD105, and "HLA-ABC + DAPI" represents a fluorescent image of HLA-ABC. As is clear from Figure 2B, the thin, spindle-shaped cells were positive for HLA-ABC and CD105, an MSC marker. In other words, the thin, spindle-shaped cells observed in the first culture step are thought to be maternal MSCs.

[0060] <Second culture step> The MSCs of the present application were selected from the cells cultured in the first culture step, and the selected MSCs were cultured in the same procedure as in the <First culture step>.

[0061] In the second culture step, the MSCs disclosed in the present application were first selected based on cell shape. Specifically, from the cells in the dish obtained in the above-mentioned <First Culture Step>, cells with spindle shape and elongated or expanded cytoplasm, as shown in Figure 2A, were removed using a scraper or chip, and the MSCs disclosed in the present application were selected on the plate. Note that the 14th day of culture shown below refers to the number of days from the enzyme treatment in the <First Culture Step>.

[0062] [Immunostaining of cells on day 14 of culture (2)] Immunostaining was carried out in the same manner as in [Immunostaining of cells on day 14 of culture (1)] above.

[0063] The results are shown in Figure 3. In the photographs of "CD105 + DAPI" and "HLA-ABC + DAPI" in Figure 3, the cells marked with arrowheads are cells that are strongly positive for the MSC marker CD105 and positive for HLA-ABC. On the other hand, the cells surrounded by dotted circles are cells that are strongly positive for the MSC marker CD105 and negative for HLA-ABC.

[0064] [Immunostaining of cells on day 14 of culture (3)] Immunostaining was performed in the same manner as in [Immunostaining of cells on day 14 of culture (1)] above, except that in place of the CD105 mouse antibody described in (5), a CD90 mouse antibody (Themo Fisher Scientific) diluted 100-fold with PBS containing 1% bovine serum albumin (BSA) was used.

[0065] The results are shown in Figure 4. The cells marked with arrowheads and the cells circled by dotted lines in the "CD90+DAPI" photograph in Figure 4 are cells that are strongly positive for the MSC marker CD90. From the results shown in Figures 3 and 4, it was confirmed that when the selected cells were subjected to the second culture step, although some HLA-ABC-positive cells were mixed in, cells that were strongly positive for the MSC markers CD90 and CD105 and negative for HLA-ABC were obtained.

[0066] [Analysis by Flow Cytometry (FCM) (1)] Next, FCM analysis was performed on the cells on the 14th day of culture after the above-mentioned <Second Culture Step>. The experimental procedure is as follows: (1) An enzyme (TrypLE) was applied to the cells adhering to the cell culture dish. TM The cells were incubated with CD105 mouse antibody (Thermo Fisher Scientific) at 4°C for 20 minutes in a 5% CO2 incubator at 37°C. (2) The cells detached from the cell culture dish were collected, washed with 10 mL of PBS, and centrifuged at 1,000 rpm for 5 minutes. (3) CD105 mouse antibody (Thermo Fisher Scientific) was diluted 100-fold with PBS containing 5% fetal bovine serum (FBS) and incubated at 4°C for 20 minutes. (4) PBS was added, and the cells were washed with centrifugation at 1,000 rpm for 5 minutes. (5) Anti-mouse PE-labeled secondary antibody (BD Pharmingen) was diluted 200-fold and HLA-ABC-FITC antibody (Beckman Coulter) was diluted 10-fold in 5% FBS-containing PBS, and the mixture was incubated at 4°C for 20 minutes. (6) PBS was added, and the mixture was washed by centrifugation at 1,000 rpm for 5 minutes. (7) Aggregated cells were removed by passing through a cell strainer with a mesh size of 35 μm. (8) Measurement was performed using a CytoFLEX flow cytometer.

[0067] The results of the FCM analysis are shown in Figure 5. The cells used in the FCM analysis will be described with reference to Figure 3. The cells marked with arrowheads in the three photographs in Figure 3 are cells that are strongly positive for the MSC marker CD105 and positive for HLA-ABC. In Figure 5, cells that are strongly positive for the MSC marker CD105 and positive for HLA-ABC are detected in the area surrounded by the dotted line in the graph for "HLA-ABC-FITC+CD105-PE" on the right side of Figure 5. On the other hand, the cells surrounded by the dotted circle in Figure 3 represent a cell population that is positive for CD105 but negative for HLA-ABC. In Figure 5, they are detected in the area surrounded by the solid line in the graph for "HLA-ABC-FITC+CD105-PE" on the right side. Note that "Negative FITC+PE" on the left side of Figure 5 shows the results obtained by using an isotype control antibody instead of the "CD105 mouse antibody" and the "HLA-ABC-FITC antibody" in (3) of the above [Analysis by flow cytometry (FCM) (1)].

[0068] [Flow Cytometry (FCM) Analysis (2)] Next, an experiment was conducted to confirm whether the cultured cells were HLA-G positive. The experimental procedure is as follows. The experiment was conducted in the same manner as in [Flow Cytometry (FCM) Analysis (1)] above, except that steps (3) to (5) described in [Flow Cytometry (FCM) Analysis (1)] above were replaced with step (3) described below. (3) HLA-G-APC-labeled antibody (Miltenyi Biotec) was diluted 50-fold with PBS containing 5% fetal bovine serum (FBS) and incubated at 4°C for 20 minutes. In addition, an isotype control antibody was used as a comparison control instead of the HLA-G-APC-labeled antibody.

[0069] The results are shown in Figure 6. Figure 6A shows the results using an isotype control antibody, and Figure 6B shows the results using an HLA-G-APC-labeled antibody. As is clear from Figure 6B, almost all cells reacted with the HLA-G-APC-labeled antibody, confirming their positivity. The results shown in Figure 6 indicate that all cells cultured in serum-free medium (cells detected by FCM surrounded by a solid line in Figure 5, all HLA-ABC-positive and -negative cells, and all CD105-positive and -negative cells) were HLA-G-positive. Therefore, it was confirmed that the isolation and culture method disclosed in the present application resulted in all MSCs being HLA-G-positive.

[0070] [Method for purifying and concentrating HLA-ABC negative, CD105 positive cells] <Example 2> Next, HLA-ABC negative and CD105 positive cells were purified from the cultured cells. The protocol is shown below. (1) Cells on the 14th day of culture that had been subjected to the above-mentioned <Second culture step> were purified by an enzyme (TrypLE TM(Select Enzyme, Thermo Fisher Scientific) was incubated for 5 minutes at 37°C in a 5% carbon dioxide incubator. (2) The cells detached from the cell culture dish were collected, and 10 mL of PBS was added and washed by centrifugation at 1,000 rpm for 5 minutes. (3) HLA-ABC-FITC-labeled antibody (Beckman Coulter) was diluted 10-fold with 5% FBS-containing PBS and incubated for 20 minutes at 4°C. (4) PBS was added, and the cells were centrifuged at 1,000 rpm for 5 minutes to wash. (5) Anti-FITC microbeads (Miltenyi Biotec) were diluted 10-fold with 5% FBS-containing PBS and incubated for 20 minutes at 4°C. (6) PBS was added, and the cells were washed by centrifugation at 1,000 rpm for 5 minutes. (7) The labeled cells were applied to a magnetic column (LD column, Miltenyi Biotec), and the fraction of cells that flowed out of the column (HLA-ABC-negative cells) was collected. (8) PBS was added, and the cells were washed by centrifugation at 1,000 rpm for 5 minutes. (9) Anti-CD105 mouse antibody (Themo Fisher Scientific) was diluted 100-fold and allowed to react at 4°C for 20 minutes. (10) PBS was added, and the cells were washed by centrifugation at 1,000 rpm for 5 minutes. (11) Anti-mouse antibody microbeads (Miltenyi Biotec) were diluted 10-fold with PBS containing 5% FBS, and allowed to react at 4°C for 20 minutes. (12) PBS was added and the cells were washed by centrifugation at 1,000 rpm for 5 minutes. (13) The labeled cells were applied to a magnetic column (MS column, Miltenyi Biotec), and the cells bound to the column (CD105-positive cells) were collected. (14) PBS was added and the cells were washed by centrifugation at 1,000 rpm for 5 minutes. (15) The cells were stained and subjected to FCM analysis using the same method as in the above [Flow cytometry (FCM) analysis (1)].

[0071] The results are shown in Figure 7. As shown in Figure 7, when HLA-ABC-negative, CD105-positive MSCs were purified and concentrated using a magnetic column and stained with HLA-ABC antibody and CD105 antibody, an MSC cell population that was 99.08% HLA-ABC-negative and 99.14% CD105-positive was successfully purified and concentrated.

[0072] Comparative Example 1 Culture was carried out for 5 days in the same manner as in the first culture step of Example 1, except that a 10% FBS-containing DMEM / F12 liquid medium (manufactured by Thermo Fisher Scientific) was used instead of the medium of Example 1.

[0073] Next, the cells of Comparative Example 1 were immunostained using the same procedure as in [Immunostaining of cells on day 14 of culture (1)]. The results are shown in the upper panel of Figure 8.

[0074] The cells of Comparative Example 1 were immunostained in the same manner as in [Immunostaining of cells on day 14 of culture (1)] in Example 1, except that in step (5) of [Immunostaining of cells on day 14 of culture (1)], a CD90 mouse antibody (Themo Fisher Scientific) diluted 100-fold with PBS containing 1% bovine serum albumin (BSA) was used instead of the CD105 mouse antibody. The results are shown in the lower panel of Figure 8.

[0075] As is clear from the upper and lower panels of Figure 8, many of the cells were positive for the MSC markers CD105 and CD90. On the other hand, as is clear from the upper panel of Figure 8, approximately 30% of the cells were positive for HLA-ABC.

[0076] [Analysis by Flow Cytometry (FCM)] Cells cultured in Comparative Example 1 were cultured using the 10% FBS-containing DMEM / F12 liquid medium of Comparative Example 1. Cells on days 5 and 12 of culture were analyzed using the same procedure as in Example 1 [Analysis by Flow Cytometry (FCM) (1)]. The results are shown in Figure 9. As is clear from the results in Figure 9, when MSCs were cultured in a medium supplemented with 10% bovine serum, the percentage of HLA-ABC-positive cells was 35.11% on day 5 of culture, but increased to 96.25% on day 12 of culture. Furthermore, on day 12 of culture, 85.12% of cells were CD105-positive, and most of the cells were HLA-G-negative.

[0077] Comparative Example 2: Culture was performed in the same manner as in Comparative Example 1, except that 0.1% FBS was used instead of the 10% FBS used in Comparative Example 1, and FCM analysis of HLA-ABC was performed. The results are shown in Figure 10. As is clear from Figure 10, although the proportion of HLA-ABC-positive cells was lower compared to the medium containing 10% bovine serum, it was confirmed that even with the addition of only 0.1% FBS to the medium, the number of HLA-ABC-positive cells increased as the number of days of culture increased.

[0078] From the above results, the following considerations can be made regarding the presence or absence of serum in the culture medium when culturing MSCs. (1) When MSCs were cultured in a serum-containing medium, HLA-G-positive MSCs, which act on immune tolerance, were not obtained. On the other hand, when MSCs were cultured in a serum-free medium, the MSCs became HLA-G-positive. Therefore, the isolation and culture method disclosed in the present application allows for the production of HLA-G-positive MSCs, which were not previously obtainable. (2) When MSCs were cultured in a serum-containing medium, the proportion of HLA-ABC-positive cells significantly increased with increasing culture time. This suggests that even if HLA-ABC-negative MSCs are isolated and preserved, their HLA-ABC characteristics may change from negative to positive during expansion culture or subculture for transplantation in a serum-containing medium. On the other hand, as shown in Example 3 below, subculture of MSCs in a serum-free medium did not result in a change from negative to positive HLA-ABC. Therefore, the use of a serum-free medium allows the HLA-ABC-negative and HLA-G-positive properties of MSCs to be maintained.

[0079] [Subculture] Example 3 The cells obtained in Example 2 that were positive for MSC markers, negative for HLA-ABC, and positive for HLA-G were isolated, and the isolated MSCs were subcultured according to the following procedure: (1) Cells adhering to a cell culture dish were treated with an enzyme (TrypLE TM(2) The cells were collected from the cell culture dish and 10 mL of PBS was added. (3) The cells were washed by centrifugation at 1,000 rpm for 5 minutes. (4) The cells were seeded onto a new culture dish and cultured using the same medium as in Example 1.

[0080] Figure 11 shows a photograph of MSCs cultured for five passages. Figure 12 shows the results of FCM analysis performed using the same procedure as in Example 1. When the expression of MSC markers (CD105), HLA-ABC, and HLA-G was examined using the same procedure as in Example 1, the MSCs cultured for five passages were positive for the MSC marker, negative for HLA-ABC, and positive for HLA-G.

[0081] From the above results, it was confirmed that MSCs that are MSC marker positive, HLA-ABC negative, and HLA-G positive obtained by the isolation and culture method disclosed in the present application can maintain the same characteristics even after passage by passage using serum-free medium.

[0082] [Confirmation of typing of HLA-A locus, HLA-B locus, and HLA-DR locus] Example 4 MSCs were produced from the placentas of six people using the same procedure as in Example 2 described above. The produced MSCs were verified to be MSC cells using the same procedure as in Example 1, and were confirmed to be negative for HLA-ABC expression and positive for HLA-G expression. Next, the typing of the HLA-A locus, HLA-B locus, and HLA-DR locus of the six types of produced MSC cells was examined using the SSOP typing method (see "SSOP typing method for HLA-A, B, and DR in the Japanese population. MHC 8(3), 175-186, 2002. doi.org / 10.12667 / mhc.8.175").

[0083] The typing of six types of MSCs (1) to (6) obtained from six placentas is shown below. (1) A*24:02, B*40:02, DRB1*08:03 (2) A*02:01, B*52:01, DRB1*08:03 (3) A*33:03, B*15:01, DRB1*04:05 (4) A*11:01, B*54:01, DRB1*04:05 (5) A*26:01, B*40:01, DRB1*08:03 (6) A*31:01, B*07:02, DRB1*09:01

[0084] The HLA-A locus subtypes, HLA-B locus subtypes, and HLA-DRB1 locus subtypes of the six types of MSCs were each included in the group of subtypes frequently found in Japanese people listed on the website of the Japan Society for Histocompatibility Research (https: / / drive.google.com / file / d / 1tn5Sgd2fkFf1bO0lnSBjmY4wiipZMgWD / view). Furthermore, among the six types of MSCs, (1) and (2) shared the same HLA-DRB1 subtype (DRB1*08:03), and (3) and (4) shared the same HLA-DRB1 subtype (DRB1*04:05), but the other subtypes were different.

[0085] As described above, the MSCs disclosed in the present application are negative for HLA-ABC expression and positive for HLA-G expression, and are therefore thought to be less likely to cause immune rejection when allografted. Furthermore, by creating a bank in which the HLA typing of the prepared MSCs is associated with typing information for at least the HLA-A locus, HLA-B locus, and HLA-DR locus and stored, it is possible to provide MSCs suitable for a patient based on the patient's HLA typing information. Information identifying each MSC contained in the bank and the HLA typing information of the MSCs can be stored, for example, in a computer, and a program can be stored that can identify the iMSCs to be provided by inputting the patient's HLA typing information.

[0086] [Confirmation of HGF secretion ability] Example 5 The amount of HGF secreted by MSCs was measured according to the following procedure. (1) Cryopreserved MSCs (prepared in Example 2) were thawed, added to 9 mL of medium (serum-free medium used in Example 1), and centrifuged at 800 G for 3 minutes. (2) After aspirating the supernatant, 10 mL of medium was added, and the number of viable cells was counted using trypan blue. (3) When the number of cells in each well of a 6-well multiplate was 5 x 10 ４ (4) 37°C, 5% CO ２ The cells were incubated under 100% humidified conditions. (5) After 3 days of culture, the medium was replaced. (6) After 7 days of culture, the cell supernatant (hereinafter referred to as "sample") was collected and stored frozen at -80°C.

[0087] Next, HGF contained in the cell supernatant was measured by the following procedure. TM The assay was performed using the HGF ELISA Kit. All reagents except for the sample were included in the kit. (7) 100 μL of each standard and sample were dispensed into appropriate wells. (8) The plate was incubated at 37°C for 2 hours. (9) The plate was washed four times with 1× Wash Buffer. (10) 100 μL of 1× Detection antibody, HRP-conjugated, was added to each well and incubated at 37°C for 40 minutes. (11) The plate was washed four times with 1× Wash Buffer. (12) 100 μL of TMB substrate solution was added to each well and incubated in the dark for 15 to 20 minutes. (13) 100 μL of Stop Solution was added to each well in the same order as the addition of the TMB substrate to stop color development. (14) Immediately after adding the stop solution, the absorbance was read at a wavelength of 450 nm using a microplate reader.

[0088] The HGF concentration in the cell supernatant was 15.5 ng / mL, and the background value measured using a standard sample not containing MSCs was 0.6 ng / mL.

[0089] Comparative Example 3: MSCs were cultured in the same manner as in Example 5, except that the serum-containing medium described in Comparative Example 1 was used, and the HGF content in the cell supernatant was measured. The HGF content in the cell supernatant was 0.6 ng / mL.

[0090] From the results of Example 5 and Comparative Example 3, it was confirmed that the MSCs disclosed in the present application secrete HGF, a growth factor, when cultured in a serum-free medium.

[0091] The value in Example 5 is approximately 26 times that of Comparative Example 3. Therefore, culturing MSCs in serum-free medium as disclosed in the present application can be applied to a method for producing HGF with high efficiency. The production method may include a culture step of culturing MSCs in serum-free medium and an HGF purification step of purifying HGF contained in the cell supernatant. The HGF purification step is not particularly limited as long as it can purify HGF contained in the cell supernatant. It is sufficient that HGF contained in the cell supernatant can be purified by using known means such as, but not limited to, ion exchange chromatography, affinity chromatography, gel filtration chromatography, etc., appropriately (combined as necessary).

[0092] The isolation and culture method disclosed in the present application allows for the production of MSCs that are MSC marker positive, HLA-ABC negative, and HLA-G positive. Furthermore, the resulting MSCs maintain their characteristics even after subculture, making them useful in the medical industry.

Claims

1. Mesenchymal stem cells isolated from the placenta, which are HLA-ABC negative and HLA-G positive.

2. The mesenchymal stem cells of claim 1, wherein said mesenchymal stem cells are isolated from placental tissue excluding the amniotic membrane.

3. The mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are cultured in a serum-free medium.

4. The mesenchymal stem cells according to claim 2, wherein the mesenchymal stem cells are cultured in a serum-free medium.

5. Mesenchymal stem cells according to any one of claims 1 to 4, wherein the placenta is a placenta from 16 weeks of pregnancy to full term.

6. A method for isolating and culturing HLA-ABC-negative and HLA-G-positive mesenchymal stem cells from a placenta, comprising: an enzyme treatment step of shredding placental tissue collected from the placenta and treating it with an enzyme; a first culture step of culturing placental tissue-derived cells obtained by the enzyme treatment in a serum-free medium; and a second culture step of selecting mesenchymal stem cells from the cells cultured in the first culture step and culturing the selected mesenchymal stem cells in a serum-free medium.

7. The method for isolating and culturing according to claim 6, wherein the placental tissue is a placental tissue excluding the amnion.

8. The method for isolating and culturing according to claim 6, wherein the placenta is from 16 weeks of pregnancy to full term.

9. A method for purifying and concentrating mesenchymal stem cells that are HLA-ABC negative and HLA-G positive, said method comprising a selection step of selecting mesenchymal stem cells that are HLA-ABC negative using an HLA-ABC antibody and selecting mesenchymal stem cells that are mesenchymal stem cell marker positive using a mesenchymal stem cell marker antibody from mesenchymal stem cells cultured by the isolation and culture method of any one of claims 6 to 8.

10. A method for isolating mesenchymal stem cells that are HLA-ABC negative and HLA-G positive, the method comprising the step of isolating the mesenchymal stem cells after carrying out the purification and concentration method described in claim 9.

11. A mesenchymal stem cell bank comprising the mesenchymal stem cells according to any one of claims 1 to 4, wherein the mesenchymal stem cell bank contains two or more types of mesenchymal stem cells that differ in at least one of the HLA typings, namely, HLA-A locus, HLA-B locus, and HLA-DR locus.

12. The mesenchymal stem cell bank contains at least one HLA-A locus typing selected from the group consisting of A*24:02, A*02:01, A*11:01, A*26:01, A*33:03, and A*31:01, at least one HLA-B locus typing selected from the group consisting of B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01, and B*07:02, and The mesenchymal stem cell bank according to claim 11, comprising mesenchymal stem cells of at least one type selected from the group consisting of HLA-DR locus typings DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03 and DRB1*01:

01.

13. The mesenchymal stem cell bank according to claim 11, wherein the mesenchymal stem cell bank contains mesenchymal stem cells having at least one haplotype selected from the group consisting of: A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:

05.

14. A mesenchymal stem cell information providing method for providing information on mesenchymal stem cells stored in a mesenchymal stem cell bank according to claim 11, wherein each mesenchymal stem cell constituting said mesenchymal stem cell bank is stored in association with typing information for at least the HLA-A locus, HLA-B locus, and HLA-DR locus among HLA typing information, and said mesenchymal stem cell information providing method comprises: an HLA typing information input step in which typing information for at least the HLA-A locus, HLA-B locus, and HLA-DR locus of a subject is input; and based on the input HLA typing information of the subject, among the stored mesenchymal stem cells, information on mesenchymal stem cells with matching haplotypes, information on mesenchymal stem cells with matching two of the typing information for the HLA-A locus, HLA-B locus, and HLA-DR locus, and information on mesenchymal stem cells with matching one of the typing information for the HLA-A locus, HLA-B locus, and HLA-DR locus, A method for providing mesenchymal stem cell information, comprising providing at least one piece of information selected from the group consisting of: