Cell population containing adherent stem cells, method for producing the same, and pharmaceutical composition

Abstract

To provide a cell population comprising safe adherent stem cells maintaining a normal karyotype, a method for producing the cell population, and a pharmaceutical composition comprising the cell population.SOLUTION: The present invention provides a production method for a cell population comprising adherent stem cells, comprising obtaining a cell population in which the proportion of KCNAB1-positive adherent stem cells in the cell population is 85% or more.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell population of adherent stem cells such as mesenchymal stem cells. The present invention relates to a method for producing the above cell population and a pharmaceutical composition containing the above cell population. Further, the present invention relates to a method for monitoring karyotypic abnormalities of adherent stem cells, a method for evaluating a donor and / or a biological sample collected from a donor, and a method for determining and / or predicting enzyme treatment conditions, using as an index the ratio of adherent stem cells expressing a specific marker in the cell population.

Background Art

[0002] Adherent stem cells such as mesenchymal stem cells, also called mesenchymal stromal cells, are somatic stem cells reported to exist in bone marrow, adipose tissue, dental pulp, etc. Recently, it has been clarified that they also exist in fetal appendages such as placenta, umbilical cord, and amniotic membrane. Further, the adherent stem cells have immunosuppressive ability, and their practical application for acute graft-versus-host disease (GVHD) and Crohn's disease, which is an inflammatory bowel disease, is progressing.

[0003] Furthermore, in recent years, there has been a demand for providing safe adherent stem cells that can withstand use for cell therapy applications. For example, Non-Patent Document 1 shows criteria for karyotype analysis of mesenchymal stem cells used for cell therapy. Non-Patent Document 2 describes the incidence of karyotypic abnormalities in bone marrow-derived mesenchymal stem cells from multiple donors and the correlation between the passage number and the incidence of karyotypic abnormalities.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non - Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors conducted studies to provide safe adherent stem cells that can withstand use for cell therapy applications. As a result, they confirmed the problem that karyotypic abnormalities frequently occur during the culture process of adherent stem cells, and karyotypic abnormalities accumulate as the passages increase. Since the above - mentioned problem leads to a risk of tumorigenicity, any adherent stem cell population containing even a small number of adherent stem cells with confirmed karyotypic abnormalities must be discarded. Furthermore, the karyotyping method of analyzing the structure of each chromosome one by one to evaluate the frequency of karyotypic abnormalities requires a great deal of time and labor for the analysis, which also causes the inability to perform rapid quality assurance. Therefore, the inventors found the problems that they must prepare a cell population without karyotypic abnormalities and must monitor the occurrence of karyotypic abnormalities over time.

[0006] To solve this problem, Non-Patent Document 1 was examined. Non-Patent Document 1 describes criteria for determining the frequency of chromosomal structural abnormalities in mesenchymal stem cells used for cell therapy. Further, Non-Patent Document 1 suggests that setting culture conditions to suppress the cell growth rate and doubling number as low as possible leads to the acquisition of a cell population without karyotypic abnormalities. However, the present inventors confirmed that karyotypic abnormalities occur frequently even when the growth rate and doubling number are decreased. Also, Non-Patent Document 1 does not describe a method for monitoring the presence or absence of karyotypic abnormalities.

[0007] Non-Patent Document 2 describes the occurrence frequency of karyotypic abnormalities and the correlation between the passage number and the occurrence frequency of karyotypic abnormalities in bone marrow-derived mesenchymal stem cells from multiple donors. Different from Non-Patent Document 1, it has been confirmed that as the number of passages increases and the doubling number increases, the frequency of karyotypic abnormalities tends to decrease. Thus, the relationship between the doubling number of cells and karyotypic abnormalities varies depending on the literature, and the mechanism of occurrence of karyotypic abnormalities has not been fully elucidated. Also, Non-Patent Document 2 mentions that karyotype analysis of mesenchymal stem cells obtained at each passage is useful for monitoring the chromosomal stability of mesenchymal stem cells during expansion culture. However, the method of karyotype analysis is to extract chromosomes from 17 to 144 cells to be subjected to karyotype analysis after obtaining mesenchymal stem cells, and observe the structure of each chromosome by the SKY (Spectral Karyotyping) method. The above analysis requires a great deal of time and labor, making it difficult to monitor the occurrence of karyotypic abnormalities over time and impossible to rapidly evaluate the presence or absence of karyotypic abnormalities. Also, there is no description of a method for preparing a cell population without karyotypic abnormalities.

[0008] In view of the above problems, an object of the present invention is to provide a means for obtaining a cell population containing adherent stem cells maintaining a normal karyotype, and for monitoring the occurrence of karyotypic abnormalities over time and rapidly evaluating the presence or absence of karyotypic abnormalities in the cell population containing adherent stem cells.

Means for Solving the Problem

[0009] As a result of intensive studies to solve the above problems, the present inventors have found that when culturing a cell population containing adherent stem cells under conditions where the ratio of adherent stem cells that are positive for KCNAB1 is maintained at a predetermined value or more, a cell population containing adherent stem cells maintaining a normal karyotype can be obtained. Furthermore, the present inventors have found that the presence or absence of karyotype abnormalities in adherent stem cells can be monitored by using, as an index, the fact that the ratio of adherent stem cells that are positive for KCNAB1 in a cell population containing adherent stem cells is at a predetermined value or more. Furthermore, from the viewpoint of efficiently obtaining adherent stem cells maintaining a normal karyotype, the quality of a donor and / or a biological sample collected from the donor can be rapidly evaluated, and furthermore, it has been found that the optimal enzyme treatment conditions for a biological sample collected from the donor can be determined and / or predicted. The present invention has been completed based on these findings.

[0010] That is, according to the present specification, the following inventions are provided. (1) A method for producing a cell population containing adherent stem cells, comprising: obtaining a cell population in which the ratio of adherent stem cells that are positive for KCNAB1 in the cell population is 85% or more. (2) A cell population containing adherent stem cells, wherein: the ratio of adherent stem cells that are positive for KCNAB1 in the cell population is 85% or more. (3) The cell population according to (2), wherein the relative expression level of the KCNAB1 gene with respect to the expression level of the SDHA gene in the cell population is 0.05 or more. (4) The cell population according to (2) or (3), wherein the relative expression level of the SULT1E1 gene with respect to the expression level of the SDHA gene in the cell population is 0.1 or more. (5) The cell population according to any one of (2) to (4), wherein the relative expression level of the MN1 gene with respect to the expression level of the SDHA gene in the cell population is 0.7 or more. (6) The cell population according to any one of (2) to (5), wherein the relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene in the cell population is 0.4 or less. (7) The cell population according to any one of (2) to (6), wherein the adherent stem cells are derived from fetal appendages.

[0011] (8) A pharmaceutical composition comprising the cell population according to any one of (2) to (7) and a pharmaceutically acceptable medium. (9) A pharmaceutical composition comprising the cell population according to any one of (2) to (7) and other administrable cells. (10) The pharmaceutical composition according to (8) or (9), wherein a single dose of the adherent stem cells for human is 10 12 cells / kg body weight or less. (11) The pharmaceutical composition according to any one of (8) to (10), wherein the pharmaceutical composition is an injectable preparation. (12) The pharmaceutical composition according to any one of (8) to (10), wherein the pharmaceutical composition is a preparation for transplantation. (13) The pharmaceutical composition according to (12), wherein the preparation for transplantation is a cell mass or a sheet-like structure. (14) A therapeutic agent for diseases selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungiform polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injuries, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart diseases, coronary heart diseases, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancers. The pharmaceutical composition according to any one of (8) to (13).

[0012] (15) In a cell population containing adherent stem cells, A method for monitoring karyotypic abnormalities of adherent stem cells using, as an index, the ratio of adherent stem cells that are positive for KCNAB1 measured in the cell population, wherein the ratio of adherent stem cells that are positive for KCNAB1 in the cell population is 85% or more. (16) A method for evaluating a donor and / or a biological sample collected from a donor, which comprises collecting a cell population containing adhesive stem cells from the donor, measuring the ratio of adhesive stem cells that are positive for KCNAB1, and evaluating using, as an index, that the ratio of adhesive stem cells that are positive for KCNAB1 in the cell population is 85% or more. (17) A method for determining and / or predicting the enzyme treatment conditions of a biological sample, which comprises measuring the ratio of adhesive stem cells that are positive for KCNAB1 in a cell population obtained by subjecting a biological sample collected from a donor to enzyme treatment, and evaluating using, as an index, that the ratio of adhesive stem cells that are positive for KCNAB1 in the cell population is 85% or more.

[0013] (21) A method for treating a disease, which comprises administering the cell population according to any one of (2) to (7) to a patient or subject in need of treatment. (22) The method according to (21), wherein a single dose of the adhesive stem cells for humans is 1×10 12 cells / kg body weight or less. (23) The method according to (21) or (22), which is an injectable preparation. (24) The method according to (21) or (22), which is a preparation for transplantation. (25) The pharmaceutical composition according to (24), wherein the preparation for transplantation is a cell mass or a sheet-like structure. (26) The disease is a disease selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscus injury, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart diseases, coronary heart diseases, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancers. The method according to any one of (21) to (25).

[0014] (31) Use of the cell population according to any one of (2) to (6) for the manufacture of a pharmaceutical composition. (32) The use according to (31), wherein the pharmaceutical composition is a pharmaceutical composition in which the single dose of adherent stem cells to a human is 1×10 12 cells / kg body weight or less. (33) The use according to (31) or (32), wherein the pharmaceutical composition is an injectable preparation. (34) The use according to (31) or (32), wherein the pharmaceutical composition is a preparation for transplantation. (35) The pharmaceutical composition according to (34), wherein the preparation for transplantation is a cell mass or a sheet-like structure. (36) The pharmaceutical composition according to any one of (31) to (35), wherein the pharmaceutical composition is a therapeutic agent for a disease selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injuries, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart diseases, coronary heart diseases, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancers.

[0015] (41) The cell population according to any one of (2) to (6) for use in the treatment of a disease. (42) The cell population according to (41), wherein the single dose of adherent stem cells to a human is 1×10 12 cells / kg body weight or less. (43) The cell population according to (41) or (42), which is an injectable preparation. (44) The cell population according to (41) or (42), which is a preparation for transplantation. (45) The pharmaceutical composition according to (44), wherein the preparation for transplantation is a cell mass or a sheet-like structure. (46) The disease is a disease selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injuries, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, trauma, epithelial injury, fibrosis, lung diseases, and cancer, and the cell population according to any one of (41) to (45).

Advantages of the Invention

[0016] According to the present invention, a cell population containing adherent stem cells maintaining a normal karyotype can be obtained. Further, according to the present invention, in a cell population containing adherent stem cells, it is possible to provide a means for monitoring the occurrence of karyotype abnormalities over time and rapidly evaluating the presence or absence of karyotype abnormalities. Furthermore, according to the present invention, the positive rate of a predetermined antigen can be used as an index for forming a cell population containing safe adherent stem cells maintaining a normal karyotype. Furthermore, according to the present invention, a safe cell preparation (pharmaceutical composition) with high chromosomal stability and suitable for clinical use can be produced.

Brief Description of the Drawings

[0017]

Figure 1

Modes for Carrying Out the Invention

[0018] Hereinafter, embodiments of the present invention will be specifically described. However, the following description is for facilitating the understanding of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments obtained by appropriately substituting the configurations of the following embodiments are also included in the scope of the present invention.

[0019] [1] Explanation of Terms As used herein, the "fetal appendage" refers to the amnion, placenta, umbilical cord, and amniotic fluid. Further, the "amnion" is a gestational sac containing the amniotic fluid of the fetus, and consists of the amnion, chorion, and decidua from the inside. Among these, the amnion and chorion originate from the fetus. The "amnion" refers to a transparent thin membrane lacking blood vessels in the innermost layer of the amnion. The inner layer of the amnion (also called the epithelial cell layer) is covered with a layer of epithelial cells with secretory function and secretes amniotic fluid. The outer layer of the amnion (also called the extracellular matrix layer, corresponding to the stroma) contains adhesive stem cells.

[0020] As used herein, the "adhesive stem cell" refers to a stem cell that satisfies the following definition, and "mesenchymal stromal cells" and "mesenchymal stem cells" are also included in the adhesive stem cells. In this specification, the "mesenchymal stem cell" may be described as "MSC".

[0021] As the "adhesive stem cell", among the somatic stem cells (tissue stem cells) that can be collected from various tissues and organs, cells that satisfy the following definition can be used. Somatic stem cells (tissue stem cells) include bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, stem cells in umbilical cord blood, umbilical cord-derived stem cells, amnion-derived stem cells, amniotic fluid stem cells, placenta villus cell-derived mesenchymal stem cells, neural stem cells, adipose tissue-derived stem cells, pancreatic stem cells, synovial mesenchymal stem cells, dental pulp stem cells, dental pulp-derived stem cells from exfoliated deciduous teeth, sperm stem cells (GS cells), testicular pluripotent stem cells (mGS cells), corneal epithelial stem cells, corneal stromal stem cells, pigment stem cells, tissue stem cells in organs, etc., but are not particularly limited.

[0022] Definition of Adhesive Stem Cells i) It shows adhesiveness to plastic under the culture conditions in a standard medium. ii) The surface antigens CD73 and CD90 are positive, and CD326 is negative.

[0023] The "adhesive stem cells" only need to satisfy the above definitions i) and ii), and there are no particular limitations on the presence or absence of the ability to differentiate into bone, cartilage, fat, etc. The "adhesive stem cells" in this specification include cells having the ability to differentiate into bone, cartilage, and fat, such as mesenchymal stem cells. In addition, the "adhesive stem cells" include cells that satisfy the above definitions but do not have the ability to differentiate into bone, cartilage, and fat. In addition, the "adhesive stem cells" include cells that satisfy the above definitions but can only differentiate into any one or two of bone, cartilage, and fat.

[0024] The "amnion-derived adhesive stem cells" in this specification refer to adhesive stem cells derived from the amnion, and also include "amnion mesenchymal stromal cells" and "amnion mesenchymal stem cells". In this specification, the "amnion mesenchymal stem cells" may sometimes be referred to as "amnion MSCs".

[0025] The "adhesive stem cell population" in this specification means a cell population containing adhesive stem cells, and its form is not particularly limited. Examples include cell pellets, cell sheets, cell aggregates, cell suspensions or cell suspensions.

[0026] The "karyotype abnormality" in this specification refers to the structural abnormality of chromosomes, which means the numerical abnormality of chromosomes or the partial structural abnormality. Examples of numerical abnormalities include "monosomy" in which there is only one chromosome that usually pairs in two, or "trisomy" in which there are three chromosomes. Examples of partial structural abnormalities include translocation, inversion, and deletion. The "normal karyotype" in this specification means that the above-mentioned karyotype abnormality is not recognized or a karyotype close to normal.

[0027] "Normal karyotype" and "abnormal karyotype" can be evaluated by karyotype analysis. Specifically, based on the characteristic band patterns of chromosomes detected by the differential staining method, each chromosome can be identified, and numerical abnormalities and partial structural abnormalities can be analyzed to evaluate "normal karyotype" and "abnormal karyotype". The type of karyotype analysis is not particularly limited, and Q-band analysis detected by fluorescent dyes such as quinacrine mustard and Hoechst, G-band analysis detected by protease treatment such as trypsin and Giemsa staining, analysis by multi-color FISH method that stains each chromosome in a different color, and simple analysis by Giemsa staining are known, but any of them may be used in the present invention. For example, in order to determine an abnormal karyotype, chromosomes can be extracted from 20 cells and subjected to karyotype analysis to evaluate the presence or absence of an abnormal karyotype. As a criterion for determining a normal karyotype, among the 20 cells subjected to the analysis, the proportion of cells having an abnormal karyotype is preferably 10% or less, more preferably 5% or less, still more preferably 4% or less, still more preferably 3% or less, still more preferably 2% or less, still more preferably 1% or less, and still more preferably 0%.

[0028] As used herein, the "proportion of adherent stem cells that are positive for KCNAB1" refers to the proportion of cells that are positive for the above antigen analyzed by flow cytometry as described in the examples below. In this specification, the "proportion of cells that are positive for an antigen" may be referred to as the "positive rate".

[0029] [2] Cell population containing adherent stem cells The cell population containing adherent stem cells provided by the present invention is characterized in that in the cell population, the proportion of adherent stem cells that are positive for KCNAB1 is 85% or more. In addition, when the cell population containing the adhesive stem cells provided by the present invention satisfies the condition that the ratio of adhesive stem cells positive for KCNAB1 is 85% or more, a cell population containing adhesive stem cells maintaining a normal karyotype is formed. Therefore, in the present invention, the above condition can be used as an index for forming a cell population containing adhesive stem cells maintaining a normal karyotype. Further, by measuring the above index over time, changes in karyotype abnormalities of adhesive stem cells can be grasped and predicted quickly and simply. Furthermore, according to the present invention, by using the above index, the quality of the donor itself and / or a biological sample collected from the donor can be evaluated. Furthermore, according to the present invention, by using the above index, it is possible to determine and / or predict whether the enzyme treatment method is optimal when the biological sample collected from the donor is enzyme-treated. In addition to the above index, the relative expression levels of the KCNAB1 gene, SULT1E1 gene, MN1 gene, and RARRES2 gene showing a specific numerical range can also be used as an index for forming a cell population containing adhesive stem cells maintaining a normal karyotype.

[0030] In the above cell population, the ratio of adhesive stem cells positive for KCNAB1 is preferably 86% or more, more preferably 87% or more, more preferably 88% or more, more preferably 89% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and most preferably 100%.

[0031] According to one aspect of the present invention, the cell population containing the adhesive stem cells provided by the present invention may satisfy the condition that the ratio of adhesive stem cells positive for CD105, CD73, and / or CD90 is 90% or more. CD105 means cluster of differentiation 105 and is a protein also known as Endoglin. CD73 refers to Cluster of Differentiation 73, a protein also known as 5-Nucleotidase, or Ecto-5’-nucleotidase. CD90 refers to Cluster of Differentiation 90, a protein also known as Thy-1.

[0032] The ratio of adherent stem cells that are positive for CD105 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The ratio of adherent stem cells that are positive for CD73 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The ratio of adherent stem cells that are positive for CD90 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0033] According to one aspect of the present invention, the cell population containing the adherent stem cells provided by the present invention may satisfy that the ratio of adherent stem cells that are positive for CD166 is 30% or more. CD166 refers to Cluster of Differentiation 166, a protein also known as Activaed leukocyte cell adhesion molecule (ALCAM).

[0034] The ratio of adherent stem cells that are positive for CD166 in the cell population may be 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0035] According to one aspect of the present invention, the cell population containing the adhesive stem cells provided by the present invention may satisfy that the ratio of adhesive stem cells that are negative for CD45, CD34, and / or CD326 is 95% or more. CD45 means cluster of differentiation 45 and is a protein also known as PTPRC (Protein tyrosine phosphatase, receptor type, C) or LCA (Leukocyte common antigen). CD34 means cluster of differentiation 34 and is a protein also known as Hematopoietic progenitor cell antigen CD34. CD326 means cluster of differentiation 326 and is a protein also known as Epithelial cell adhesion molecule encoded by the EPCAM gene.

[0036] The ratio of adhesive stem cells that are negative for CD45 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The ratio of adhesive stem cells that are negative for CD34 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The ratio of adhesive stem cells that are negative for CD326 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0037] Various antigens including KCNAB1, CD73, CD90, CD166, CD34, CD45, and CD326 can be detected by any detection method known in the art. Examples of methods for detecting these antigens include, but are not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, when cells that emit stronger fluorescence compared to a negative control (isotype control) are detected, the cells are determined to be "positive" for the marker. Any antibody known in the art can be used as the fluorescently labeled antibody. Examples include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. In cell staining, when cells that are stained or emit fluorescence are observed under a microscope, the cells are determined to be "positive" for the marker. Cell staining may be immunocytochemical staining using an antibody or non-immunocytochemical staining without using an antibody.

[0038] The ratio (positive rate) of cells presenting positive for KCNAB1 can specifically be measured by the following procedures (1) to (8) using dot plot unfolding analysis of flow cytometry. (1) Thaw the cryopreserved cell population and recover it by centrifugation. Wash the recovered cell population with phosphate buffer (PBS) and recover it by centrifugation. (2) After fixing and permeabilizing the cells using a solution prepared by adding polyoxyethylene (10) octylphenyl ether (Triton-X) to 4% paraformaldehyde to a final concentration of 0.1%, wash the cells with phosphate buffer (PBS) and prepare a cell suspension at a concentration of 2.0×10 6 cells / mL with 0.5% BSA / PBS. Aliquot 100 μL of the cell suspension. (3) Centrifuge the dispensed cell suspension, and add 100 μL of 0.5% BSA / PBS to the obtained cell pellet one by one. Then, add the antibody corresponding to each antigen marker or the antibody for its isotype control. After vortexing each reaction solution, let it stand at 4°C for 20 minutes. (4) Add 0.5% BSA / PBS, wash the cells by centrifugation, then suspend the cells in 0.5% BSA / PBS and filter them through a cell strainer (35 μm nylon mesh filter) (Corning / Product No.: 352235). (5) Analyze the cell suspension obtained by filtration using a BD Accuri TM C6 Flow Cytometer (Becton Dickinson) (ALL Event 10000). (6) Develop the measurement results in a dot plot with SSC (side scatter) on the vertical axis and FSC (forward scatter) on the horizontal axis. (7) In the dot plot development diagram, select all regions (gates) where the proportion of cell populations with stronger fluorescence intensity is 0.5% or less among the total cells measured with the antibody for isotype control. (8) Calculate the proportion of cells contained within the gate selected in (7) among the total cells measured with the antibody corresponding to the antigen marker.

[0039] Note that the ratio (negative rate) of cells negative for each surface antigen is calculated by the following formula. Negative rate (%) = 100 - Positive rate

[0040] The timing for detecting KCNAB1 described above is not particularly limited. For example, it can be immediately after separating cells from a biological sample, during the culture process, after purification in the culture process, immediately after n passages (n represents an integer of 1 or more), during maintenance culture, before cryopreservation, after thawing, or before formulating as a pharmaceutical composition.

[0041] The cell population containing adherent stem cells provided by the present invention preferably satisfies that the relative expression level of the KCNAB1 gene with respect to the expression level of the SDHA gene is 0.05 or more. The relative expression level of the KCNAB1 gene with respect to the expression level of the SDHA gene may be 0.75 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.25 or more, 0.3 or more, 0.35 or more, or 0.4 or more. The upper limit of the relative expression level of the KCNAB1 gene with respect to the expression level of the SDHA gene is not particularly limited, and may be, for example, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, 0.9 or less, 0.8 or less, 0.7 or less, or 0.6 or less.

[0042] The cell population containing adherent stem cells provided by the present invention preferably satisfies that the relative expression level of the SULT1E1 gene with respect to the expression level of the SDHA gene is 0.1 or more. The relative expression level of the SULT1E1 gene with respect to the expression level of the SDHA gene may be 0.13 or more, 0.15 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, or 0.6 or more. The upper limit of the relative expression level of the SULT1E1 gene with respect to the expression level of the SDHA gene is not particularly limited, and may be, for example, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less.

[0043] The cell population containing adherent stem cells provided by the present invention preferably satisfies that the relative expression level of the MN1 gene with respect to the expression level of the SDHA gene is 0.7 or more. The relative expression level of the MN1 gene with respect to the expression level of the SDHA gene may be 0.8 or more, 0.9 or more, 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, or 1.7 or more. The upper limit of the relative expression level of the MN1 gene with respect to the expression level of the SDHA gene is not particularly limited, and may be, for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0044] The cell population containing adherent stem cells provided by the present invention preferably satisfies that the relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene is 0.4 or less. The relative expression level of the RARRES2 gene to the expression level of the SDHA gene may be 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, or 0.003 or less. The lower limit of the relative expression level of the RARRES2 gene to the expression level of the SDHA gene is not particularly limited, and may be, for example, 0.001 or more, or 0.002 or more.

[0045] The method for detecting each gene and / or measuring its expression level is not particularly limited as long as it is a method known in the art, and for example, microarray, RT-PCR, quantitative RT-PCR, or Northern blot hybridization can be used. A microarray can be used to measure the relative expression level of each gene to the expression level of the SDHA gene. Specifically, the microarray can be performed according to the following steps (1) to (5). The following steps (3) to (5) can be carried out by entrusting them to Riken Genesis Co., Ltd. (1) The frozen cell population is thawed and collected by centrifugation. The collected cell population is washed with phosphate buffered saline (PBS) and the cells are collected by centrifugation. (2) Total RNA is extracted and purified using an RNA extraction kit (RNeasy Plus Mini kit (QIAGEN)). (3) cDNA is synthesized by reverse transcription using the purified total RNA as a template, and the synthesized cDNA is then transcribed into cRNA by in vitro transcription and labeled with biotin. (4) Add the biotin-labeled cRNA to the hybridization buffer and perform hybridization on a Human Gene Genome U133A 2.0 Array (manufactured by Affymetrix) for 16 hours. Wash with a GeneChip Fluidics Station 450 (manufactured by Affymetrix), stain with phycoerythrin, scan with a GeneChip Scanner 3000 7G (manufactured by Affymetrix), perform image analysis with AGCC (Affymetrix GeneChip Command Console Software) (manufactured by Affymetrix), and digitize using Affymetrix Expression Console (manufactured by Affymetrix). (5) Compare and analyze the numerical data file using analysis software GeneSpring GX (manufactured by Agilent Technologies). Calculate the relative expression level of each gene with respect to the expression level of the SDHA gene in each cell. As a method for measuring the relative expression level of each gene with respect to the expression level of the SDHA gene, quantitative PCR can be used. Specifically, quantitative PCR can be performed according to the following procedures (1) to (5). (1) Thaw the cryopreserved cell population and recover by centrifugation. Wash the recovered cell population with phosphate buffer (PBS) and recover the cells by centrifugation. (2) Extract and purify total RNA using an RNA extraction kit (RNeasy Plus Mini kit (manufactured by QIAGEN)). (3) Synthesize cDNA by reverse transcription reaction using the purified total RNA as a template. Further, mix the synthesized cDNA, Taqman Fast Advanced Master Mix (manufactured by Applied Biosystems), and primer (Taqman Gene Expression Assay, manufactured by Thermo Fisher) and inject into a 96-well plate to perform quantitative PCR. (4) Analyze the ΔCt values for SDHA in each sample using the StepOnePlus Real-Time PCR System (manufactured by Applied Biosystems), and calculate the relative expression levels (2^(-ΔCt)) of each gene relative to the expression level of the SDHA gene in each cell.

[0046] SDHA (Succinate dehydrogenase complex, subunit A) is a type of housekeeping gene, and its gene sequence is registered as ID: 6389 in the gene database of the National Center for Biotechnology Information. SDHA is a gene encoding a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0047] The sequence of the KCNAB1 (potassium channel, voltage gated subfamily A regulatory beta subunit 1) gene is registered as ID: 7881 in the gene database of the National Center for Biotechnology Information. KCNAB1 is a gene encoding a gene consisting of the nucleotide sequence shown in SEQ ID NO: 3 or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4. The sequence of the SULT1E1 (sulfotransferase family 1E member 1) gene is registered as ID: 6783 in the gene database of the National Center for Biotechnology Information. SULT1E1 is a gene encoding a gene consisting of the nucleotide sequence shown in SEQ ID NO: 5 or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6. The sequence of the MN1 (meningioma (disrupted in balanced translocation) 1) gene is registered as ID: 4330 in the gene database of the National Center for Biotechnology Information. MN1 is a gene that encodes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 7, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8. The sequence of the RARRES2 (retinoic acid receptor responder (tazarotene induced) 2) gene is registered as ID: 5919 in the gene database of the National Center for Biotechnology Information. RARRES2 is a gene that encodes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 9, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0048] The timing for measuring the gene expression level described above is not particularly limited. For example, it may be immediately after separating cells from a biological sample, during the culture process, after purification in the culture process, immediately after n passages (n represents an integer of 1 or more), during maintenance culture, before cryopreservation, after thawing, or before formulating as a pharmaceutical composition.

[0049] In the cell population of the present invention, adherent stem cells can be cultured without growth arrest while maintaining a normal karyotype after the start of in vitro culture, preferably after 20 days, more preferably after 25 days, after 30 days, after 35 days, after 40 days, after 45 days, after 50 days, after 55 days, after 60 days, after 65 days, after 70 days, after 75 days, after 80 days, after 85 days, after 90 days, after 95 days, after 100 days, after 105 days, or after 110 days.

[0050] The number of passages of the adherent stem cells in the cell population of the present invention can be cultured while maintaining a normal karyotype for 1 or more times, preferably 2 or more times, more preferably 3 or more times, still more preferably 4 or more times, still more preferably 5 or more times, still more preferably 6 or more times, still more preferably 8 or more times, still more preferably 10 or more times, still more preferably 12 or more times, still more preferably 14 or more times, still more preferably 16 or more times, still more preferably 18 or more times, still more preferably 20 or more times, still more preferably 22 or more times, still more preferably 24 or more times, and still more preferably up to 25 or more times. The upper limit of the number of passages is not particularly limited, but for example, it is 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less.

[0051] The cell population containing the adherent stem cells provided by the present invention can preferably double the population 10 or more times, more preferably 20 or more times, 30 or more times, 40 or more times, 50 or more times, or 60 or more times while maintaining a normal karyotype. Further, the cell population containing the adherent stem cells provided by the present invention can double the population, for example, 100 or less, 90 or less, 80 or less, or 70 or less, but is not limited thereto. The population doubling number is the number of times the cell population has divided during a certain culture period, and is calculated by the formula [log 10 (the number of cells at the end of the culture) - log 10 (the number of cells at the start of the culture)] / log 10 (2). When subculture is performed, after calculating the population doubling number for each passage using the above formula, the total population doubling number is calculated by accumulation.

[0052] The cell viability of the cell population containing the adherent stem cells provided by the present invention can be measured, for example, by trypan blue staining, PI (Propidium iodide) staining, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium Bromide) assay, etc., but is not limited thereto.

[0053] The cell viability of the cell population containing adhesive stem cells provided by the present invention is preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0054] The origin of the adhesive stem cells is not particularly limited. For example, adhesive stem cells derived from fetal appendages, bone marrow, adipose tissue, or dental pulp can be used. The adhesive stem cells are preferably adhesive stem cells derived from fetal appendages, and more preferably adhesive stem cells derived from amniotic membrane. The adhesive stem cells are adhesive stem cells isolated from autologous, allogeneic, or xenogeneic biological samples, and are preferably adhesive stem cells isolated from allogeneic biological samples.

[0055] The adhesive stem cells are either genetically modified or non-genetically modified adhesive stem cells, and are preferably non-genetically modified adhesive stem cells.

[0056] The cell population of the present invention can contain any number of adhesive stem cells. The cell population of the present invention can contain, for example, 1.0×10 1 cells, 1.0×10 2 cells, 1.0×10 3 cells, 1.0×10 4 cells, 1.0×10 5 cells, 1.0×10 6 cells, 1.0×10 7 cells, 1.0×10 8 cells, 1.0×10 9 cells, 1.0×10 10 cells, 1.0×10 11 cells, 1.0×10 12 cells, 1.0×10 13 cells or more or less, but are not limited thereto.

[0057] In addition to adherent stem cells, the cell population of the present invention may contain any components. Examples of the above components include salts (e.g., physiological saline, Ringer's solution, bicarbonate infusion), polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, medium components (e.g., components contained in RPMI1640 medium, etc.), etc., but are not limited thereto.

[0058] The cell population of the present invention can be stored in a frozen state until immediately before use. In addition to adherent stem cells, the above cell population may contain a cryopreservation solution. As the above cryopreservation solution, a commercially available cryopreservation solution may be used. For example, CP-1 (registered trademark) (manufactured by Kanto Chemical Co., Inc.), BAMBANKER (manufactured by Lymphotec Co., Ltd.), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), ReproCryo RM (manufactured by ReproCell Inc.), CryoNovo (manufactured by Akron Biotechnology Inc.), MSC Freezing Solution (manufactured by Biological Industries), CryoStor (manufactured by HemaCare), etc. can be mentioned, but are not limited thereto.

[0059] The cell population of the present invention may be provided as a composition combined with a medium. As the medium, preferably a liquid medium (e.g., medium, dimethyl sulfoxide (DMSO), cryopreservation solution, or a pharmaceutically acceptable medium described below, etc.) can be used.

[0060] The composition containing the cell population of the present invention and the medium can have any cell concentration. The cell concentration of the composition containing the cell population of the present invention and the medium is, for example, 1.0×10 1 cells / mL, 1.0×10 2 cells / mL, 1.0×10 3 cells / mL, 1.0×10 4 cells / mL, 1.0×10 5 cells / mL, 1.0×10 6 cells / mL, 1.0×10 7 cells / mL, 1.0×10 8cells / mL, 1.0×10 9 cells / mL, 1.0×10 10 The cell concentration can be, but is not limited to, 1.0×10

[0061] [3] Method for producing a cell population containing adherent stem cells The method for producing a cell population containing adherent stem cells according to the present invention includes obtaining a cell population in which the ratio of adherent stem cells that are positive for KCNAB1 is 85% or more, in a cell population containing cells collected from a biological tissue or organ such as a fetal appendage. Further, the method for producing a cell population containing adherent stem cells according to the present invention is a method including a step of culturing a cell population containing cells collected from a biological tissue or organ such as a fetal appendage under conditions that maintain the ratio of adherent stem cells that are positive for KCNAB1 at 85% or more. The above conditions are indicators for the formation of a cell population containing adherent stem cells maintaining a normal karyotype, and the culturing method of the present invention is not particularly limited as long as the above indicators are satisfied.

[0062] The production method of the present invention may, for example, include a cell population acquisition step of obtaining a cell population containing adherent stem cells by subjecting a fetal appendage such as amniotic membrane to enzymatic treatment. The above cell population acquisition step may be a step including a step of obtaining amniotic membrane by cesarean section. Further, the above cell population acquisition step may be a step including a step of washing a biological sample containing adherent stem cells.

[0063] The amniotic membrane is composed of an epithelial cell layer and an extracellular matrix layer, and the latter contains amniotic membrane adherent stem cells. Amniotic epithelial cells, like other epithelial cells, characteristically express an epithelial adhesion factor (EpCAM: CD326), whereas amniotic membrane adherent stem cells do not express the epithelial-specific surface antigen marker of CD326 and can be easily distinguished by flow cytometry. The above cell population acquisition step may be a step including a step of obtaining amniotic membrane by cesarean section.

[0064] The cell population containing adhesive stem cells in the present invention is preferably a cell population obtained by treating a biological sample containing an epithelial cell layer and an adhesive stem cell layer collected from a fetal appendage with at least collagenase.

[0065] The enzymatic treatment of a biological sample collected from a fetal appendage (preferably a biological sample containing an epithelial cell layer and an adhesive stem cell layer) is preferably a treatment with an enzyme (or a combination thereof) that can release the adhesive stem cells contained in the extracellular matrix layer of the fetal appendage and does not decompose the epithelial cell layer. Such enzymes are not particularly limited, and examples thereof include collagenase and / or metalloprotease. Examples of the metalloprotease include, but are not particularly limited to, thermolysin and / or dispase, which are metalloproteases that cleave the N-terminal side of non-polar amino acids.

[0066] The active concentration of collagenase is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, and still more preferably 200 PU / ml or more. The active concentration of collagenase is not particularly limited, and examples thereof include 1000 PU / ml or less, 900 PU / ml or less, 800 PU / ml or less, 700 PU / ml or less, 600 PU / ml or less, and 500 PU / ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that degrades 1 μg of FITC-collagen in 1 minute at pH 7.5 and 30°C.

[0067] The activity concentration of the metalloprotease (for example, thermolysin and / or dispase) is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, still more preferably 200 PU / ml or more. Also, the activity concentration of the metalloprotease is preferably 1000 PU / ml or less, more preferably 900 PU / ml or less, still more preferably 800 PU / ml or less, still more preferably 700 PU / ml or less, still more preferably 600 PU / ml or less, still more preferably 500 PU / ml or less. Here, in the embodiment where dispase is used as the metalloprotease, PU (Protease Unit) is defined as the amount of enzyme that liberates amino acids equivalent to 1 μg of tyrosine per minute from casein lactate at pH 7.5 and 30°C. Within the above enzyme concentration range, adhesive stem cells contained in the extracellular matrix layer can be efficiently liberated while preventing contamination of epithelial cells contained in the epithelial cell layer of the fetal appendage. The preferred combination of the concentrations of collagenase and / or metalloprotease can be determined by microscopic observation of the fetal appendage after enzyme treatment and flow cytometry of the obtained cells.

[0068] From the viewpoint of efficiently recovering viable cells, it is preferable to treat the fetal appendage by combining collagenase and metalloprotease. More preferably, the fetal appendage is treated simultaneously and collectively by the above combination. As the metalloprotease in this case, thermolysin and / or dispase can be used, but it is not limited thereto. By treating the fetal appendage only once with an enzyme solution containing collagenase and metalloprotease, adhesive stem cells can be easily obtained. Also, by treating simultaneously and collectively, the risk of contamination such as bacteria and viruses can be reduced.

[0069] For the enzymatic treatment of fetal appendages, it is preferable to immerse the amniotic membrane washed with a washing solution such as physiological saline or Hank's balanced salt solution in an enzyme solution and perform the treatment while stirring with a stirring means. As such a stirring means, from the viewpoint of efficiently releasing the adhesive stem cells contained in the extracellular matrix layer of the fetal appendages, for example, a stirrer or a shaker can be used, but it is not limited thereto. The stirring speed is not particularly limited, but when a stirrer or a shaker is used, for example, it is 10 rpm or more, 30 rpm or more, or 50 rpm or more. Also, the stirring speed is not particularly limited, but when a stirrer or a shaker is used, for example, it is 100 rpm or less, 80 rpm or less, or 60 rpm or less. The enzymatic treatment time is not particularly limited, but for example, it is 10 minutes or more, 30 minutes or more, 50 minutes or more, 70 minutes or more, or 90 minutes or more. Also, the enzymatic treatment time is not particularly limited, but for example, it is 6 hours or less, 4 hours or less, 2 hours or less, 100 minutes or less. The enzymatic treatment temperature is not particularly limited, but for example, it is 16°C or more, 20°C or more, 24°C or more, 28°C or more, 32°C or more, or 36°C or more. Also, the enzymatic treatment temperature is not particularly limited, but for example, it is 40°C or less, 39°C or less, or 38°C or less.

[0070] In the production method of the present invention, if desired, adherent stem cells released can be separated and / or recovered from an enzyme solution containing the released adherent stem cells by known methods such as filtration, centrifugation, hollow fiber separation membranes, cell sorters, etc. Preferably, an enzyme solution containing the released adherent stem cells is filtered through a filter. In the mode of filtering the enzyme solution through a filter, only the released cells pass through the filter, and the undegraded epithelial cell layer cannot pass through the filter and remains on the filter. Therefore, not only can the released adherent stem cells be easily separated and / or recovered, but also the risk of contamination such as bacteria and viruses can be reduced. The filter is not particularly limited, and for example, a mesh filter can be mentioned. The pore size (mesh size) of the mesh filter is not particularly limited, and for example, it is 40 μm or more, 60 μm or more, 80 μm or more, or 90 μm or more. Also, the pore size of the mesh filter is not particularly limited, and for example, it is 200 μm or less, 180 μm or less, 160 μm or less, 140 μm or less, 120 μm or less, or 100 μm or less. Regarding the filtration rate, it is not particularly limited, but by setting the pore size of the mesh filter within the above range, the enzyme solution containing adherent stem cells can be filtered by natural drop, thereby preventing a decrease in cell viability.

[0071] As the material of the mesh filter, nylon is preferably used. Tubes containing nylon mesh filters of 40 μm, 70 μm, 95 μm or 100 μm such as Falcon cell strainers commonly used for research can be used. Also, medical mesh cloths (nylon and polyester) used in hemodialysis etc. can be used. Furthermore, arterial filters (polyester mesh filters, pore size: 40 μm or more and 120 μm or less) used during extracorporeal circulation can also be used. Other materials, for example, stainless steel mesh filters etc. can also be used.

[0072] When passing adherent stem cells through a filter, natural dropping (free fall) is preferred. Although forced passage through a filter such as suction using a pump is also possible, it is desirable to apply as weak a pressure as possible in order to avoid damaging the cells.

[0073] The adherent stem cells that have passed through the filter can be recovered by centrifugation after diluting the filtrate with a medium or balanced salt buffer in an amount twice or more the volume. As the balanced salt buffer, physiological saline, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), phosphate buffer (PBS), etc. can be used, but are not limited thereto.

[0074] The cell population obtained in the above cell population acquisition step is cultured under conditions that maintain a ratio of adherent stem cells that are positive for KCNAB1 in the cell population of 85% or more. The above conditions are useful as an index when obtaining a cell population containing adherent stem cells maintaining a normal karyotype. The culturing method is not particularly limited as long as it satisfies the above index. Such methods include, for example, sorting a cell population that satisfies the above index using a cell sorter, and culturing a cell population under conditions that satisfy the above index.

[0075] As a culturing method that satisfies the above index, for example, a step of repeatedly seeding and culturing a cell population in an uncoated plastic culture vessel at a density of 100 to 20,000 cells / cm 2 can be mentioned. The lower limit of the density when seeding the cell population is more preferably 200 cells / cm 2 or more, more preferably 400 cells / cm 2 or more, more preferably 600 cells / cm 2 or more, more preferably 800 cells / cm 2 or more, more preferably 1000 cells / cm 2 or more, more preferably 1200 cells / cm 2 or more, more preferably 1400 cells / cm 2or more, more preferably 1,600 cells / cm 2 or more, more preferably 1,800 cells / cm 2 or more, more preferably 2,000 cells / cm 2 or more. The upper limit of the density when seeding the cell population is more preferably 18,000 cells / cm 2 or less, more preferably 16,000 cells / cm 2 or less, more preferably 14,000 cells / cm 2 or less, more preferably 12,000 cells / cm 2 or less, more preferably 10,000 cells / cm 2 or less, more preferably 8,000 cells / cm 2 or less.

[0076] As another culture method that satisfies the above index, for example, a step of seeding a cell population at a density of 100 to 20,000 cells / cm 2 in a plastic culture vessel coated with a coating agent and repeating the culture a plurality of times can be mentioned. The preferable conditions for the density when seeding the cell population are the same as the above-mentioned conditions.

[0077] Examples of the coating agent include, but are not limited to, extracellular matrix, fibronectin, vitronectin, osteopontin, laminin, entactin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, gelatin, poly-L-ornithine, poly-D-lysine, Matrigel (registered trademark) matrix.

[0078] The medium used for the above culture can be prepared by using an arbitrary liquid medium for animal cell culture as a basal medium and appropriately adding other components (albumin, serum, serum replacement reagent, growth factor, human platelet lysate, etc.) as necessary.

[0079] As the basal medium, media such as BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove’s Modified Dulbecco’s Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco’s Modified Eagle’s Medium), Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer’s medium, and mixed media thereof (for example, DMEM / F12 medium (Dulbecco’s Modified Eagle’s Medium / Nutrient Mixture F-12 Ham)) can be used, but it is not particularly limited.

[0080] In addition, a commercially available serum-free medium may be used as the medium for the above culture. For example, STK1 or STK2 (manufactured by DS Pharma Biomedical Co., Ltd.), EXPREP MSC Medium (manufactured by Biomimetics Sympathies Co., Ltd.), Corning stemgro human adherent stem cell medium (manufactured by Corning Inc.), etc. can be mentioned, but it is not particularly limited.

[0081] Examples of other components to be added to the basal medium include albumin, serum, serum replacement reagents, growth factors, or human platelet lysate. In the mode of adding albumin to the basal medium, the concentration of albumin is preferably more than 0.05% and 5% or less. In the mode of adding serum to the basal medium, the concentration of serum is preferably 5% or more. In the mode of adding growth factors, a reagent (such as a protein, gel, polysaccharide, etc. like heparin) for stabilizing the growth factor in the medium may be further added in addition to the growth factor, or a pre-stabilized growth factor may be added to the basal medium. Growth factors can be, for example, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and their families, but are not particularly limited.

[0082] Examples of still other culture methods that satisfy the above criteria include, for example, adding human platelet lysate (hPL) to the basal medium used for culture and then culturing. The human platelet lysate is preferably inactivated and / or sterilized for bacteria and viruses. As the above human platelet lysate, commercially available human platelet lysate may be used. Examples include, but are not limited to, Stemulate (manufactured by Cook Regentec), PLTMax (manufactured by Mill Creek Life Science), UltraGRO (manufactured by AventaCell BioMedial), PLUS (manufactured by Compass Biomedical), etc.

[0083] The final concentration of human platelet lysate in the medium is preferably 1% or more, more preferably 2% or more, still more preferably 3% or more, still more preferably 4% or more, and still more preferably 5% or more. The final concentration of platelet lysate in the medium is preferably 20% or less, more preferably 18% or less, still more preferably 16% or less, still more preferably 14% or less, still more preferably 12% or less, still more preferably 10% or less, still more preferably 9% or less, still more preferably 8% or less, still more preferably 7% or less, and still more preferably 6% or less.

[0084] The timing of adding human platelet lysate is not particularly limited. For example, it can be at the beginning of the culture process, during the culture process, after purification in the culture process, immediately after n passages (n represents an integer of 1 or more), during maintenance culture, before cryopreservation, or after thawing.

[0085] The culture of adherent stem cells can be carried out, for example, in the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture vessel and cultured using the medium at a CO2 concentration of 3% or more and 5% or less and an environment of 37 °C so that the confluence rate is 95% or less. Examples of the above-mentioned medium include, but are not limited to, αMEM, M199, or a medium based on these. The cells obtained by the above-mentioned culture are cells cultured once.

[0086] Examples of the culture period of the above-mentioned single culture include, for example, 2 to 15 days, and more specifically, 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, 12 days, 14 days, or 15 days.

[0087] The cells cultured once above can be further passaged and cultured as follows, for example. First, the cells cultured once are treated with cell detachment means to detach them from the plastic culture vessel. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Finally, the cells are seeded in a plastic culture vessel and cultured using a medium at a CO2 concentration of 3% or more and 5% or less and at an environment of 37°C so that the confluence rate is 95% or less. Examples of the above medium include, but are not limited to, αMEM, M199, or a medium based on these. The cells obtained by the above passage and culture are cells passaged once. By performing the same passage and culture, cells passaged n times can be obtained (n represents an integer of 1 or more). From the viewpoint of mass-producing cells, the lower limit of the number of passages n is, for example, 1 or more, preferably 2 or more, more preferably 4 or more, still more preferably 6 or more, still more preferably 8 or more, still more preferably 10 or more, still more preferably 12 or more, still more preferably 14 or more, still more preferably 16 or more, still more preferably 18 or more, still more preferably 20 or more, still more preferably 25 or more. Also, from the viewpoint of suppressing cell aging, the upper limit of the number of passages n is preferably 50 or less, 40 or less, 30 or less, for example. As the above cell detachment means, for example, a cell detachment agent may be used. As the cell detachment agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA), etc. can be used, but it is not particularly limited. A commercially available cell detachment agent may be used as the cell detachment agent. Examples include, but are not limited to, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technologies), Accumax (manufactured by Stemcell Technologies), etc. Also, as the cell detachment means, physical cell detachment means may be used. For example, a cell scraper (manufactured by Corning) can be used, but it is not limited thereto.The cell detachment means may be used alone or in combination of a plurality thereof.

[0088] According to the production method of the present invention, adherent stem cells maintaining a normal karyotype can be obtained, and thereby a safe cell preparation (pharmaceutical composition) can be produced. The lower limit of the number of cells obtained per batch of culture (the number of cells obtained per unit surface area and per unit culture days) varies depending on the number of seeded cells, seeding density, etc. For example, 5.0×10 3 (cells / cm 2 / day) or more, 6.0×10 3 (cells / cm 2 / day) or more, 8.0×10 3 (cells / cm 2 / day) or more, 1.0×10 4 (cells / cm 2 / day) or more, 1.1×10 4 (cells / cm 2 / day) or more, or 1.2×10 4 (cells / cm 2 / day) or more. Also, the upper limit of the number of cells obtained per batch of culture is not particularly limited. For example, 1.0×10 5 (cells / cm 2 / day) or less, 8.0×10 4 (cells / cm 2 / day) or less, 6.0×10 4 (cells / cm 2 / day) or less, 4.0×10 4 (cells / cm 2 / day) or less, or 2.0×10 4 (cells / cm 2 / day) or less.

[0089] According to the production method of the present invention, adherent stem cells maintaining a normal karyotype can be obtained. Thus, the adherent stem cells obtained by the production method of the present invention can be cultured without growth arrest while maintaining a normal karyotype after the start of in vitro culture, preferably after 20 days, more preferably after 30 days, after 40 days, after 50 days, after 60 days, after 70 days, after 80 days, after 90 days, after 100 days, or after 110 days.

[0090] In addition, the adherent stem cells obtained by the production method of the present invention can be cultured without growth arrest while maintaining a normal karyotype until the doubling times reach preferably 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, or 60 times or more after the start of in vitro culture.

[0091] The production method of the present invention may include a discrimination step of discriminating a population containing adherent stem cells maintaining a normal karyotype using, as an index, the ratio of adherent stem cells positive for KCNAB1 being 85% or more in a cell population containing adherent stem cells.

[0092] The means for discriminating the cell population containing the adherent stem cells is preferably flow cytometry, microarray, RT-PCR, and / or quantitative RT-PCR.

[0093] The timing for performing the above discrimination is not particularly limited. For example, it may be immediately after separating cells from a biological sample, during the culture process, after purification in the culture process, immediately after n passages (n represents an integer of 1 or more), during maintenance culture, before cryopreservation, after thawing, or before formulating as a pharmaceutical composition.

[0094] In addition, the production method of the present invention can include a step of selectively separating a cell population containing the adherent stem cells after identifying the cell population containing the adherent stem cells using, as an index, the ratio of the adherent stem cells showing positive for KCNAB1 being 85% or more. The means for selectively separating the identified cell population is not particularly limited, and examples include cell sorting by a cell sorter, purification of the cell population by culture, and the like.

[0095] In addition, the production method of the present invention can include a step of cryopreserving the cell population containing the adherent stem cells. In an embodiment including the step of cryopreserving the cell population, after thawing the cell population, the cell population may be separated, recovered, and / or cultured as necessary. Also, after thawing the cell population, it may be used as it is.

[0096] The means for cryopreserving the cell population containing the adherent stem cells is not particularly limited, and examples include a programmable freezer, a deep freezer, immersion in liquid nitrogen, and the like. The temperature during freezing is preferably -30°C or lower, -40°C or lower, -50°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -150°C or lower, -180°C or lower, or -196°C (liquid nitrogen temperature) or lower. A preferable freezing rate during freezing is, for example, -1°C / min, -2°C / min, -5°C / min, -9°C / min, -10°C / min, -11°C / min, or -15°C / min. When a programmable freezer is used as such a freezing means, for example, the temperature can be lowered to a temperature between -50°C and -30°C (for example, -40°C) at a freezing rate of -2°C / min or more and -1°C / min or less, and further lowered to a temperature between -100°C and -80°C (for example, -90°C) at a freezing rate of -11°C / min or more and -9°C / min or less (for example, -10°C / min). Also, when immersion in liquid nitrogen is used as the above freezing means, for example, the temperature can be rapidly lowered to -196°C for freezing, and then cryopreserved in liquid nitrogen (vapor phase).

[0097] When freezing by the above-described freezing means, the above cell population may be frozen while contained in an arbitrary storage container. Examples of such storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, transfusion bags, and the like.

[0098] When freezing by the above-described freezing means, the above cell population may be frozen in an arbitrary cryopreservation solution. As the above cryopreservation solution, a commercially available cryopreservation solution may be used. For example, CP-1 (registered trademark) (manufactured by Kanto Chemical Co., Inc.), BAMBANKER (manufactured by Rintech, Inc.), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), ReproCryo RM (manufactured by ReproCell Inc.), CryoNovo (manufactured by Akron Biotechnology), MSC Freezing Solution (manufactured by Biological Industries), CryoStor (manufactured by HemaCare), and the like can be mentioned, but are not limited thereto.

[0099] The above cryopreservation solution can contain a polysaccharide at a predetermined concentration. Preferred concentrations of the polysaccharide are, for example, 1% by mass or more, 2% by mass or more, 4% by mass or more, or 6% by mass or more. Also, preferred concentrations of the polysaccharide are, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, or 13% by mass or less. Examples of the polysaccharide include, but are not limited to, hydroxyethyl starch (HES) or dextran (such as Dextran40).

[0100] The above cryopreservation solution can contain dimethyl sulfoxide (DMSO) at a predetermined concentration. Preferred concentrations of DMSO are, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, or 5% by mass or more. Also, preferred concentrations of DMSO are, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, 12% by mass or less, or 10% by mass or less.

[0101] The above cryopreservation solution may contain albumin at a predetermined concentration greater than 0% by mass. Preferred concentrations of albumin are, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, or 4% by mass or more. Also, preferred concentrations of albumin are, for example, 30% by mass or less, 20% by mass or less, 10% by mass or less, or 9% by mass or less. Examples of albumin include, but are not limited to, bovine serum albumin (BSA), mouse albumin, human albumin, and the like.

[0102] The production method of the present invention can include a step of washing a cell population containing the adherent stem cells. Examples of the washing solution used in the step of washing the cell population containing the above adherent stem cells include, but are not limited to, physiological saline, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), phosphate buffer (PBS), and the like. By washing the cell population, allergens, endotoxins, etc. can be reduced or removed. Examples of the above allergens include, but are not limited to, bovine serum albumin (BSA), porcine trypsin, porcine heparin, and the like.

[0103] The production method of the present invention can include a step of removing unwanted cell aggregates from the cell population containing the adherent stem cells as needed. The step of removing unwanted cell aggregates from the cell population containing the above adherent stem cells may be a step including a step of filter-filtering a cell population (cell suspension) containing adherent stem cells.

[0104] The production method of the present invention can include a step of filling a storage container with the cell population containing the adherent stem cells. Examples of the above storage container include, but are not limited to, cryotubes, cryovials, freezing bags, infusion bags, and the like.

[0105] [4] Method for monitoring karyotype abnormality of adherent stem cells, method for evaluating a donor and / or a biological sample collected from a donor, and method for determining and / or predicting optimal enzyme treatment conditions In the present invention, by measuring, using as an index that the ratio of adhesive stem cells that are positive for KCNAB1 in a cell population containing adhesive stem cells is 85% or more (preferably by measuring over time), the karyotype abnormality of the adhesive stem cells can be monitored. As the steps that require the said monitoring, for example, are the steps of culturing, cryopreserving and / or formulating.

[0106] In the step of culturing, by measuring the index over time, the change in the karyotype abnormality of the adhesive stem cells can be grasped and predicted quickly and simply. In a cell population containing adhesive stem cells that satisfy the above index, it can be understood that the adhesive stem cells maintain a normal karyotype. On the other hand, when the culture state deviating from the above index continues, it can be predicted that the karyotype abnormality of the adhesive stem cells is increasing. When it is read from the index that the karyotype abnormality is increasing, by appropriately changing the culture conditions (such as changes in seeding density, medium, growth factor, serum, etc.) as necessary, the expression of the karyotype abnormality of the adhesive stem cells can be suppressed. Also, when the above index is not satisfied, for example, by using cell sorting technology, a cell population containing adhesive stem cells that satisfy the above index can be separated. By reseeding the adhesive stem cells in the said cell population and performing subculture, the expression of the karyotype abnormality of the adhesive stem cells can be suppressed. In the initial stage of culturing, the culture conditions (such as changes in seeding density, medium, growth factor, serum, etc.) are designed so as to satisfy the said index at the final stage of the step, and it is sufficient that the above index can be satisfied at least at the final stage.

[0107] In the present invention, a cell population containing adhesive stem cells is obtained from a donor, the ratio of adhesive stem cells that are positive for KCNAB1 is measured, and the quality of the donor itself and / or a biological sample collected from the donor is evaluated by using as an index that the ratio of adhesive stem cells that are positive for KCNAB1 in the cell population is 85% or more. When a cell population containing adhesive stem cells that meets the above index is obtained (preferably easily obtained), it can be confirmed that the quality of the donor and / or a biological sample collected from the donor is good. On the other hand, when the above ratio in the cell population containing the adhesive stem cells deviates from the above index, since the quality of the biological sample collected from the donor is poor, the occurrence of karyotypic abnormalities in the adhesive stem cells can be suppressed by appropriately changing the culture conditions (such as changes in seeding density, medium, growth factors, serum). Further, when the above ratio in the cell population containing the adhesive stem cells deviates from the above index, for example, by using cell sorting technology, a cell population containing adhesive stem cells that meets the above index is fractionated, and the adhesive stem cells in the cell population are seeded and cultured, whereby the karyotypic abnormalities of the adhesive stem cells can be reduced. Alternatively, by discarding the biological sample that has deviated from the above index without culturing, the risk of obtaining a large amount of adhesive stem cells with poor quality can be reduced. In the initial stage of culture, the culture conditions (such as changes in seeding density, medium, growth factors, serum) are designed so as to meet the above index at the final stage of the process, and it is only necessary to be able to meet the above index at least at the final stage. In addition, when confirming the quality of a biological sample collected from a donor, the preparation and treatment method of the biological sample and the culture method of the cell population are not particularly limited, and any method can be adopted.

[0108] In the present invention, for a cell population obtained by enzymatically treating a biological sample collected from a donor, the ratio of adherent stem cells that are positive for KCNAB1 is measured, and by evaluating using as an index that the ratio of adherent stem cells that are positive for KCNAB1 in the cell population is 85% or more, the optimal enzymatic treatment conditions for the biological sample can be determined and / or predicted. When a cell population containing adherent stem cells that satisfy the above index is obtained (preferably easily obtained), it can be determined and / or predicted that the enzymatic treatment method for the biological sample collected from the donor is appropriate. On the other hand, when a culture state deviating from the above index continues, it can be determined and / or predicted that the enzymatic treatment method for the biological sample collected from the donor is inappropriate. In addition, when determining and / or predicting the optimal enzymatic treatment method, the preparation and treatment method of the biological sample and the culture method of the cell population are not particularly limited, and any method can be adopted.

[0109] The above index may be measured at the necessary timing and is not particularly limited, but examples include immediately after separating cells from the biological sample, during the culture process, after purification in the culture process, immediately after n passages (n represents an integer of 1 or more), during maintenance culture, before cryopreservation, after thawing, or before formulating as a pharmaceutical composition.

[0110] [5] Pharmaceutical composition The cell population containing adherent stem cells according to the present invention can be used as a pharmaceutical composition. That is, according to the present invention, there is provided a pharmaceutical composition comprising the cell population containing adherent stem cells according to the present invention and a pharmaceutically acceptable medium. Further, according to the present invention, there is provided a pharmaceutical composition comprising the cell population containing adherent stem cells according to the present invention and other administrable cells.

[0111] The pharmaceutical composition of the present invention can be used as a cell therapy agent, for example, as a therapeutic agent for intractable diseases. The pharmaceutical composition of the present invention can be used as a therapeutic agent for diseases selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injuries, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancer. By administering the pharmaceutical composition of the present invention in an amount capable of measuring the effect at the treatment site, the above diseases can be treated.

[0112] According to the present invention, there is provided a cell population containing adherent stem cells according to the present invention for use in a pharmaceutical composition. According to the present invention, there is provided a cell population containing adherent stem cells according to the present invention for use in a cell therapy agent.

[0113] According to the present invention, there is provided a cell population containing adherent stem cells according to the present invention for use in the treatment of diseases selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injuries, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancer.

[0114] According to the present invention, there is provided a cell population comprising adherent stem cells according to the present invention, which is administered to a patient or subject and used for myocardial regeneration, cardiomyocyte production, angiogenesis, blood vessel repair, or suppression of immune response.

[0115] According to the present invention, there are provided a method for transplanting cells into a patient or subject, which comprises administering a therapeutically effective amount of a cell population comprising adherent stem cells according to the present invention to the patient or subject, and a method for treating a disease of the patient or subject.

[0116] According to the present invention, there is provided the use of a cell population comprising adherent stem cells according to the present invention for the manufacture of a pharmaceutical composition. According to the present invention, there is provided the use of a cell population comprising adherent stem cells according to the present invention for the manufacture of a cell therapy agent.

[0117] According to the present invention, there is provided the use of a cell population comprising adherent stem cells according to the present invention for the manufacture of a therapeutic agent for a disease selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscus injury, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial damage, fibrosis, lung diseases, and cancer.

[0118] According to the present invention, there is provided the use of a cell population comprising adherent stem cells according to the present invention for the manufacture of a therapeutic agent necessary for myocardial regeneration, cardiomyocyte production, angiogenesis, blood vessel repair, or suppression of immune response, which is administered to a patient or subject.

[0119] The pharmaceutical composition of the present invention may be a cell population containing adhesive stem cells diluted with a pharmaceutically acceptable medium. The above-mentioned pharmaceutically acceptable medium is not particularly limited as long as it is a solution that can be administered to a patient or subject. The pharmaceutically acceptable medium may be an infusion preparation, for example, water for injection, physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, amino acid solution, starting solution (solution No. 1), dehydration replenishment solution (solution No. 2), maintenance infusion (solution No. 3), postoperative recovery solution (solution No. 4), Plasma-Lyte A (registered trademark), etc., but is not limited thereto.

[0120] As used herein, "patient or subject" typically refers to a human, but may also be other animals. Examples of other animals include mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese macaques), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), etc., and birds such as chickens, quails, etc., but are not limited thereto.

[0121] As used herein, "treatment" includes, for example, significantly improving at least one of the life prognosis, functional prognosis, survival rate, weight loss, anemia, diarrhea, hematochezia, abdominal pain, fever, anorexia, malnutrition, vomiting, fatigue, rash, inflammation, ulcer, erosion, fistula, stricture, intestinal obstruction, internal bleeding, rectal bleeding, spasm, pain, decreased liver function, decreased heart function, decreased lung function, or blood test items of a patient or subject, but is not limited thereto.

[0122] The pharmaceutical composition of the present invention may contain any components used in the treatment of a patient or subject. Examples of the above components include salts (e.g., physiological saline, Ringer's solution, bicarbonate infusion), polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, medium components (e.g., components contained in RPMI1640 medium, etc.), etc., but are not limited thereto.

[0123] The pharmaceutical composition of the present invention may contain various additives for increasing storage stability, isotonicity, absorbability and / or viscosity, such as emulsifiers, dispersants, buffers, preservatives, wetting agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters, etc. Examples of the thickener include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, etc. The concentration of the thickener can be arbitrarily set within a range that is safe when administered to a patient or subject and achieves the desired viscosity, depending on the thickener selected.

[0124] In addition to adhesive stem cells, the pharmaceutical composition of the present invention may contain one or more other pharmaceuticals. Examples of the above-mentioned other pharmaceuticals include, but are not limited to, antibiotics, albumin preparations, vitamin preparations, anti-inflammatory agents, etc. Examples of the above anti-inflammatory agents include, but are not limited to, 5-aminosalicylic acid preparations, steroid preparations, immunosuppressants, biological preparations, etc. Examples of the above 5-aminosalicylic acid preparations include, but are not limited to, sulfasalazine, mesalazine, etc. Examples of the above steroid preparations include, but are not limited to, cortisone, prednisolone, methylprednisolone, etc. Examples of the above immunosuppressants include, but are not limited to, tacrolimus, cyclosporine, methotrexate, azathioprine, 6-mercaptopurine, etc. Examples of the above biological preparations include, but are not limited to, infliximab, adalimumab, ustekinumab, secukinumab, ixekizumab, brodalumab, tocilizumab, vedolizumab, filgotinib, golimumab, certolizumab pegol, abatacept, etanercept, etc.

[0125] In addition, the other medicaments described above may be other administrable cells. Examples of other administrable cells include, but are not particularly limited to, blood-derived cells (leukocytes, erythrocytes, monocytes, etc.), vascular endothelial cells, vascular endothelial progenitor cells, pericytes, vascular wall cells, fibroblasts, skeletal myoblasts, epithelial cells, stromal cells, mature adipocytes, and the like.

[0126] The pH of the pharmaceutical composition of the present invention can be a pH near neutrality, for example, pH 5.5 or higher, pH 6.5 or higher, or pH 7.0 or higher, and can also be pH 10.5 or lower, pH 9.5 or lower, pH 8.5 or lower, or pH 8.0 or lower, but is not limited thereto.

[0127] The cell concentration of the pharmaceutical composition of the present invention is such that when administered to a patient or subject, a cell concentration can be obtained that provides a therapeutic effect against the disease as compared to a patient or subject who has not been administered. The specific cell concentration can be appropriately determined depending on the administration form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject. The lower limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited, but for example, 1.0×10 5 cells / mL or more, 1.0×10 6 cells / mL or more, 1.2×10 6 cells / mL or more, 1.4×10 6 cells / mL or more, 1.6×10 6 cells / mL or more, 1.8×10 6 cells / mL or more, 2.0×10 6 cells / mL or more, 3.0×10 6 cells / mL or more, 4.0×10 6 cells / mL or more, 5.0×10 6 cells / mL or more, 6.0×10 6 cells / mL or more, 7.0×10 6 cells / mL or more, 8.0×10 6 cells / mL or more, 9.0×10 6 cells / mL or more, 9.5×10 6 cells / mL or more, or 1.0×10 7 cells / mL or more. The upper limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited, but for example, 1.0×10 10 cells / mL or less, 1.0×109 cells / mL or less, 8.0×10 8 cells / mL or less, 6.0×10 8 cells / mL or less, 4.0×10 8 cells / mL or less, 2.0×10 8 cells / mL or less, or 1.0×10 8 cells / mL or less.

[0128] The pharmaceutical composition of the present invention is preferably a liquid preparation, more preferably a liquid preparation for injection. As the liquid preparation for injection, for example, in International Publication WO2011 / 043136, Japanese Patent Application Laid-Open No. 2013-256510, etc., liquid preparations suitable for injection are known. The pharmaceutical composition of the present invention can also be the liquid preparation for injection described in the above documents. Further, the above liquid preparation may be a cell suspension or a liquid preparation in which cells are dispersed in the liquid preparation. Furthermore, the form of the cells contained in the liquid preparation is not particularly limited, and for example, it may be single cells or cell aggregates.

[0129] When the pharmaceutical composition of the present invention is a liquid preparation for injection, the lower limit of the cell concentration of the liquid preparation for injection is 1.0×10 6 cells / mL or more, 1.2×10 6 cells / mL or more, 1.4×10 6 cells / mL or more, 1.6×10 6 cells / mL or more, 1.8×10 6 cells / mL or more, 2.0×10 6 cells / mL or more, 3.0×10 6 cells / mL or more, 4.0×10 6 cells / mL or more, 5.0×10 6 cells / mL or more, 6.0×10 6 cells / mL or more, 7.0×10 6 cells / mL or more, 8.0×10 6 cells / mL or more, 9.0×10 6 cells / mL or more, 9.5×10 6 cells / mL or more, or 1.0×10 7 cells / mL or more is preferable. Also, the upper limit of the cell concentration of the liquid preparation for injection is 1.0×109 cells / mL or less, 8.0×10 8 cells / mL or less, 6.0×10 8 cells / mL or less, 4.0×10 8 cells / mL or less, 2.0×10 8 cells / mL or less, or 1.0×10 8 cells / mL or less is preferred.

[0130] Further, according to one aspect of the present invention, the pharmaceutical composition of the present invention may be a preparation for transplantation. The preparation for transplantation is a solid or gel preparation. For example, as the solid preparation for transplantation, a preparation for transplantation having a sheet structure or a pellet structure can be mentioned. Further, as the preparation for transplantation having a gel structure, for example, in International Publication WO2017 / 126549, a preparation for transplantation containing a gel obtained by adhering isolated cells with an adhesive (for example, fibrinogen) is known. Further, according to one aspect of the present invention, the pharmaceutical composition of the present invention may be a gel preparation in which cells and an arbitrary gel are mixed. As the gel preparation, for example, in Japanese Patent Application Laid-Open No. 2017-529362, a cell therapy agent composed of an adhesive stem cell-hydrogel composition is known. The pharmaceutical composition of the present invention can also be made into a gel preparation by using, for example, the method described in the above-mentioned document. Further, as the preparation for transplantation having a sheet structure, for example, in International Publication WO2006 / 080434, Japanese Patent Application Laid-Open No. 2016-52272, etc., a cell sheet obtained by culturing using a temperature-responsive culture dish (for example, UpCell (registered trademark) (manufactured by CellSeed)), a laminate of a sheet-like cell culture and a fibrin gel, a cell-coated sheet obtained by applying a cell suspension to a sheet-like substrate, etc. are known. The pharmaceutical composition of the present invention can also be made into various preparations for transplantation having a sheet structure by using, for example, the method described in the above-mentioned document.

[0131] The administration method of the pharmaceutical composition of the present invention is not particularly limited. For example, it includes subcutaneous injection, intradermal injection, intramuscular injection, intranodal injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, direct injection locally, direct application, or direct transplantation locally, etc. According to one aspect of the present invention, an injection solution is filled into a syringe and administered intravenously, intraarterially, intramyocardially, intraarticularly, intrahepatically, intramuscularly, epidurally, gingivally, intraventricularly, subcutaneously, intradermally, intraperitoneally, or into the portal vein through an injection needle or catheter, but it is not limited thereto. Regarding the administration method of the pharmaceutical composition, for example, in JP-A-2015-61520, Onken JE, et al. American College of Gastroenterology Conference 2006 Las Vegas, NV, Abstract 121., Garcia-Olmo D, et al. Dis Colon Rectum 2005;48:1416-23., etc., intravenous injection, intravenous drip injection, direct injection locally, direct transplantation locally, etc. are known. The pharmaceutical composition of the present invention can also be administered by various methods described in the above-mentioned documents.

[0132] As the dosage of the pharmaceutical composition of the present invention, it is an amount of cells that can obtain a therapeutic effect on a disease when administered to a patient or subject, as compared with a patient or subject who has not been administered. The specific dosage can be appropriately determined according to the administration form, administration method, purpose of use, and age, weight, symptoms, etc. of the patient or subject. The single dosage of adherent stem cells for humans is not particularly limited. For example, it is 1×10 4 cells / kg body weight or more, 1×10 5 cells / kg body weight or more, 5×10 5 cells / kg body weight or more, 1×10 6 cells / kg body weight or more, 2×10 6 cells / kg body weight or more, 4×10 6 cells / kg body weight or more, 6×10 6 cells / kg body weight or more, or 8×10 6 cells / kg body weight or more. Also, the single dosage of adherent stem cells for humans is not particularly limited. For example, it is 1×10 12 cells / kg body weight or less, 1×10 11cells / kg body weight or less, 1×10 10 cells / kg body weight or less, 1×10 9 cells / kg body weight or less, 5×10 8 cells / kg body weight or less, 1×10 8 cells / kg body weight or less, 8×10 7 cells / kg body weight or less, 6×10 7 cells / kg body weight or less, 4×10 7 cells / kg body weight or less, or 2×10 7 cells / kg body weight or less.

[0133] When the pharmaceutical composition of the present invention is a liquid preparation for injection, the single dose of adherent stem cells for injection into humans, from the viewpoint of enhancing the therapeutic effect on the disease, is 1×10 5 cells / kg body weight or more, 5×10 5 cells / kg body weight or more, 1×10 6 cells / kg body weight or more, 2×10 6 cells / kg body weight or more, 4×10 6 cells / kg body weight or more, 6×10 6 cells / kg body weight or more, or 8×10 6 cells / kg body weight or more is preferable. Also, the single dose of adherent stem cells for injection into humans in the liquid preparation for injection, from the viewpoint of facilitating the preparation and administration of the liquid preparation for injection, is 1×10 9 cells / kg body weight or less, 5×10 8 cells / kg body weight or less, 1×10 8 cells / kg body weight or less, 8×10 7 cells / kg body weight or less, 6×10 7 cells / kg body weight or less, 4×10 7 cells / kg body weight or less, or 2×10 7 cells / kg body weight or less is preferable.

[0134] The dosing frequency of the pharmaceutical composition of the present invention is a frequency that can achieve a therapeutic effect on the disease when administered to a patient or subject. The specific dosing frequency can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject, etc. For example, it can be once every 4 weeks, once every 3 weeks, once every 2 weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week.

[0135] The dosing period of the pharmaceutical composition of the present invention is a period that can achieve a therapeutic effect on the disease when administered to a patient or subject. The specific dosing period can be appropriately determined depending on the dosage form, administration method, purpose of use, and the age, weight, and symptoms of the patient or subject, etc. For example, it can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0136] The timing of administering the pharmaceutical composition of the present invention to a patient or subject is not particularly limited. For example, it can be immediately after the onset, within n days from the onset (n represents an integer of 1 or more), immediately after diagnosis, within n days from diagnosis (n represents an integer of 1 or more), before remission, during remission, after remission, before relapse, during relapse, after relapse, etc.

[0137] The pharmaceutical composition of the present invention can be stored in a frozen state until immediately before use. The temperature for frozen storage is preferably -30°C or lower, -40°C or lower, -50°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -150°C or lower, -180°C or lower, or -196°C (liquid nitrogen temperature) or lower. When administering the pharmaceutical composition of the present invention to a patient or subject, it can be rapidly thawed at 37°C and then used.

[0138] Other examples of diseases and the like that can be treated using a cell population containing adhesive stem cells in a patient or subject, further specific examples of the diseases and the like, and specific procedures for treatment can be found in Hare et al., J. Am. Coll. Cardiol., December 8, 2009; 54(24): 2277-2286, Honmou et al., Brain 2011: 134; 1790-1807, Makhoul et al., Ann. Thorac. Surg. 2013; 95: 1827-1833, Japanese Patent No. 590577, JP 2010-518096 A, JP 2012-509087 T, JP 2014-501249 T, JP 2013-256515 A, JP 2014-185173 A, JP 2010-535715 T, JP 2015-038059 A, JP 2015-110659 A, JP 2006-521121 T, JP 2009-542727 T, JP 2014-224117 A, JP 2015-061862 A, JP 2002-511094 T, JP 2004-507454 T, JP 2010-505764 T, JP 2011-514901 T, JP 2013-064003 A, JP 2015-131795 A, etc.

[0139] The present invention will be specifically described in the following examples, but the present invention is not limited by the examples.

Examples

[0140] <Comparative Example 1> In Comparative Example 1 and Example 1 shown below, indices for obtaining a cell population containing adhesive stem cells with high karyotype stability were examined. (Step 1-1: Collection of amniotic membrane) From pregnant women with elective cesarean section cases who had given informed consent, the fetal membranes and placenta, which are fetal appendages, were aseptically collected. The obtained fetal membranes and placenta were placed in a sterile bath containing physiological saline, and the amniotic membrane was manually peeled off from the cut end of the fetal membrane. The amniotic membrane was washed with Hank's balanced salt solution (without Ca·Mg) to remove the attached blood and blood clots.

[0141] (Step 1-2: Enzymatic treatment of amniotic membrane and recovery of amniotic membrane-adherent stem cells) The amniotic membrane containing an epithelial cell layer and an adherent stem cell layer was immersed in Hank's balanced salt solution (containing Ca·Mg) containing 240 PU / mL of collagenase and 200 PU / mL of dispase I, and the amniotic membrane was enzymatically treated by shaking and stirring at 37 °C for 90 minutes under the condition of 50 rpm. The undigested matter of the amniotic membrane was removed by filtering the solution after enzymatic treatment through a nylon mesh with an aperture of 95 μm, and a cell suspension containing amniotic membrane-adherent stem cells was recovered.

[0142] (Step 1-3: Culture of amniotic membrane-adherent stem cells) A cell population containing amniotic membrane-adherent stem cells obtained by the above-mentioned "enzymatic treatment of amniotic membrane and recovery of amniotic membrane-adherent stem cells" was seeded at a density of 6,000 cells / cm 2 in a CellSTACK (registered trademark) (manufactured by Corning) of a culture vessel, and adhered and cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL of basic fibroblast growth factor (bFGF) at a final concentration until sub-confluent. Then, the passage 0 cells were detached using TrypLE Select, and 1 / 5 volume of the cells was seeded in a CellSTACK (registered trademark) of the same scale as the previous culture to perform passage culture. The medium was changed once every 2 to 4 days. When sub-confluence was reached, the passage 1 cells were detached using TrypLE Select, and RPMI1640 was added so that the cell concentration became 2×10 7 cells / mL. An equal volume of CP-1 (registered trademark) solution (CP-1 (registered trademark): a solution mixed at a ratio of 25% human serum albumin = 34:16) was added thereto, transferred to cryovials at 1 mL each, slowly frozen to -80 °C, and then cryopreserved under liquid nitrogen for 1 day. Then, it was thawed and seeded at about 15,000 to 18,000 cells / cm 2The cells of the second passage were seeded into CellSTACK (registered trademark) at a density of [density value], and adherent culture was performed in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing fetal bovine serum (FBS) at a final concentration of 10% and basic fibroblast growth factor (bFGF) at 10 ng / mL until sub-confluent. Then, the cells of the second passage were detached using TrypLE Select, and subculture was performed by seeding 1 / 5 of the cells into CellSTACK (registered trademark) of the same scale as the previous culture. Medium exchange was carried out at a frequency of once every 2 - 4 days. When sub-confluence was reached, the cells of the third passage were detached using TrypLE Select, and RPMI1640 was added to make the cell concentration 4×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (CP-1 (registered trademark): a solution mixed at a ratio of 25% human serum albumin = 34:16) was added, transferred to cryovials at 1 mL each, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen for 1 day. Then, after thawing, the cells of the fourth passage were seeded into CellSTACK (registered trademark) at a density of approximately 6,000 cells / cm 2 and adherent culture was performed in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing fetal bovine serum (FBS) at a final concentration of 10% and basic fibroblast growth factor (bFGF) at 10 ng / mL until sub-confluent. Then, the cells of the fourth passage were detached using TrypLE Select, and subculture was performed by seeding 1 / 5 of the cells into CellSTACK (registered trademark) of the same scale as the previous culture. Medium exchange was carried out at a frequency of once every 2 - 4 days. When sub-confluence was reached, the cells of the fifth passage were detached using TrypLE Select, and RPMI1640 was added to make the cell concentration 4×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (CP-1 (registered trademark): a solution mixed at a ratio of 25% human serum albumin = 34:16) was added, transferred to cryovials at 1 mL each, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen.

[0143] (Project 1-4: Antigen Analysis of Amniotic Adherent Stem Cells) Regarding the 5th passage amniotic adherent stem cells cultured by the above culture method, various surface antigens (positive rate of CD73 known as an MSC marker, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rates of CD73, CD90, and CD105 were all 90% or more (specifically, CD73: 100%, CD90: 97%, CD105: 100%), and the positive rates of CD166 were all 30% or more (specifically, CD166: 98%). The negative rates of CD45, CD34, and CD326 were all 95% or more (specifically, CD45: 100%, CD34: 100%, CD326: 100%). From the above results, it was confirmed that the cells cultured by the above culture method were adherent stem cells. In addition, fixation and membrane permeabilization treatments were performed on the 3rd and 5th passage amniotic adherent stem cells cultured by the above culture method, and the ratio of cells positive for the KCNAB1 antigen was analyzed using a flow cytometer. As a result, it was less than 85% in any passage number (3rd passage: 78%, 5th passage: 81%).

[0144] In this measurement, as isotype control antibodies, FITC Mouse IgG1, κ Isotype Control (BD, catalog number: 550616) and PE Mouse IgG1, κ Isotype Control (BD, catalog number: 555749) were used. As the antibody against CD73 antigen, FITC Mouse Anti-Human CD73 (BD, catalog number: 561254) was used. As the antibody against CD90 antigen, FITC Mouse Anti-Human CD90 (BD, catalog number: 555595) was used. As the antibody against CD105 antigen, Anti-Human Antibodies FITC Conjugate (BioLegend, catalog number: 323203) was used. As the antibody against CD166 antigen, PE Mouse Anti-Human CD166 (BD, catalog number: 559263) was used. As the antibody against CD45 antigen, FITC Mouse Anti-Human CD45 (BD, catalog number: 555482) was used. As the antibody against CD34 antigen, PE Mouse Anti-Human CD34 (BD, catalog number: 555822) was used. As the antibody against CD326 antigen, FITC Mouse Anti-Human EpCAM (BD, catalog number: 347197) was used. As the antibody against KCNAB1 antigen, Kcnab1 monoclonal antibody (FITC) (Abnova, catalog number: MAB11866) was used. Surface antigen analysis and antigen analysis were performed using BD Accuri TM C6 Flow Cytometer of Becton Dickinson (BD). The measurement conditions were as follows: the number of analyzed cells was 5,000 cells, and the flow rate setting was Slow (14 μL / min). The ratio of positive cells for each antigen was calculated according to the following procedure. (1) The measurement results were plotted on a dot plot with side scatter (SSC) on the vertical axis and forward scatter (FSC) on the horizontal axis. (2) In the dot plot, all regions (gates) where the cell population with stronger fluorescence intensity was 0.5% or less among the total cells measured with the isotype control antibody were selected. (3) Of the total cells measured with an antibody corresponding to the antigen marker, the proportion of cells contained within the gate selected in (2) was calculated.

[0145] (Step 1-5: Gene expression analysis of amniotic membrane adherent stem cells) Regarding the 5th passage of amniotic membrane adherent stem cells cultured by the above culture method, gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis using a microarray was commissioned to RIKEN GENESIS Co., Ltd. Microarray analysis was performed according to the following procedures (1) to (4). Note that the following procedures (2) to (4) were performed by RIKEN GENESIS Co., Ltd. (1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with phosphate buffer (PBS), and the cells were recovered by centrifugation. Thereafter, total RNA was extracted and purified using an RNeasy Plus Mini Kit (manufactured by QIAGEN). (2) cDNA was synthesized from 100 ng of total RNA by reverse transcription reaction, and transcribed into cRNA by in vitro transcription and biotin-labeled (using the 3’IVT PLUS Reagent Kit). (3) 10.0 μg of labeled cRNA was added to hybridization buffer, and hybridization was performed on a Human GeneGenome U133A 2.0 Array (manufactured by Affymetrix) for 16 hours. After washing and phycoerythrin staining with a GeneChip Fluidics Station 450 (manufactured by Affymetrix), scanning was performed with a GeneChip Scanner 3000 7G (manufactured by Affymetrix), image analysis was performed with AGCC (Affymetrix GeneChip Command Console Software) (manufactured by Affymetrix), and quantification was performed using Affymetrix Expression Console (manufactured by Affymetrix). (4) The numerical data file was analyzed using analysis software GeneSpring GX (manufactured by Agilent Technologies). The expression levels of the respective genes were determined as relative expression levels with respect to the expression level of the SDHA gene. As a result, the KCNAB1 gene was 0.03, the SULT1E1 gene was 0.05, the MN1 gene was 0.69, and the RARRES2 gene was 0.41.

[0146] (Step 1-6: Karyotype analysis of amnion-adherent stem cells) Regarding the 3rd and 5th passage amnion-adherent stem cells cultured by the above culture method, karyotype analysis by the G-banding method was entrusted to Nippon Gene Research Laboratories, Inc. The cells frozen at the 3rd and 5th passages were thawed and seeded at a density of about 8,000 cells / cm 2 into two T25 flasks each, and 5 mL of αMEM containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) at the final concentration was added, followed by culturing for 24 hours. Then, the T25 flasks were filled with αMEM containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) at the final concentration and transported to Nippon Gene Research Laboratories, Inc. (Sendai City, Miyagi Prefecture) at room temperature. At Nippon Gene Research Laboratories, Inc., any 20 cells were collected, chromosomes were extracted from them, each chromosome was identified based on the band pattern characteristic of the chromosomes detected by the differential staining method, and the presence or absence of karyotype abnormalities such as aneuploidy and translocation was analyzed. As a result, karyotype abnormalities were observed in the cells of both the 3rd and 5th passages. Specifically, in the 3rd passage, karyotype abnormality was observed in 1 out of 20 cells, and there was an increase in the isochromosome of the long arm of chromosome 2 (+i(2)(q10)). In the 5th passage, karyotype abnormality was observed in 5 out of 20 cells, and trisomy of chromosome 2 was observed in all 5 cells.

[0147] <Example 1> (Step 2-1: Collection of amnion) Amnion was obtained by the same method as in Comparative Example 1.

[0148] (Step 2-2: Enzymatic treatment of amnion and recovery of amnion-adherent stem cells) A cell population containing amnion-adherent stem cells was obtained by the same method as in Comparative Example 1.

[0149] (Procedure 2-3: Culture of Amniotic Adhesion Stem Cells) A cell population containing amniotic adhesion stem cells obtained by the above-mentioned "enzymatic treatment of amniotic membrane and recovery of amniotic adhesion stem cells" was seeded in CellSTACK (registered trademark) at a density of 6,000 cells / cm 2 and adherently cultured in αMEM containing 5% human platelet lysate (hPL) at the final concentration until sub-confluent. Then, the passage 0 cells were detached using TrypLE Select, and 1 / 5 volume of the cells were seeded in CellSTACK (registered trademark) at the same scale as the previous culture to perform passage culture. The medium was changed once every 2 - 4 days. When sub-confluent was reached, the passage 1 cells were detached using TrypLE Select, and RPMI1640 was added to make the cell concentration 2×10 7 cells / mL. An equal volume of CP-1 (registered trademark) solution (CP-1 (registered trademark): a solution mixed at a ratio of 34:16 with 25% human serum albumin) was added, transferred to cryovials at 1 mL each, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen for 1 day. Then, after thawing, the passage 2 cells were seeded in CellSTACK (registered trademark) at a density of about 15,000 - 18,000 cells / cm 2 and adherently cultured in αMEM containing 5% human platelet lysate (hPL) at the final concentration until sub-confluent. Then, the passage 2 cells were detached using TrypLE Select, and 1 / 5 volume of the cells were seeded in CellSTACK (registered trademark) at the same scale as the previous culture to perform passage culture. The medium was changed once every 2 - 4 days. When sub-confluent was reached, the passage 3 cells were detached using TrypLE Select, and RPMI1640 was added to make the cell concentration 4×10 6 cells / mL. An equal volume of CP-1 solution (registered trademark) (CP-1 (registered trademark): a solution mixed at a ratio of 34:16 with 25% human serum albumin) was added, transferred to cryovials at 1 mL each, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen for 1 day. Then, after thawing, about 6,000 cells / cm 2The cells of the fourth passage were seeded into CellSTACK (registered trademark) at a density of [density value], and adherent culture was performed in αMEM containing 5% human platelet lysate (hPL) at a final concentration until sub-confluence. Then, the cells of the fourth passage were detached using TrypLE Select, and subculture was performed by seeding 1 / 5 volume of the cells into CellSTACK (registered trademark) at the same scale as the previous culture. Medium exchange was carried out once every 2 - 4 days. When sub-confluence was reached, the cells of the fifth passage were detached using TrypLE Select, and RPMI1640 was added so that the cell concentration became 4×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added, transferred to cryovials at 1 mL each, slowly frozen to -80°C, and then stored frozen under liquid nitrogen. After the sixth passage, all cells were seeded into CellSTACK (registered trademark) at a density of approximately 6,000 cells / cm 2 , and adherent culture was performed in αMEM containing 5% human platelet lysate (hPL) at a final concentration until sub-confluence. Then, detachment was performed using TrypLE Select, and subculture was repeated up to the ninth passage.

[0150] (Step 2-4: Antigen analysis of amniotic adherent stem cells) Regarding the amniotic adherent stem cells of the fifth passage cultured by the above culture method, various surface antigens (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rates of CD73, CD90, and CD105 were all 50% or more (specifically, CD73: 99%, CD90: 100%, CD105: 100%). The positive rates of CD166 were all 30% or more (specifically, CD166: 100%). The negative rates of CD45, CD34, and CD326 were all 95% or more (specifically, CD45: 100%, CD34: 100%, CD326: 100%). From the above results, it was confirmed that the cells cultured by the above culture method were adherent stem cells. Regarding the 3rd and 5th passage amniotic membrane adherent stem cells cultured by the above culture method, the ratio of cells positive for the KCNAB1 antigen was analyzed using a flow cytometer. As a result, it was 85% or more in any passage number (3rd passage: 91%, 5th passage: 90%). Therefore, it was found that the 5th passage amniotic membrane adherent stem cells of Example 1 satisfy the condition that the ratio of adherent stem cells positive for KCNAB1 is 85% or more. In addition, the measurement method and reagent of this measurement are the same as those of Comparative Example 1.

[0151] (Step 2-5: Gene expression analysis of amniotic membrane adherent stem cells) Regarding the 5th passage amniotic membrane adherent stem cells cultured by the above culture method, the gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis by microarray was entrusted to RIKEN Genesis Co., Ltd. Microarray analysis was performed in the same manner as in Comparative Example 1. The expression level of each gene was determined as the relative expression level with respect to the expression level of the SDHA gene. As a result, it was KCNAB1 gene: 0.44, SULT1E1 gene: 0.68, MN1 gene: 1.77, RARRES2 gene: 0.003.

[0152] (Step 2-6: Karyotype analysis of amniotic membrane adherent stem cells) Regarding the 3rd and 5th passage amniotic membrane adherent stem cells cultured by the above culture method, karyotype analysis by the G-banding method was entrusted to Japan Gene Research Laboratories, Inc. The cells frozen at the 3rd and 5th passages were thawed, and about 8,000 cells / cm 2Seeded into two T25 flasks each at a density of , added 5 mL of αMEM containing 5% human platelet lysate (hPL) at the final concentration, and cultured for 24 hours. Thereafter, the T25 flasks were filled with αMEM containing 5% human platelet lysate (hPL) at the final concentration and transported to Japan Gene Research Institute Co., Ltd. (Sendai City, Miyagi Prefecture) at room temperature. At Japan Gene Research Institute Co., Ltd., any 20 cells were collected, chromosomes were extracted therefrom, each chromosome was identified based on the band pattern characteristic of the chromosomes detected by the differential staining method, and the presence or absence of karyotype abnormalities such as aneuploidy and translocation was analyzed. As a result, all cells at the 3rd and 5th passages maintained a normal karyotype.

[0153] Table 1 summarizes the positive rate of KCNAB1 and the results of karyotype analysis in Comparative Example 1 and Example 1.

[0154]

Table 1

[0155] From the above results, it was found that cell populations with a positive rate of KCNAB1 of 85% or more maintained a normal karyotype. Also, it was suggested that the condition that the positive rate of KCNAB1 is 85% or more is effective as an index for obtaining a cell population containing adherent stem cells that maintain a normal karyotype. That is, according to the present invention, by using the condition that the positive rate of KCNAB1 is 85% or more as an index, a cell population containing adherent stem cells that maintain a normal karyotype can be obtained. Thereby, a safe cell preparation suitable for clinical use can be manufactured.

[0156] Also, if the condition that the positive rate of KCNAB1 is 85% or more is used as an index, it is possible to evaluate (judge and / or predict) the karyotype stability of a biological sample without performing karyotype analysis that usually requires an evaluation period of about one month. Furthermore, according to the present invention, in a cell population containing adhesive stem cells, by using the positive rate of KCNAB1 being 85% or more as an index, it is possible to simply and quickly monitor the karyotype stability of a biological sample (the presence or absence of karyotype abnormalities in adhesive stem cells) over time. This enables a reduction in the cost required for quality evaluation of biological samples and a shortening of the period required for quality evaluation, leading to a reduction in the manufacturing cost of cell preparations.

[0157] <Example 2> From pregnant women with elective cesarean section cases who had obtained informed consent from two donors (#1, #2) different from the donors in Comparative Example 1 and Example 1, the fetal membranes and placenta, which are fetal appendages, were aseptically collected, and each fetal appendage was processed according to "Step 1-1: Collection of amniotic membrane" and "Step 1-2: Enzymatic treatment of amniotic membrane and recovery of amniotic membrane adhesive stem cells" to obtain amniotic membrane adhesive stem cells. Cell populations containing amniotic membrane adhesive stem cells obtained from donors #1 and #2 were cultured by the method of "Example 1: Culture of amniotic membrane adhesive stem cells" to obtain cell populations at the 5th passage.

[0158] Regarding the amniotic membrane adhesive stem cells cultured by the above culture method, in the same manner as in "Step 1-4: Antigen analysis of amniotic membrane adhesive stem cells", the ratio of cells that are positive for KCNAB1 can be analyzed using a flow cytometer. The positive rate of KCNAB1 was 85% or more for #1 (specifically, #1: 94%) and less than 85% for #2 (specifically, 49%). Also, regarding the amniotic membrane adhesive stem cells cultured by the above culture method, the karyotype can be analyzed in the same manner as in "Step 1-6: Karyotype analysis of amniotic membrane adhesive stem cells". As a result of karyotype analysis, all cells of #1 maintained a normal karyotype, but karyotype abnormalities were observed in #2 (specifically, karyotype abnormalities were observed in 1 out of 20 cells, and there was a structural abnormality of chromosome 13 (add(13)(p11.2))). Table 2 summarizes the positive rate of KCNAB1 and the results of karyotype analysis in Example 2.

[0159]

Table 2

[0160] <Example 3> In Example 3 shown below, amniotic adherent stem cells with different donors, enzyme treatment conditions, and culture conditions were obtained compared with Comparative Example 1, Example 1, and Example 2. Amniotic membranes and placentas, which are fetal appendages, were aseptically collected from pregnant women with elective cesarean section cases who had obtained informed consent from three different donors (#3 to #5) different from those in Comparative Example 1, Example 1, and Example 2.

[0161] (Step 3-1: Collection of amniotic membrane) The amniotic membrane was obtained in the same manner as in Comparative Example 1.

[0162] (Step 3-2: Enzyme treatment of amniotic membrane and recovery of amniotic adherent stem cells) The amniotic membrane containing an epithelial cell layer and an adherent stem cell layer was immersed in Hank's balanced salt solution (containing Ca·Mg) containing 480 PU / mL collagenase and 400 PU / mL dispase I, and the amniotic membrane was enzyme-treated by shaking and stirring at 37 °C for 90 minutes under the condition of 50 rpm. The solution after the enzyme treatment was filtered through a nylon mesh with an aperture of 95 μm to remove undigested matter of the amniotic membrane, and a cell suspension containing amniotic adherent stem cells was recovered.

[0163] (Step 3-3: Culture of amniotic adherent stem cells) The cell population containing amniotic adherent stem cells obtained in the above-mentioned "Enzyme treatment of amniotic membrane and recovery of amniotic adherent stem cells" was seeded in CellSTACK (registered trademark) at a density of 1,000 cells / cm 2 and adherently cultured in αMEM containing 5% human platelet lysate (hPL) at a final concentration until sub-confluent. The medium was changed once every 3 to 5 days. Then, the passage 0 cells were detached using TrypLE Select, and the cell concentration was 2×10 7Physiological saline was added to achieve a concentration of cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added thereto, and 1 mL aliquots were transferred to cryovials and slowly frozen to -80°C, and then stored frozen under liquid nitrogen for 1 day. Thereafter, it was thawed and seeded onto CellSTACK (registered trademark) at a density of approximately 1,000 cells / cm 2 and cultured adherently for 5 days in αMEM containing 5% human platelet lysate (hPL) at a final concentration until sub-confluent. Thereafter, the passage 1 cells were detached using TrypLE Select, and physiological saline was added to achieve a cell concentration of 2×10 7 cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution mixed at a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added thereto, and 1 mL aliquots were transferred to cryovials and slowly frozen to -80°C, and then stored frozen under liquid nitrogen for 1 day. Thereafter, it was thawed and seeded onto CellSTACK (registered trademark) at a density of approximately 1,000 cells / cm 2 and cultured adherently for 5 days in αMEM containing 5% human platelet lysate (hPL) at a final concentration until sub-confluent. Thereafter, the passage 2 cells were detached using TrypLE Select, and physiological saline was added to achieve a cell concentration of 4×10 6 cells / mL. An equal volume of CP-1 solution (registered trademark) (a solution mixed at a ratio of CP-1 (registered trademark): 25% human serum albumin = 34:16) was added thereto, and 1 mL aliquots were transferred to cryovials and slowly frozen to -80°C, and then stored frozen under liquid nitrogen.

[0164] (Step 3-4: Antigen analysis of amniotic adherent stem cells) Regarding the second-passage amniotic adherent stem cells cultured by the above culture method, various surface antigens (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rates of CD73, CD90, and CD105 were all 50% or more (specifically, all were 100% for #3 to #5). The positive rates of CD166 were all 30% or more (specifically, #3: 99%, #4: 100%, #5: 99%). The negative rates of CD45, CD34, and CD326 were all 95% or more (specifically, #3: 99%, #4: 100%, #5: 100%). From the above results, it was confirmed that the cells cultured by the above culture method are adherent stem cells. Also, regarding the second-passage amniotic adherent stem cells cultured by the above culture method, the ratio of cells positive for the KCNAB1 antigen was analyzed using a flow cytometer. As a result, it was 85% or more in all donors (#3: 98.2%, #4: 99.9%, #5: 99.7%). Therefore, it was found that the second-passage amniotic adherent stem cells of Example 3 satisfy the condition that the ratio of adherent stem cells positive for KCNAB1 is 85% or more for all donors from #3 to #5. The method and reagent for this measurement are the same as those in Comparative Example 1.

[0165] (Step 3-5: Gene Expression Analysis of Amniotic Adherent Stem Cells) Regarding the second-passage amniotic adherent stem cells cultured by the above culture method, gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis by microarray was entrusted to RIKEN GENESIS Co., Ltd. Microarray analysis was performed in the same manner as Comparative Example 1. The expression level of each gene was determined as the relative expression level relative to the expression level of the SDHA gene. As a result, for #3, the KCNAB1 gene was 0.15, the SULT1E1 gene was 0.36, the MN1 gene was 0.81, and the RARRES2 gene was 0.01. For #4, the KCNAB1 gene was 0.07, the SULT1E1 gene was 0.16, the MN1 gene was 0.75, and the RARRES2 gene was 0.001. For #5, the KCNAB1 gene was 0.11, the SULT1E1 gene was 0.27, the MN1 gene was 1.38, and the RARRES2 gene was 0.001.

[0166] Furthermore, with respect to the passage 2 amniotic adhesion stem cells cultured by the above culture method, gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis by quantitative PCR was performed. Gene expression analysis by quantitative PCR was performed according to the following procedures (1) to (4). (1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with phosphate buffer (PBS), and the cells were recovered by centrifugation. Then, total RNA was extracted and purified using an RNeasy Plus Mini Kit (manufactured by QIAGEN). (2) ReverTra Ace qPCR RT Master Mix (manufactured by Toyobo Co., Ltd.) was added to the purified total RNA, and cDNA was synthesized by reverse transcription reaction using the total RNA as a template. (3) The synthesized cDNA was mixed with Taqman Fast Advanced Master Mix (manufactured by Applied Biosystems) and primers (Taqman Gene Expression Assay, manufactured by Thermo Fisher, Assay ID for SDHA primers: Hs00188166_m1, Assay ID for KCNAB1 primers: Hs00185764_m1, Assay ID for SULT1E1 primers: Hs00960938_m1, Assay ID for MN1 primers: Hs00159202_m1, Assay ID for RARRES2 primers: Hs00414615_m1), and the mixture was injected into a 96-well plate for quantitative PCR. (4) The ΔCt values for SDHA in each sample were analyzed using the StepOnePlus Real-Time PCR System (manufactured by Applied Biosystems), and the relative expression levels (2^(-ΔCt)) of each gene relative to the expression level of the SDHA gene in each cell were calculated. As a result, for #3, the KCNAB1 gene was 0.77, the SULT1E1 gene was 0.37, the MN1 gene was 1.1, and the RARRES2 gene was 0.0026. For #4, the KCNAB1 gene was 0.72, the SULT1E1 gene was 0.13, the MN1 gene was 1.8, and the RARRES2 gene was 0.0029. For #5, the KCNAB1 gene was 4.4, the SULT1E1 gene was 0.50, the MN1 gene was 5.9, and the RARRES2 gene was 0.0033.

[0167] (Steps 3-6: Karyotype analysis of amniotic membrane adherent stem cells) Regarding the second-passage amniotic membrane adherent stem cells cultured by the above culture method, karyotype analysis by the G-banding method was commissioned to Nippon Gene Research Laboratories, Inc. The cells frozen at the second passage were thawed, and about 8,000 cells / cm 2Seeded into two T25 flasks each at a density of [density value], added 5 mL of αMEM containing 5% human platelet lysate (hPL) at the final concentration, and cultured for 24 hours. Subsequently, filled the T25 flasks with αMEM containing 5% human platelet lysate (hPL) at the final concentration and transported them at room temperature to the Gene Research Institute of Japan, Inc. (Sendai City, Miyagi Prefecture). At the Gene Research Institute of Japan, Inc., randomly collected 20 cells, extracted chromosomes from them, identified each chromosome based on the band pattern characteristic of the chromosomes detected by the differential staining method, and analyzed the presence or absence of karyotype abnormalities such as aneuploidy and translocation. As a result, all cells at the second passage maintained a normal karyotype.

[0168] From the above results, it was found that a cell population with a positive rate of KCNAB1 of 85% or more maintained a normal karyotype. Also, it was suggested that even when amniotic adherent stem cells are collected and cultured from amniotic membranes by different methods, the quality of the donor itself and biological samples collected from the donor can be evaluated by examining that the positive rate of KCNAB1 is 85% or more. That is, according to the present invention, by using the positive rate of KCNAB1 being 85% or more as an index, a biological sample with a high content of amniotic adherent stem cells without karyotype abnormalities can be selected (donor screening). Furthermore, by using the positive rate of KCNAB1 being 85% or more as an index, the enzyme treatment conditions and culture conditions can be optimized (improvement of enzyme treatment conditions and culture conditions). Thereby, the period for quality evaluation can be shortened, and it becomes possible to reduce the period required for improvement of the manufacturing method such as enzyme treatment conditions and culture conditions.

[0169] <Example 4> A part of the amniotic adherent stem cells obtained in Example 1 above is used for the preparation of a pharmaceutical composition. Prepare a pharmaceutical composition (cell preparation) containing 2.0×10 8 cells, 6.8 mL of CP-1 solution (registered trademark), 3.2 mL of 25% human serum albumin solution, and 10 mL of RPMI1640 medium. Enclose the pharmaceutical composition in a freezing bag and store it in a frozen state. Incidentally, when in use, the pharmaceutical composition can be thawed and provided to patients.

[0170] <Reference Example> (Step 4-1: Cultivation of Bone Marrow-Derived Mesenchymal Stem Cells) Three donors (#6 - #8) of human bone marrow-derived mesenchymal stem cells (hMSC mesenchymal stem cells, manufactured by Lonza) were purchased, thawed respectively, and seeded in a φ15 cm dish at a density of 6,000 cells / cm 2 and adherently cultured until sub-confluent in a dedicated medium manufactured by Lonza. Medium replacement was carried out at a frequency of once every 3 - 5 days. Thereafter, the cells were detached using TrypLE Select, and physiological saline was added so that the cell concentration became 2×10 6 cells / mL. An equal volume of CP-1 (registered trademark) solution (CP-1 (registered trademark): a solution mixed at a ratio of 25% human serum albumin = 34:16) was added thereto, transferred 1 mL at a time into cryovials, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen.

[0171] (Step 4-2: Antigen Analysis of Bone Marrow-Derived Mesenchymal Stem Cells) Regarding the bone marrow-derived mesenchymal stem cells cultured by the above cultivation method, various surface antigens (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, negative rate of CD326) were analyzed using a flow cytometer. As a result, the positive rates of CD73, CD90, and CD105 were all 50% or more. The positive rates of CD166 were all 30% or more. CD45, CD34, and CD326 were all less than 5%. Also, the ratio of cells positive for the KCNAB1 antigen was analyzed using a flow cytometer. As a result, it was less than 85% in any donor (#6: 66%, #7: 71%, #8: 57%). The method and reagent for this measurement are the same as those in Comparative Example 1.

[0172] (Step 4-3: Gene Expression Analysis of Bone Marrow-Derived Mesenchymal Stem Cells) Regarding the bone marrow-derived mesenchymal stem cells cultured by the above-described culture method, gene expression analysis (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) was performed by quantitative PCR. As a result, for #6, the KCNAB1 gene was 0.0015, the SULT1E1 gene: the expression level was too low to be detected, the MN1 gene was 0.54, and the RARRES2 gene was 0.0026. For #7, the KCNAB1 gene was 0.0020, the SULT1E1 gene: the expression level was too low to be detected, the MN1 gene was 0.90, and the RARRES2 gene was 0.0029. For #8, the KCNAB1 gene was 0.0033, the SULT1E1 gene: the expression level was too low to be detected, the MN1 gene was 0.52, and the RARRES2 gene was 0.0033. The method and reagents for this measurement were the same as those in Example 3. Table 3 summarizes the results of quantitative PCR gene expression analysis in Example 3 and the reference example.

[0173]

Table 3

[0174] SEQUENCE LISTING <110> kaneka corporation <120> CELL POPULATION THAT INCLUDES MESENCHYMAL STEM CELLS, METHOD FOR PRODUCING THE SAME, AND PHARMACEUTICAL COMPOSITION <130> B170810 <150> JP 2017-253879 <151> 2017-12-28 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 1851 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(1851) <400> 1 atg tcg ggg gtc cgg ggc ctg tcg cgg ctg ctg agc gct cgg cgc ctg 48 Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg Leu 1 5 10 15 gcg ctg gcc aag gcg tgg cca aca gtg ttg caa aca gga acc cga ggt 96 Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr Arg Gly 20 25 30 ttt cac ttc act gtt gat ggg aac aag agg gca tct gct aaa gtt tca 144 Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala Lys Val Ser 35 40 45 gat tcc att tct gct cag tat cca gta gtg gat cat gaa ttt gat gca 192 Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His Glu Phe Asp Ala 50 55 60 gtg gtg gta ggc gct gga ggg gca ggc ttg cga gct gca ttt ggc ctt 240 Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg Ala Ala Phe Gly Leu 65 70 75 80 tct gag gca ggg ttt aat aca gca tgt gtt acc aag ctg ttt cct acc 288 Ser Glu Ala Gly Phe Asn Thr Ala Cys Val Thr Lys Leu Phe Pro Thr 85 90 95 agg tca cac act gtt gca gca cag cta gaa aat tat ggc atg ccg ttt 336 Arg Ser His Thr Val Ala Ala Gln Leu Glu Asn Tyr Gly Met Pro Phe 100 105 110 agc aga act gaa gat ggg aag att tat cag cgt gca ttt ggt gga cag 384 Ser Arg Thr Glu Asp Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln 115 120 125 agc ctc aag ttt gga aag ggc ggg cag gcc cat cgg tgc tgc tgt gtg 432 Ser Leu Lys Phe Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val 130 135 140 gct gat cgg act ggc cac tcg cta ttg cac acc tta tat gga agg tct 480 Ala Asp Arg Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser 145 150 155 160 ctg cga tat gat acc agc tat ttt gtg gag tat ttt gcc ttg gat ctc 528 Leu Arg Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu 165 170 175 ctg atg gag aat ggg gag tgc cgt ggt gtc atc gca ctg tgc ata gag 576 Leu Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu 180 185 190 gac ggg tcc atc cat cgc ata aga gca aag aac act gtt gtt gcc aca 624 Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala Thr 195 200 205 gga ggc tac ggg cgc acc tac ttc agc tgc acg tct gcc cac acc agc 672 Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser 210 215 220 act ggc gac ggc acg gcc atg atc acc agg gca ggc ctt cct tgc cag 720 Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu Pro Cys Gln 225 230 235 240 gac cta gag ttt gtt cag ttc cac cct aca ggc ata tat ggt gct ggt 768 Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile Tyr Gly Ala Gly 245 250 255 tgt ctc att acg gaa gga tgt cgt gga gag gga ggc att ctc att aac 816 Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly Gly Ile Leu Ile Asn 260 265 270 agt caa ggc gaa agg ttt atg gag cga tac gcc cct gtc gcg aag gac 864 Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Val Ala Lys Asp 275 280 285 ctg gcg tct aga gat gtg gtg tct cgg tcc atg act ctg gag atc cga 912 Leu Ala Ser Arg Asp Val Val Ser Arg Ser Met Thr Leu Glu Ile Arg 290 295 300 gaa gga aga ggc tgt ggc cct gag aaa gat cac gtc tac ctg cag ctg 960 Glu Gly Arg Gly Cys Gly Pro Glu Lys Asp His Val Tyr Leu Gln Leu 305 310 315 320 cac cac cta cct cca gag cag ctg gcc acg cgc ctg cct ggc att tca 1008 His His Leu Pro Pro Glu Gln Leu Ala Thr Arg Leu Pro Gly Ile Ser 325 330 335 gag aca gcc atg atc ttc gct ggc gtg gac gtc acg aag gag ccg atc 1056 Glu Thr Ala Met Ile Phe Ala Gly Val Asp Val Thr Lys Glu Pro Ile 340 345 350 cct gtc ctc ccc acc gtg cat tat aac atg ggc ggc att ccc acc aac 1104 Pro Val Leu Pro Thr Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn 355 360 365 tac aag ggg cag gtc ctg agg cac gtg aat ggc cag gat cag att gtg 1152 Tyr Lys Gly Gln Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val 370 375 380 ccc ggc ctg tac gcc tgt ggg gag gcc gcc tgt gcc tcg gta cat ggt 1200 Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly 385 390 395 400 gcc aac cgc ctc ggg gca aac tcg ctc ttg gac ctg gtt gtc ttt ggt 1248 Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly 405 410 415 cgg gca tgt gcc ctg agc atc gaa gag tca tgc agg cct gga gat aaa 1296 Arg Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys 420 425 430 gtc cct cca att aaa cca aac gct ggg gaa gaa tct gtc atg aat ctt 1344 Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu 435 440 445 gac aaa ttg aga ttt gct gat gga agc ata aga aca tcg gaa ctg cga 1392 Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu Leu Arg 450 455 460 ctc agc atg cag aag tca atg caa aat cat gct gcc gtg ttc cgt gtg 1440 Leu Ser Met Gln Lys Ser Met Gln Asn His Ala Ala Val Phe Arg Val 465 470 475 480 gga agc gtg ttg caa gaa ggt tgt ggg aaa atc agc aag ctc tat gga 1488 Gly Ser Val Leu Gln Glu Gly Cys Gly Lys Ile Ser Lys Leu Tyr Gly 485 490 495 gac cta aag cac ctg aag acg ttc gac cgg gga atg gtc tgg aac acg 1536 Asp Leu Lys His Leu Lys Thr Phe Asp Arg Gly Met Val Trp Asn Thr 500 505 510 gac ctg gtg gag acc ctg gag ctg cag aac ctg atg ctg tgt gcg ctg 1584 Asp Leu Val Glu Thr Leu Glu Leu Gln Asn Leu Met Leu Cys Ala Leu 515 520 525 cag acc atc tac gga gca gag gca cgg aag gag tca cgg ggc gcg cat 1632 Gln Thr Ile Tyr Gly Ala Glu Ala Arg Lys Glu Ser Arg Gly Ala His 530 535 540 gcc agg gaa gac tac aag gtg cgg att gat gag tac gat tac tcc aag 1680 Ala Arg Glu Asp Tyr Lys Val Arg Ile Asp Glu Tyr Asp Tyr Ser Lys 545 550 555 560 ccc atc cag ggg caa cag aag aag ccc ttt gag gag cac tgg agg aag 1728 Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His Trp Arg Lys 565 570 575 cac acc ctg tcc tat gtg gac gtt ggc act ggg aag gtc act ctg gaa 1776 His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu 580 585 590 tat aga ccc gtg atc gac aaa act ttg aac gag gct gac tgt gcc acc 1824 Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu Ala Asp Cys Ala Thr 595 600 605 gtc ccg cca gcc att cgc tcc tac tga 1851 Val Pro Pro Ala Ile Arg Ser Tyr 610 615 <210> 2 <211> 616 <212> PRT <213> Homo sapiens <400> 2 Met Ser Gly Val Arg Gly Leu Ser Arg Leu Leu Ser Ala Arg Arg Leu 1 5 10 15 Ala Leu Ala Lys Ala Trp Pro Thr Val Leu Gln Thr Gly Thr Arg Gly 20 25 30 Phe His Phe Thr Val Asp Gly Asn Lys Arg Ala Ser Ala Lys Val Ser 35 40 45 Asp Ser Ile Ser Ala Gln Tyr Pro Val Val Asp His Glu Phe Asp Ala 50 55 60 Val Val Val Gly Ala Gly Gly Ala Gly Leu Arg Ala Ala Phe Gly Leu 65 70 75 80 Ser Glu Ala Gly Phe Asn Thr Ala Cys Val Thr Lys Leu Phe Pro Thr 85 90 95 Arg Ser His Thr Val Ala Ala Gln Leu Glu Asn Tyr Gly Met Pro Phe 100 105 110 Ser Arg Thr Glu Asp Gly Lys Ile Tyr Gln Arg Ala Phe Gly Gly Gln 115 120 125 Ser Leu Lys Phe Gly Lys Gly Gly Gln Ala His Arg Cys Cys Cys Val 130 135 140 Ala Asp Arg Thr Gly His Ser Leu Leu His Thr Leu Tyr Gly Arg Ser 145 150 155 160 Leu Arg Tyr Asp Thr Ser Tyr Phe Val Glu Tyr Phe Ala Leu Asp Leu 165 170 175 Leu Met Glu Asn Gly Glu Cys Arg Gly Val Ile Ala Leu Cys Ile Glu 180 185 190 Asp Gly Ser Ile His Arg Ile Arg Ala Lys Asn Thr Val Val Ala Thr 195 200 205 Gly Gly Tyr Gly Arg Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser 210 215 220 Thr Gly Asp Gly Thr Ala Met Ile Thr Arg Ala Gly Leu Pro Cys Gln 225 230 235 240 Asp Leu Glu Phe Val Gln Phe His Pro Thr Gly Ile Tyr Gly Ala Gly 245 250 255 Cys Leu Ile Thr Glu Gly Cys Arg Gly Glu Gly Gly Ile Leu Ile Asn 260 265 270 Ser Gln Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Val Ala Lys Asp 275 280 285 Leu Ala Ser Arg Asp Val Val Ser Arg Ser Met Thr Leu Glu Ile Arg 290 295 300 Glu Gly Arg Gly Cys Gly Pro Glu Lys Asp His Val Tyr Leu Gln Leu 305 310 315 320 His His Leu Pro Pro Glu Gln Leu Ala Thr Arg Leu Pro Gly Ile Ser 325 330 335 Glu Thr Ala Met Ile Phe Ala Gly Val Asp Val Thr Lys Glu Pro Ile 340 345 350 Pro Val Leu Pro Thr Val His Tyr Asn Met Gly Gly Ile Pro Thr Asn 355 360 365 Tyr Lys Gly Gln Val Leu Arg His Val Asn Gly Gln Asp Gln Ile Val 370 375 380 Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly 385 390 395 400 Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly 405 410 415 Arg Ala Cys Ala Leu Ser Ile Glu Glu Ser Cys Arg Pro Gly Asp Lys 420 425 430 Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu 435 440 445 Asp Lys Leu Arg Phe Ala Asp Gly Ser Ile Arg Thr Ser Glu Leu Arg 450 455 460 Leu Ser Met Gln Lys Ser Met Gln Asn His Ala Ala Val Phe Arg Val 465 470 475 480 Gly Ser Val Leu Gln Glu Gly Cys Gly Lys Ile Ser Lys Leu Tyr Gly 485 490 495 Asp Leu Lys His Leu Lys Thr Phe Asp Arg Gly Met Val Trp Asn Thr 500 505 510 Asp Leu Val Glu Thr Leu Glu Leu Gln Asn Leu Met Leu Cys Ala Leu 515 520 525 Gln Thr Ile Tyr Gly Ala Glu Ala Arg Lys Glu Ser Arg Gly Ala His 530 535 540 Ala Arg Glu Asp Tyr Lys Val Arg Ile Asp Glu Tyr Asp Tyr Ser Lys 545 550 555 560 Pro Ile Gln Gly Gln Gln Lys Lys Pro Phe Glu Glu His Trp Arg Lys 565 570 575 His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu 580 585 590 Tyr Arg Pro Val Ile Asp Lys Thr Leu Asn Glu Ala Asp Cys Ala Thr 595 600 605 Val Pro Pro Ala Ile Arg Ser Tyr 610 615 <210> 3 <211> 1173 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(1173) <400> 3 atg ctg gca gcc cgg aca ggg gca gcg ggg agt cag atc tca gag gag 48 Met Leu Ala Ala Arg Thr Gly Ala Ala Gly Ser Gln Ile Ser Glu Glu 1 5 10 15 aac acc aag tta agg aga cag tct ggg ttt tct gta gca ggg aaa gac 96 Asn Thr Lys Leu Arg Arg Gln Ser Gly Phe Ser Val Ala Gly Lys Asp 20 25 30 aaa tct ccc aag aaa gcc tca gaa aac gct aaa gac agc agc ctt agt 144 Lys Ser Pro Lys Lys Ala Ser Glu Asn Ala Lys Asp Ser Ser Leu Ser 35 40 45 ccc tca ggg gaa agc cag ctc agg gcg cgt caa ctg gct ctg ctg cgc 192 Pro Ser Gly Glu Ser Gln Leu Arg Ala Arg Gln Leu Ala Leu Leu Arg 50 55 60 gaa gtg gag atg aac tgg tac cta aag ctc tgc gac ctg tcc agc gag 240 Glu Val Glu Met Asn Trp Tyr Leu Lys Leu Cys Asp Leu Ser Ser Glu 65 70 75 80 cac acc acc gtc tgc acc aca ggc atg ccg cac agg aat ctt gga aaa 288 His Thr Thr Val Cys Thr Thr Gly Met Pro His Arg Asn Leu Gly Lys 85 90 95 tca gga ctc aga gtt tct tgc ttg ggt ctt gga aca tgg gtg aca ttt 336 Ser Gly Leu Arg Val Ser Cys Leu Gly Leu Gly Thr Trp Val Thr Phe 100 105 110 gga ggt caa att tca gat gag gtt gct gaa cgg ctg atg acc atc gcc 384 Gly Gly Gln Ile Ser Asp Glu Val Ala Glu Arg Leu Met Thr Ile Ala 115 120 125 tat gaa agt ggt gtt aac ctc ttt gat act gcc gaa gtc tat gct gct 432 Tyr Glu Ser Gly Val Asn Leu Phe Asp Thr Ala Glu Val Tyr Ala Ala 130 135 140 gga aag gct gaa gtg att ctg ggg agc atc atc aag aag aaa ggc tgg 480 Gly Lys Ala Glu Val Ile Leu Gly Ser Ile Ile Lys Lys Lys Gly Trp 145 150 155 160 agg agg tcc agt ctg gtc ata aca acc aaa ctc tac tgg ggt gga aaa 528 Arg Arg Ser Ser Leu Val Ile Thr Thr Lys Leu Tyr Trp Gly Gly Lys 165 170 175 gct gaa aca gaa aga ggg ctg tca aga aag cat att att gaa gaa att 576 Ala Glu Thr Glu Arg Gly Leu Ser Arg Lys His Ile Ile Glu Glu Ile 180 185 190 gtc cga gcc atg aca cat gtg ata aac caa ggc atg gcg atg tac tgg 624 Val Arg Ala Met Thr His Val Ile Asn Gln Gly Met Ala Met Tyr Trp 195 200 205 ggc acc tcg aga tgg agt gct atg gag atc atg gaa gcc tat tct gta 672 Gly Thr Ser Arg Trp Ser Ala Met Glu Ile Met Glu Ala Tyr Ser Val 210 215 220 gca aga cag ttc aat atg atc cca ccg gtc tgt gaa caa gct gag tac 720 Ala Arg Gln Phe Asn Met Ile Pro Pro Val Cys Glu Gln Ala Glu Tyr 225 230 235 240 cat ctt ttc cag aga gag aaa gtg gag gtc cag ctg cca gag ctc tac 768 His Leu Phe Gln Arg Glu Lys Val Glu Val Gln Leu Pro Glu Leu Tyr 245 250 255 cac aaa ata ggt gtt ggc gca atg aca tgg tct cca ctt gcc tgt gga 816 His Lys Ile Gly Val Gly Ala Met Thr Trp Ser Pro Leu Ala Cys Gly 260 265 270 atc atc tca gga aaa tac gga aac ggg gtg cct gaa agt tcc agg gct 864 Ile Ile Ser Gly Lys Tyr Gly Asn Gly Val Pro Glu Ser Ser Arg Ala 275 280 285 tca ctg aag tgc tac cag tgg ttg aaa gaa aga att gta agt gaa gaa 912 Ser Leu Lys Cys Tyr Gln Trp Leu Lys Glu Arg Ile Val Ser Glu Glu 290 295 300 ggg aga aaa cag caa aac aag cta aaa gac ctt tcc cca att gcg gag 960 Gly Arg Lys Gln Gln Asn Lys Leu Lys Asp Leu Ser Pro Ile Ala Glu 305 310 315 320 cgt ctg gga tgc aca cta cct cag cta gct gtt gcg tgg tgc ctg aga 1008 Arg Leu Gly Cys Thr Leu Pro Gln Leu Ala Val Ala Trp Cys Leu Arg 325 330 335 aat gaa ggt gtg agt tct gtg ctc ctg gga tca tcc act cct gaa caa 1056 Asn Glu Gly Val Ser Ser Val Leu Leu Gly Ser Ser Thr Pro Glu Gln 340 345 350 ctc att gaa aac ctt ggt gcc att cag gtt ctc cca aag atg aca tca 1104 Leu Ile Glu Asn Leu Gly Ala Ile Gln Val Leu Pro Lys Met Thr Ser 355 360 365 cat gtg gta aat gag att gat aac ata ctg cgc aac aag ccc tac agc 1152 His Val Val Asn Glu Ile Asp Asn Ile Leu Arg Asn Lys Pro Tyr Ser 370 375 380 aag aag gac tat aga tca taa 1173 Lys Lys Asp Tyr Arg Ser 385 390 <210> 4 <211> 390 <212> PRT <213> Homo sapiens <400> 4 Met Leu Ala Ala Arg Thr Gly Ala Ala Gly Ser Gln Ile Ser Glu Glu 1 5 10 15 Asn Thr Lys Leu Arg Arg Gln Ser Gly Phe Ser Val Ala Gly Lys Asp 20 25 30 Lys Ser Pro Lys Lys Ala Ser Glu Asn Ala Lys Asp Ser Ser Leu Ser 35 40 45 Pro Ser Gly Glu Ser Gln Leu Arg Ala Arg Gln Leu Ala Leu Leu Arg 50 55 60 Glu Val Glu Met Asn Trp Tyr Leu Lys Leu Cys Asp Leu Ser Ser Glu 65 70 75 80 His Thr Thr Val Cys Thr Thr Gly Met Pro His Arg Asn Leu Gly Lys 85 90 95 Ser Gly Leu Arg Val Ser Cys Leu Gly Leu Gly Thr Trp Val Thr Phe 100 105 110 Gly Gly Gln Ile Ser Asp Glu Val Ala Glu Arg Leu Met Thr Ile Ala 115 120 125 Tyr Glu Ser Gly Val Asn Leu Phe Asp Thr Ala Glu Val Tyr Ala Ala 130 135 140 Gly Lys Ala Glu Val Ile Leu Gly Ser Ile Ile Lys Lys Lys Gly Trp 145 150 155 160 Arg Arg Ser Ser Leu Val Ile Thr Thr Lys Leu Tyr Trp Gly Gly Lys 165 170 175 Ala Glu Thr Glu Arg Gly Leu Ser Arg Lys His Ile Ile Glu Glu Ile 180 185 190 Val Arg Ala Met Thr His Val Ile Asn Gln Gly Met Ala Met Tyr Trp 195 200 205 Gly Thr Ser Arg Trp Ser Ala Met Glu Ile Met Glu Ala Tyr Ser Val 210 215 220 Ala Arg Gln Phe Asn Met Ile Pro Pro Val Cys Glu Gln Ala Glu Tyr 225 230 235 240 His Leu Phe Gln Arg Glu Lys Val Glu Val Gln Leu Pro Glu Leu Tyr 245 250 255 His Lys Ile Gly Val Gly Ala Met Thr Trp Ser Pro Leu Ala Cys Gly 260 265 270 Ile Ile Ser Gly Lys Tyr Gly Asn Gly Val Pro Glu Ser Ser Arg Ala 275 280 285 Ser Leu Lys Cys Tyr Gln Trp Leu Lys Glu Arg Ile Val Ser Glu Glu 290 295 300 Gly Arg Lys Gln Gln Asn Lys Leu Lys Asp Leu Ser Pro Ile Ala Glu 305 310 315 320 Arg Leu Gly Cys Thr Leu Pro Gln Leu Ala Val Ala Trp Cys Leu Arg 325 330 335 Asn Glu Gly Val Ser Ser Val Leu Leu Gly Ser Ser Thr Pro Glu Gln 340 345 350 Leu Ile Glu Asn Leu Gly Ala Ile Gln Val Leu Pro Lys Met Thr Ser 355 360 365 His Val Val Asn Glu Ile Asp Asn Ile Leu Arg Asn Lys Pro Tyr Ser 370 375 380 Lys Lys Asp Tyr Arg Ser 385 390 <210> 5 <211> 885 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(885) <400> 5 atg aat tct gaa ctt gac tat tat gaa aag ttt gaa gaa gtc cat ggg 48 Met Asn Ser Glu Leu Asp Tyr Tyr Glu Lys Phe Glu Glu Val His Gly 1 5 10 15 att cta atg tat aaa gat ttt gtc aaa tat tgg gat aat gtg gaa gcg 96 Ile Leu Met Tyr Lys Asp Phe Val Lys Tyr Trp Asp Asn Val Glu Ala 20 25 30 ttc cag gca aga cca gat gat ctt gtc att gcc acc tac cct aaa tct 144 Phe Gln Ala Arg Pro Asp Asp Leu Val Ile Ala Thr Tyr Pro Lys Ser 35 40 45 ggt aca acc tgg gtt agt gaa att gtg tat atg atc tat aaa gag ggt 192 Gly Thr Thr Trp Val Ser Glu Ile Val Tyr Met Ile Tyr Lys Glu Gly 50 55 60 gat gtg gaa aag tgc aaa gaa gat gta att ttt aat cga ata cct ttc 240 Asp Val Glu Lys Cys Lys Glu Asp Val Ile Phe Asn Arg Ile Pro Phe 65 70 75 80 ctg gaa tgc aga aaa gaa aac ctc atg aat gga gta aaa caa tta gat 288 Leu Glu Cys Arg Lys Glu Asn Leu Met Asn Gly Val Lys Gln Leu Asp 85 90 95 gag atg aat tct cct aga att gtg aag act cat ttg cca cct gaa ctt 336 Glu Met Asn Ser Pro Arg Ile Val Lys Thr His Leu Pro Pro Glu Leu 100 105 110 ctt cct gcc tca ttt tgg gaa aag gat tgt aag ata atc tat ctt tgc 384 Leu Pro Ala Ser Phe Trp Glu Lys Asp Cys Lys Ile Ile Tyr Leu Cys 115 120 125 cgg aat gca aag gat gtg gct gtt tcc ttt tat tat ttc ttt cta atg 432 Arg Asn Ala Lys Asp Val Ala Val Ser Phe Tyr Tyr Phe Phe Leu Met 130 135 140 gtg gct ggt cat cca aat cct gga tcc ttt cca gag ttt gtg gag aaa 480 Val Ala Gly His Pro Asn Pro Gly Ser Phe Pro Glu Phe Val Glu Lys 145 150 155 160 ttc atg caa gga cag gtt cct tat ggt tcc tgg tat aaa cat gta aaa 528 Phe Met Gln Gly Gln Val Pro Tyr Gly Ser Trp Tyr Lys His Val Lys 165 170 175 tct tgg tgg gaa aag gga aag agt cca cgt gta cta ttt ctt ttc tac 576 Ser Trp Trp Glu Lys Gly Lys Ser Pro Arg Val Leu Phe Leu Phe Tyr 180 185 190 gaa gac ctg aaa gag gat atc aga aaa gag gtg ata aaa ttg ata cat 624 Glu Asp Leu Lys Glu Asp Ile Arg Lys Glu Val Ile Lys Leu Ile His 195 200 205 ttc ctg gaa agg aag cca tca gag gag ctt gtg gac agg att ata cat 672 Phe Leu Glu Arg Lys Pro Ser Glu Glu Leu Val Asp Arg Ile Ile His 210 215 220 cat act tcg ttc caa gag atg aag aac aat cca tcc aca aat tac aca 720 His Thr Ser Phe Gln Glu Met Lys Asn Asn Pro Ser Thr Asn Tyr Thr 225 230 235 240 aca ctg cca gac gaa att atg aac cag aaa ttg tcg ccc ttc atg aga 768 Thr Leu Pro Asp Glu Ile Met Asn Gln Lys Leu Ser Pro Phe Met Arg 245 250 255 aag gga att aca gga gac tgg aaa aat cac ttt aca gta gcc ctg aat 816 Lys Gly Ile Thr Gly Asp Trp Lys Asn His Phe Thr Val Ala Leu Asn 260 265 270 gaa aaa ttt gat aaa cat tat gag cag caa atg aag gaa tct aca ctg 864 Glu Lys Phe Asp Lys His Tyr Glu Gln Gln Met Lys Glu Ser Thr Leu 275 280 285 aag ttt cga act gag atc taa 885 Lys Phe Arg Thr Glu Ile 290 <210> 6 <211> 294 <212> PRT <213> Homo sapiens <400> 6 Met Asn Ser Glu Leu Asp Tyr Tyr Glu Lys Phe Glu Glu Val His Gly 1 5 10 15 Ile Leu Met Tyr Lys Asp Phe Val Lys Tyr Trp Asp Asn Val Glu Ala 20 25 30 Phe Gln Ala Arg Pro Asp Asp Leu Val Ile Ala Thr Tyr Pro Lys Ser 35 40 45 Gly Thr Thr Trp Val Ser Glu Ile Val Tyr Met Ile Tyr Lys Glu Gly 50 55 60 Asp Val Glu Lys Cys Lys Glu Asp Val Ile Phe Asn Arg Ile Pro Phe 65 70 75 80 Leu Glu Cys Arg Lys Glu Asn Leu Met Asn Gly Val Lys Gln Leu Asp 85 90 95 Glu Met Asn Ser Pro Arg Ile Val Lys Thr His Leu Pro Pro Glu Leu 100 105 110 Leu Pro Ala Ser Phe Trp Glu Lys Asp Cys Lys Ile Ile Tyr Leu Cys 115 120 125 Arg Asn Ala Lys Asp Val Ala Val Ser Phe Tyr Tyr Phe Phe Leu Met 130 135 140 Val Ala Gly His Pro Asn Pro Gly Ser Phe Pro Glu Phe Val Glu Lys 145 150 155 160 Phe Met Gln Gly Gln Val Pro Tyr Gly Ser Trp Tyr Lys His Val Lys 165 170 175 Ser Trp Trp Glu Lys Gly Lys Ser Pro Arg Val Leu Phe Leu Phe Tyr 180 185 190 Glu Asp Leu Lys Glu Asp Ile Arg Lys Glu Val Ile Lys Leu Ile His 195 200 205 Phe Leu Glu Arg Lys Pro Ser Glu Glu Leu Val Asp Arg Ile Ile His 210 215 220 His Thr Ser Phe Gln Glu Met Lys Asn Asn Pro Ser Thr Asn Tyr Thr 225 230 235 240 Thr Leu Pro Asp Glu Ile Met Asn Gln Lys Leu Ser Pro Phe Met Arg 245 250 255 Lys Gly Ile Thr Gly Asp Trp Lys Asn His Phe Thr Val Ala Leu Asn 260 265 270 Glu Lys Phe Asp Lys His Tyr Glu Gln Gln Met Lys Glu Ser Thr Leu 275 280 285 Lys Phe Arg Thr Glu Ile 290 <210> 7 <211> 3963 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(3963) <400> 7 atg ttt ggg ctg gac caa ttc gag ccc cag gtc aac agc agg aac gct 48 Met Phe Gly Leu Asp Gln Phe Glu Pro Gln Val Asn Ser Arg Asn Ala 1 5 10 15 ggc cag ggc gag agg aac ttt aac gag acc gga ctg agc atg aac acc 96 Gly Gln Gly Glu Arg Asn Phe Asn Glu Thr Gly Leu Ser Met Asn Thr 20 25 30 cac ttt aag gcc ccg gct ttc cac act ggg ggg ccc cct ggc cct gtg 144 His Phe Lys Ala Pro Ala Phe His Thr Gly Gly Pro Pro Gly Pro Val 35 40 45 gat cct gct atg agc gcg ctg ggc gaa ccc ccg atc ttg ggc atg aac 192 Asp Pro Ala Met Ser Ala Leu Gly Glu Pro Pro Ile Leu Gly Met Asn 50 55 60 atg gag ccc tac ggc ttc cac gcg cgc ggc cac tcg gag ttg cac gca 240 Met Glu Pro Tyr Gly Phe His Ala Arg Gly His Ser Glu Leu His Ala 65 70 75 80 ggg ggg ctg caa gcg cag cct gtg cac ggc ttc ttt ggc ggc cag cag 288 Gly Gly Leu Gln Ala Gln Pro Val His Gly Phe Phe Gly Gly Gln Gln 85 90 95 cct cac cac ggc cac ccg gga agt cat cat ccc cac cag cat cac ccc 336 Pro His His Gly His Pro Gly Ser His His Pro His Gln His His Pro 100 105 110 cac ttt ggg ggc aac ttc ggt ggc ccg gac ccc ggg gcc tcg tgc ctg 384 His Phe Gly Gly Asn Phe Gly Gly Pro Asp Pro Gly Ala Ser Cys Leu 115 120 125 cac ggg ggt cgc ctg ctc ggc tac ggc ggc gca gcc gga ggc ctg ggc 432 His Gly Gly Arg Leu Leu Gly Tyr Gly Gly Ala Ala Gly Gly Leu Gly 130 135 140 agc cag ccg ccc ttc gcc gag ggc tat gag cac atg gcg gag agc cag 480 Ser Gln Pro Pro Phe Ala Glu Gly Tyr Glu His Met Ala Glu Ser Gln 145 150 155 160 ggg cct gag agc ttc ggc ccg cag cga ccg ggg aac ctc ccg gac ttc 528 Gly Pro Glu Ser Phe Gly Pro Gln Arg Pro Gly Asn Leu Pro Asp Phe 165 170 175 cac agt tca ggt gcc tcc agc cac gcc gtg ccg gcc cca tgc ctg ccg 576 His Ser Ser Gly Ala Ser Ser His Ala Val Pro Ala Pro Cys Leu Pro 180 185 190 ctg gac cag agc cct aac cga gcc gcc tcc ttc cac ggc ctg ccg tcc 624 Leu Asp Gln Ser Pro Asn Arg Ala Ala Ser Phe His Gly Leu Pro Ser 195 200 205 tcc agc ggc tcc gat tcc cac agt ctg gag cca cgg agg gtg acg aac 672 Ser Ser Gly Ser Asp Ser His Ser Leu Glu Pro Arg Arg Val Thr Asn 210 215 220 caa gga gcc gtc gac tcg ctg gaa tac aat tac ccg ggc gag gcg ccc 720 Gln Gly Ala Val Asp Ser Leu Glu Tyr Asn Tyr Pro Gly Glu Ala Pro 225 230 235 240 tcg gga cat ttt gac atg ttt tcg ccc tct gac tcc gaa ggg cag ctg 768 Ser Gly His Phe Asp Met Phe Ser Pro Ser Asp Ser Glu Gly Gln Leu 245 250 255 cct cat tat gca gcg ggt cgc cag gtt cct ggg ggc gct ttc ccg ggc 816 Pro His Tyr Ala Ala Gly Arg Gln Val Pro Gly Gly Ala Phe Pro Gly 260 265 270 gcc tcg gcc atg ccc aga gct gcg ggc atg gtg ggc ttg tcc aaa atg 864 Ala Ser Ala Met Pro Arg Ala Ala Gly Met Val Gly Leu Ser Lys Met 275 280 285 cac gcc cag cca ccg cag cag cag ccc cag cag cag cag cag ccc cag 912 His Ala Gln Pro Pro Gln Gln Gln Pro Gln Gln Gln Gln Gln Pro Gln 290 295 300 cag cag cag cag cag cat ggt gtg ttc ttt gag agg ttc agt ggg gcc 960 Gln Gln Gln Gln Gln His Gly Val Phe Phe Glu Arg Phe Ser Gly Ala 305 310 315 320 aga aag atg cct gtg ggt ctg gag ccc tca gtg ggc tcc agg cac ccg 1008 Arg Lys Met Pro Val Gly Leu Glu Pro Ser Val Gly Ser Arg His Pro 325 330 335 tta atg cag cct ccc cag cag gcc ccg cca ccc cct cag cag cag ccc 1056 Leu Met Gln Pro Pro Gln Gln Ala Pro Pro Pro Pro Gln Gln Gln Pro 340 345 350 ccg cag cag ccg cca cag cag cag ccg ccg ccg cca ccc ggg ctt cta 1104 Pro Gln Gln Pro Pro Gln Gln Gln Pro Pro Pro Pro Pro Gly Leu Leu 355 360 365 gtc cga caa aat tcg tgc ccg cct gcg ctc cct cgg ccc cag cag ggc 1152 Val Arg Gln Asn Ser Cys Pro Pro Ala Leu Pro Arg Pro Gln Gln Gly 370 375 380 gag gcg ggc acg ccc agc ggc ggc ctg cag gac gga ggc ccc atg ctg 1200 Glu Ala Gly Thr Pro Ser Gly Gly Leu Gln Asp Gly Gly Pro Met Leu 385 390 395 400 ccc agc cag cac gcg caa ttc gag tat ccc atc cac cgg ctg gag aac 1248 Pro Ser Gln His Ala Gln Phe Glu Tyr Pro Ile His Arg Leu Glu Asn 405 410 415 cgg agc atg cac cct tat tcc gag cct gtt ttc agc atg cag cat cct 1296 Arg Ser Met His Pro Tyr Ser Glu Pro Val Phe Ser Met Gln His Pro 420 425 430 cct ccg cag cag gcg ccc aac cag cgg ctg cag cat ttc gac gcg ccc 1344 Pro Pro Gln Gln Ala Pro Asn Gln Arg Leu Gln His Phe Asp Ala Pro 435 440 445 ccc tac atg aac gtg gcc aag agg ccg cgc ttc gac ttt ccg ggc agc 1392 Pro Tyr Met Asn Val Ala Lys Arg Pro Arg Phe Asp Phe Pro Gly Ser 450 455 460 gcg gga gtg gac cgc tgc gct tcg tgg aac ggc agc atg cac aac ggc 1440 Ala Gly Val Asp Arg Cys Ala Ser Trp Asn Gly Ser Met His Asn Gly 465 470 475 480 gct ctg gat aat cac ctc tcc cct tcc gcc tac cca ggc cta ccc ggc 1488 Ala Leu Asp Asn His Leu Ser Pro Ser Ala Tyr Pro Gly Leu Pro Gly 485 490 495 gag ttc aca ccg cct gtg ccc gac agc ttc cct tcg ggg ccg ccc ctg 1536 Glu Phe Thr Pro Pro Val Pro Asp Ser Phe Pro Ser Gly Pro Pro Leu 500 505 510 cag cat ccg gcc ccg gac cac cag tcc ctg caa cag cag cag cag cag 1584 Gln His Pro Ala Pro Asp His Gln Ser Leu Gln Gln Gln Gln Gln 515 520 525 cag cag cag cag caa cag cag cag cag cag cag caa cag caa cag caa 1632 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 530 535 540 cag cag cag cag cag cag cgc caa aac gcg gcc ctc atg att aag cag 1680 Gln Gln Gln Gln Gln Gln Arg Gln Asn Ala Ala Leu Met Ile Lys Gln 545 550 555 560 atg gcg tcg cgg aat cag cag cag cgg ctg cgc cag ccc aac ctg gct 1728 Met Ala Ser Arg Asn Gln Gln Gln Arg Leu Arg Gln Pro Asn Leu Ala 565 570 575 cag cta ggc cac ccc ggg gac gtg ggc cag ggc ggc ctg gtg cat ggc 1776 Gln Leu Gly His Pro Gly Asp Val Gly Gln Gly Gly Leu Val His Gly 580 585 590 ggc ccg gtg ggc ggc ttg gcc cag ccg aac ttt gag cgc gaa ggc ggc 1824 Gly Pro Val Gly Gly Leu Ala Gln Pro Asn Phe Glu Arg Glu Gly Gly 595 600 605 agc acg ggc gcc ggg cgt ctg ggc acc ttc gag cag cag gcg ccg cac 1872 Ser Thr Gly Ala Gly Arg Leu Gly Thr Phe Glu Gln Gln Ala Pro His 610 615 620 ttg gcg caa gag agc gcg tgg ttc tca ggt ccg cat ccg ccg ccc gga 1920 Leu Ala Gln Glu Ser Ala Trp Phe Ser Gly Pro His Pro Pro Pro Gly 625 630 635 640 gac ctg ctg ccc cgt agg atg ggc ggc tcg ggt ctg ccc gct gac tgt 1968 Asp Leu Leu Pro Arg Arg Met Gly Gly Ser Gly Leu Pro Ala Asp Cys 645 650 655 ggc ccg cac gac ccc agc ctg gcg ccc cct cct ccg cct ggt ggc tcg 2016 Gly Pro His Asp Pro Ser Leu Ala Pro Pro Pro Pro Pro Gly Gly Ser 660 665 670 ggg gtg ctg ttc cgg ggc cct ctg cag gag ccg atg agg atg ccc gga 2064 Gly Val Leu Phe Arg Gly Pro Leu Gln Glu Pro Met Arg Met Pro Gly 675 680 685 gag ggc cac gtg ccc gcg ctg cct tca ccg ggc ctg cag ttc ggg ggc 2112 Glu Gly His Val Pro Ala Leu Pro Ser Pro Gly Leu Gln Phe Gly Gly 690 695 700 agt ctg gga ggc ctg ggt cag ctg cag tcg ccc ggg gcg ggc gtg ggg 2160 Ser Leu Gly Gly Leu Gly Gln Leu Gln Ser Pro Gly Ala Gly Val Gly 705 710 715 720 ctc ccc agc gct gct tcg gag cgc cgg ccc ccg ccg ccg gac ttt gct 2208 Leu Pro Ser Ala Ala Ser Glu Arg Arg Pro Pro Pro Pro Asp Phe Ala 725 730 735 acg tct gcg ctc ggg ggc cag ccg ggc ttt ccg ttt ggt gca gcc ggc 2256 Thr Ser Ala Leu Gly Gly Gln Pro Gly Phe Pro Phe Gly Ala Ala Gly 740 745 750 cgg cag tcc acg ccg cac agc ggt cca ggc gtg aac tcg ccc ccc agc 2304 Arg Gln Ser Thr Pro His Ser Gly Pro Gly Val Asn Ser Pro Pro Ser 755 760 765 gcg gga ggg ggc ggt ggc agc tct ggt ggc ggc ggt ggc ggg ggt gcc 2352 Ala Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala 770 775 780 tac ccg ccg cag cct gat ttc cag ccc agc cag cgc acc tcg gcc agt 2400 Tyr Pro Pro Gln Pro Asp Phe Gln Pro Ser Gln Arg Thr Ser Ala Ser 785 790 795 800 aaa ttg ggc gcg ctc tcg ctg ggc tcc ttc aac aag ccc agc tcc aag 2448 Lys Leu Gly Ala Leu Ser Leu Gly Ser Phe Asn Lys Pro Ser Ser Lys 805 810 815 gac aac ctg ttc ggc cag agc tgc ctg gct gcg ctc tcc acc gct tgc 2496 Asp Asn Leu Phe Gly Gln Ser Cys Leu Ala Ala Leu Ser Thr Ala Cys 820 825 830 cag aac atg atc gcc agc ctc ggg gcc ccc aac ctc aac gtg acc ttc 2544 Gln Asn Met Ile Ala Ser Leu Gly Ala Pro Asn Leu Asn Val Thr Phe 835 840 845 aac aag aag aac ccg cca gag ggc aag agg aaa ctg agc cag aac gag 2592 Asn Lys Lys Asn Pro Pro Glu Gly Lys Arg Lys Leu Ser Gln Asn Glu 850 855 860 acc gac ggc gcg gca gtg gcc ggc aac ccg ggc tcg gat tac ttc cca 2640 Thr Asp Gly Ala Ala Val Ala Gly Asn Pro Gly Ser Asp Tyr Phe Pro 865 870 875 880 gga ggg act gct cct ggg gcc cca gga ccc gga ggc ccg tcc ggg acc 2688 Gly Gly Thr Ala Pro Gly Ala Pro Gly Pro Gly Gly Pro Ser Gly Thr 885 890 895 agt agc agc ggc tcc aaa gcc tcg ggg ccg ccc aac cct cca gcc cag 2736 Ser Ser Ser Gly Ser Lys Ala Ser Gly Pro Pro Asn Pro Pro Ala Gln 900 905 910 ggg gac ggc acc agc ctc tcc ccc aac tac acc ctg gaa tcc acg tcg 2784 Gly Asp Gly Thr Ser Leu Ser Pro Asn Tyr Thr Leu Glu Ser Thr Ser 915 920 925 ggg aat gac ggc aag ccg gtc tcc ggg ggc ggc ggc cgg gga cgg ggt 2832 Gly Asn Asp Gly Lys Pro Val Ser Gly Gly Gly Gly Arg Gly Arg Gly 930 935 940 cgc aga aaa agg gac agt ggt cac gtg agc cct ggc acc ttc ttt gac 2880 Arg Arg Lys Arg Asp Ser Gly His Val Ser Pro Gly Thr Phe Phe Asp 945 950 955 960 aag tac tcg gcg gct ccg gac agc ggg ggc gca cct ggg gtg agc cca 2928 Lys Tyr Ser Ala Ala Pro Asp Ser Gly Gly Ala Pro Gly Val Ser Pro 965 970 975 ggg cag cag caa gcg tca ggc gca gcc gtc ggg gga agc tcc gca ggc 2976 Gly Gln Gln Gln Ala Ser Gly Ala Ala Val Gly Gly Ser Ser Ala Gly 980 985 990 gag acg cgc ggg gca ccg acg ccc cac gaa aag gcg ctc acg tcg cca 3024 Glu Thr Arg Gly Ala Pro Thr Pro His Glu Lys Ala Leu Thr Ser Pro 995 1000 1005 tcc tgg ggg aag ggg gct gag ttg ctc ctg ggg gat cag ccg gac 3069 Ser Trp Gly Lys Gly Ala Glu Leu Leu Leu Gly Asp Gln Pro Asp 1010 1015 1020 ctc att ggg tcc ctg gac ggc ggg gcc aag tcg gac agt agt tcg 3114 Leu Ile Gly Ser Leu Asp Gly Gly Ala Lys Ser Asp Ser Ser Ser 1025 1030 1035 cca aac gtg ggt gag ttc gcc tcg gac gag gtg agc acg agc tac 3159 Pro Asn Val Gly Glu Phe Ala Ser Asp Glu Val Ser Thr Ser Tyr 1040 1045 1050 gcc aat gag gac gag gtg tcg tcc agc tct gac aac ccc cag gca 3204 Ala Asn Glu Asp Glu Val Ser Ser Ser Ser Asp Asn Pro Gln Ala 1055 1060 1065 cta gtt aaa gcg agc agg agt ccc ctg gtg acc ggc tcg ccc aaa 3249 Leu Val Lys Ala Ser Arg Ser Pro Leu Val Thr Gly Ser Pro Lys 1070 1075 1080 ctc cct ccc cgt ggg gta ggc gcc ggg gaa cac gga ccg aag gcg 3294 Leu Pro Pro Arg Gly Val Gly Ala Gly Glu His Gly Pro Lys Ala 1085 1090 1095 ccc ccg ccc gcc ctc ggc ctg ggc atc atg tct aac tct acc tcg 3339 Pro Pro Pro Ala Leu Gly Leu Gly Ile Met Ser Asn Ser Thr Ser 1100 1105 1110 acc cct gac agc tac ggc ggc ggt ggg ggc ccg ggc cat ccg ggc 3384 Thr Pro Asp Ser Tyr Gly Gly Gly Gly Gly Pro Gly His Pro Gly 1115 1120 1125 act ccg ggc ctg gag cag gtc cgc acc ccg acg agc agc agc ggc 3429 Thr Pro Gly Leu Glu Gln Val Arg Thr Pro Thr Ser Ser Ser Gly 1130 1135 1140 gcc ccg cca ccc gac gag atc cac ccc ctg gag atc ctt cag gcg 3474 Ala Pro Pro Pro Asp Glu Ile His Pro Leu Glu Ile Leu Gln Ala 1145 1150 1155 cag atc cag cta cag agg cag cag ttc agc atc tcc gag gac cag 3519 Gln Ile Gln Leu Gln Arg Gln Gln Phe Ser Ile Ser Glu Asp Gln 1160 1165 1170 cct ctg ggg ctg aag ggt ggc aag aag ggt gag tgc gcc gtc ggg 3564 Pro Leu Gly Leu Lys Gly Gly Lys Lys Gly Glu Cys Ala Val Gly 1175 1180 1185 gcc tca ggg gcg cag aat ggc gac agc gag ctg ggc agc tgc tgc 3609 Ala Ser Gly Ala Gln Asn Gly Asp Ser Glu Leu Gly Ser Cys Cys 1190 1195 1200 tcc gag gcg gtc aag agc gcc atg agc acc att gac ctg gac tcg 3654 Ser Glu Ala Val Lys Ser Ala Met Ser Thr Ile Asp Leu Asp Ser 1205 1210 1215 ctg atg gca gag cac agc gct gcc tgg tac atg ccc gct gac aag 3699 Leu Met Ala Glu His Ser Ala Ala Trp Tyr Met Pro Ala Asp Lys 1220 1225 1230 gcc ctg gtg gac agc gcg gac gac gac aag acg ttg gcg ccc tgg 3744 Ala Leu Val Asp Ser Ala Asp Asp Asp Lys Thr Leu Ala Pro Trp 1235 1240 1245 gag aag gcc aaa ccc cag aac ccc aac agc aaa gaa gcc cac gac 3789 Glu Lys Ala Lys Pro Gln Asn Pro Asn Ser Lys Glu Ala His Asp 1250 1255 1260 ctc cct gca aac aag gcc tca gca tcc cag cct ggc agc cac ttg 3834 Leu Pro Ala Asn Lys Ala Ser Ala Ser Gln Pro Gly Ser His Leu 1265 1270 1275 cag tgc ctg tct gtc cac tgc aca gac gac gtg ggt gac gcc aag 3879 Gln Cys Leu Ser Val His Cys Thr Asp Asp Val Gly Asp Ala Lys 1280 1285 1290 gct cga gcc tcc gtg ccc acc tgg cgg tcc ctg cat tct gac atc 3924 Ala Arg Ala Ser Val Pro Thr Trp Arg Ser Leu His Ser Asp Ile 1295 1300 1305 tcc aac aga ttt ggg aca ttc gtg gct gcc cta act tga 3963 Ser Asn Arg Phe Gly Thr Phe Val Ala Ala Leu Thr 1310 1315 1320 <210> 8 <211> 1320 <212> PRT <213> Homo sapiens <400> 8 Met Phe Gly Leu Asp Gln Phe Glu Pro Gln Val Asn Ser Arg Asn Ala 1 5 10 15 Gly Gln Gly Glu Arg Asn Phe Asn Glu Thr Gly Leu Ser Met Asn Thr 20 25 30 His Phe Lys Ala Pro Ala Phe His Thr Gly Gly Pro Pro Gly Pro Val 35 40 45 Asp Pro Ala Met Ser Ala Leu Gly Glu Pro Pro Ile Leu Gly Met Asn 50 55 60 Met Glu Pro Tyr Gly Phe His Ala Arg Gly His Ser Glu Leu His Ala 65 70 75 80 Gly Gly Leu Gln Ala Gln Pro Val His Gly Phe Phe Gly Gly Gln Gln 85 90 95 Pro His His Gly His Pro Gly Ser His His Pro His Gln His His Pro 100 105 110 His Phe Gly Gly Asn Phe Gly Gly Pro Asp Pro Gly Ala Ser Cys Leu 115 120 125 His Gly Gly Arg Leu Leu Gly Tyr Gly Gly Ala Ala Gly Gly Leu Gly 130 135 140 Ser Gln Pro Pro Phe Ala Glu Gly Tyr Glu His Met Ala Glu Ser Gln 145 150 155 160 Gly Pro Glu Ser Phe Gly Pro Gln Arg Pro Gly Asn Leu Pro Asp Phe 165 170 175 His Ser Ser Gly Ala Ser Ser His Ala Val Pro Ala Pro Cys Leu Pro 180 185 190 Leu Asp Gln Ser Pro Asn Arg Ala Ala Ser Phe His Gly Leu Pro Ser 195 200 205 Ser Ser Gly Ser Asp Ser His Ser Leu Glu Pro Arg Arg Val Thr Asn 210 215 220 Gln Gly Ala Val Asp Ser Leu Glu Tyr Asn Tyr Pro Gly Glu Ala Pro 225 230 235 240 Ser Gly His Phe Asp Met Phe Ser Pro Ser Asp Ser Glu Gly Gln Leu 245 250 255 Pro His Tyr Ala Ala Gly Arg Gln Val Pro Gly Gly Ala Phe Pro Gly 260 265 270 Ala Ser Ala Met Pro Arg Ala Ala Gly Met Val Gly Leu Ser Lys Met 275 280 285 His Ala Gln Pro Pro Gln Gln Gln Pro Gln Gln Gln Gln Gln Pro Gln 290 295 300 Gln Gln Gln Gln Gln His Gly Val Phe Phe Glu Arg Phe Ser Gly Ala 305 310 315 320 Arg Lys Met Pro Val Gly Leu Glu Pro Ser Val Gly Ser Arg His Pro 325 330 335 Leu Met Gln Pro Pro Gln Gln Ala Pro Pro Pro Pro Gln Gln Gln Pro 340 345 350 Pro Gln Gln Pro Pro Gln Gln Gln Pro Pro Pro Pro Pro Gly Leu Leu 355 360 365 Val Arg Gln Asn Ser Cys Pro Pro Ala Leu Pro Arg Pro Gln Gln Gly 370 375 380 Glu Ala Gly Thr Pro Ser Gly Gly Leu Gln Asp Gly Gly Pro Met Leu 385 390 395 400 Pro Ser Gln His Ala Gln Phe Glu Tyr Pro Ile His Arg Leu Glu Asn 405 410 415 Arg Ser Met His Pro Tyr Ser Glu Pro Val Phe Ser Met Gln His Pro 420 425 430 Pro Pro Gln Gln Ala Pro Asn Gln Arg Leu Gln His Phe Asp Ala Pro 435 440 445 Pro Tyr Met Asn Val Ala Lys Arg Pro Arg Phe Asp Phe Pro Gly Ser 450 455 460 Ala Gly Val Asp Arg Cys Ala Ser Trp Asn Gly Ser Met His Asn Gly 465 470 475 480 Ala Leu Asp Asn His Leu Ser Pro Ser Ala Tyr Pro Gly Leu Pro Gly 485 490 495 Glu Phe Thr Pro Pro Val Pro Asp Ser Phe Pro Ser Gly Pro Pro Leu 500 505 510 Gln His Pro Ala Pro Asp His Gln Ser Leu Gln Gln Gln Gln Gln Gln 515 520 525 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 530 535 540 Gln Gln Gln Gln Gln Gln Arg Gln Asn Ala Ala Leu Met Ile Lys Gln 545 550 555 560 Met Ala Ser Arg Asn Gln Gln Gln Arg Leu Arg Gln Pro Asn Leu Ala 565 570 575 Gln Leu Gly His Pro Gly Asp Val Gly Gln Gly Gly Leu Val His Gly 580 585 590 Gly Pro Val Gly Gly Leu Ala Gln Pro Asn Phe Glu Arg Glu Gly Gly 595 600 605 Ser Thr Gly Ala Gly Arg Leu Gly Thr Phe Glu Gln Gln Ala Pro His 610 615 620 Leu Ala Gln Glu Ser Ala Trp Phe Ser Gly Pro His Pro Pro Pro Gly 625 630 635 640 Asp Leu Leu Pro Arg Arg Met Gly Gly Ser Gly Leu Pro Ala Asp Cys 645 650 655 Gly Pro His Asp Pro Ser Leu Ala Pro Pro Pro Pro Pro Gly Gly Ser 660 665 670 Gly Val Leu Phe Arg Gly Pro Leu Gln Glu Pro Met Arg Met Pro Gly 675 680 685 Glu Gly His Val Pro Ala Leu Pro Ser Pro Gly Leu Gln Phe Gly Gly 690 695 700 Ser Leu Gly Gly Leu Gly Gln Leu Gln Ser Pro Gly Ala Gly Val Gly 705 710 715 720 Leu Pro Ser Ala Ala Ser Glu Arg Arg Pro Pro Pro Pro Asp Phe Ala 725 730 735 Thr Ser Ala Leu Gly Gly Gln Pro Gly Phe Pro Phe Gly Ala Ala Gly 740 745 750 Arg Gln Ser Thr Pro His Ser Gly Pro Gly Val Asn Ser Pro Pro Ser 755 760 765 Ala Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala 770 775 780 Tyr Pro Pro Gln Pro Asp Phe Gln Pro Ser Gln Arg Thr Ser Ala Ser 785 790 795 800 Lys Leu Gly Ala Leu Ser Leu Gly Ser Phe Asn Lys Pro Ser Ser Lys 805 810 815 Asp Asn Leu Phe Gly Gln Ser Cys Leu Ala Ala Leu Ser Thr Ala Cys 820 825 830 Gln Asn Met Ile Ala Ser Leu Gly Ala Pro Asn Leu Asn Val Thr Phe 835 840 845 Asn Lys Lys Asn Pro Pro Glu Gly Lys Arg Lys Leu Ser Gln Asn Glu 850 855 860 Thr Asp Gly Ala Ala Val Ala Gly Asn Pro Gly Ser Asp Tyr Phe Pro 865 870 875 880 Gly Gly Thr Ala Pro Gly Ala Pro Gly Pro Gly Gly Pro Ser Gly Thr 885 890 895 Ser Ser Ser Gly Ser Lys Ala Ser Gly Pro Pro Asn Pro Pro Ala Gln 900 905 910 Gly Asp Gly Thr Ser Leu Ser Pro Asn Tyr Thr Leu Glu Ser Thr Ser 915 920 925 Gly Asn Asp Gly Lys Pro Val Ser Gly Gly Gly Gly Arg Gly Arg Gly 930 935 940 Arg Arg Lys Arg Asp Ser Gly His Val Ser Pro Gly Thr Phe Phe Asp 945 950 955 960 Lys Tyr Ser Ala Ala Pro Asp Ser Gly Gly Ala Pro Gly Val Ser Pro 965 970 975 Gly Gln Gln Gln Ala Ser Gly Ala Ala Val Gly Gly Ser Ser Ala Gly 980 985 990 Glu Thr Arg Gly Ala Pro Thr Pro His Glu Lys Ala Leu Thr Ser Pro 995 1000 1005 Ser Trp Gly Lys Gly Ala Glu Leu Leu Leu Gly Asp Gln Pro Asp 1010 1015 1020 Leu Ile Gly Ser Leu Asp Gly Gly Ala Lys Ser Asp Ser Ser Ser 1025 1030 1035 Pro Asn Val Gly Glu Phe Ala Ser Asp Glu Val Ser Thr Ser Tyr 1040 1045 1050 Ala Asn Glu Asp Glu Val Ser Ser Ser Ser Asp Asn Pro Gln Ala 1055 1060 1065 Leu Val Lys Ala Ser Arg Ser Pro Leu Val Thr Gly Ser Pro Lys 1070 1075 1080 Leu Pro Pro Arg Gly Val Gly Ala Gly Glu His Gly Pro Lys Ala 1085 1090 1095 Pro Pro Pro Ala Leu Gly Leu Gly Ile Met Ser Asn Ser Thr Ser 1100 1105 1110 Thr Pro Asp Ser Tyr Gly Gly Gly Gly Gly Pro Gly His Pro Gly 1115 1120 1125 Thr Pro Gly Leu Glu Gln Val Arg Thr Pro Thr Ser Ser Ser Gly 1130 1135 1140 Ala Pro Pro Pro Asp Glu Ile His Pro Leu Glu Ile Leu Gln Ala 1145 1150 1155 Gln Ile Gln Leu Gln Arg Gln Gln Phe Ser Ile Ser Glu Asp Gln 1160 1165 1170 Pro Leu Gly Leu Lys Gly Gly Lys Lys Gly Glu Cys Ala Val Gly 1175 1180 1185 Ala Ser Gly Ala Gln Asn Gly Asp Ser Glu Leu Gly Ser Cys Cys 1190 1195 1200 Ser Glu Ala Val Lys Ser Ala Met Ser Thr Ile Asp Leu Asp Ser 1205 1210 1215 Leu Met Ala Glu His Ser Ala Ala Trp Tyr Met Pro Ala Asp Lys 1220 1225 1230 Ala Leu Val Asp Ser Ala Asp Asp Asp Lys Thr Leu Ala Pro Trp 1235 1240 1245 Glu Lys Ala Lys Pro Gln Asn Pro Asn Ser Lys Glu Ala His Asp 1250 1255 1260 Leu Pro Ala Asn Lys Ala Ser Ala Ser Gln Pro Gly Ser His Leu 1265 1270 1275 Gln Cys Leu Ser Val His Cys Thr Asp Asp Val Gly Asp Ala Lys 1280 1285 1290 Ala Arg Ala Ser Val Pro Thr Trp Arg Ser Leu His Ser Asp Ile 1295 1300 1305 Ser Asn Arg Phe Gly Thr Phe Val Ala Ala Leu Thr 1310 1315 1320 <210> 9 <211> 399 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(399) <400> 9 atg cga cgg ctg ctg atc cct ctg gcc ctg tgg ctg ggt gcg gtg ggc 48 Met Arg Arg Leu Leu Ile Pro Leu Ala Leu Trp Leu Gly Ala Val Gly 1 5 10 15 gtg ggc gtc gcc gag ctc acg gaa gcc cag cgc cgg ggc ctg cag gtg 96 Val Gly Val Ala Glu Leu Thr Glu Ala Gln Arg Arg Gly Leu Gln Val 20 25 30 gcc ctg gag gaa ttt cac aag cac ccg ccc gtg cag tgg gcc ttc cag 144 Ala Leu Glu Glu Phe His Lys His Pro Pro Val Gln Trp Ala Phe Gln 35 40 45 gag acc agt gtg gag agc gcc gtg gac acg ccc ttc cca gct gga ata 192 Glu Thr Ser Val Glu Ser Ala Val Asp Thr Pro Phe Pro Ala Gly Ile 50 55 60 ttt gtg agg ctg gaa ttt aag ctg cag cag aca agc tgc cgg aag agg 240 Phe Val Arg Leu Glu Phe Lys Leu Gln Gln Thr Ser Cys Arg Lys Arg 65 70 75 80 gac tgg aag aaa ccc gag tgc aaa gtc agg ccc aat ggg agg aaa cgg 288 Asp Trp Lys Lys Pro Glu Cys Lys Val Arg Pro Asn Gly Arg Lys Arg 85 90 95 aaa tgc ctg gcc tgc atc aaa ctg ggc tct gag gac aaa gtt ctg ggc 336 Lys Cys Leu Ala Cys Ile Lys Leu Gly Ser Glu Asp Lys Val Leu Gly 100 105 110 cgg ttg gtc cac tgc ccc ata gag acc caa gtt ctg cgg ttt tgg gca 384 Arg Leu Val His Cys Pro Ile Glu Thr Gln Val Leu Arg Phe Trp Ala 115 120 125 ctg gca gga ggc tga 399 Leu Ala Gly Gly 130 <210> 10 <211> 132 <212> PRT <213> Homo sapiens <400> 10 Met Arg Arg Leu Leu Ile Pro Leu Ala Leu Trp Leu Gly Ala Val Gly 1 5 10 15 Val Gly Val Ala Glu Leu Thr Glu Ala Gln Arg Arg Gly Leu Gln Val 20 25 30 Ala Leu Glu Glu Phe His Lys His Pro Pro Val Gln Trp Ala Phe Gln 35 40 45 Glu Thr Ser Val Glu Ser Ala Val Asp Thr Pro Phe Pro Ala Gly Ile 50 55 60 Phe Val Arg Leu Glu Phe Lys Leu Gln Gln Thr Ser Cys Arg Lys Arg 65 70 75 80 Asp Trp Lys Lys Pro Glu Cys Lys Val Arg Pro Asn Gly Arg Lys Arg 85 90 95 Lys Cys Leu Ala Cys Ile Lys Leu Gly Ser Glu Asp Lys Val Leu Gly 100 105 110 Arg Leu Val His Cys Pro Ile Glu Thr Gln Val Leu Arg Phe Trp Ala 115 120 125 Leu Ala Gly Gly 130

Claims

1. A cell population containing amniotic-derived stem cells, wherein the positive rates of CD90, CD73, and CD105 are all 90% or more, and the positive rate of CD45 is less than 5%, and further having the following cell characteristics (i) to (iv): (i) The ratio of adherent stem cells showing positive for KCNAB1 is 85% or more; (ii) The relative expression level of the SULT1E1 gene with respect to the expression level of the SDHA gene is 0.1 or more; (iii) The relative expression level of the MN1 gene with respect to the expression level of the SDHA gene is 0.7 or more; and (iv) The relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene is 0.4 or less.

2. A pharmaceutical composition comprising the cell population according to Claim 1 and a pharmaceutically acceptable medium.

3. A pharmaceutical composition comprising the cell population according to Claim 1 and other administrable cells.

4. The pharmaceutical composition according to claim 2 or 3, wherein a single dose of the adhesive stem cells for humans is 10 １２ cells / kg body weight or less.

5. The pharmaceutical composition according to any one of Claims 2 to 4, wherein the pharmaceutical composition is an injectable preparation.

6. The pharmaceutical composition according to any one of Claims 2 to 4, wherein the pharmaceutical composition is a preparation for transplantation of cell masses or sheet-like structures.

7. A therapeutic agent for diseases selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, fungating polyps, scleroderma, diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injuries, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis deformans, inflammatory arthritis, rheumatoid arthritis, eye diseases, angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular disease, wounds, epithelial injuries, fibrosis, lung diseases, and cancer. The pharmaceutical composition according to any one of Claims 2 to 6.

8. A method for producing a cell population containing amniotic-derived stem cells, comprising: (a) enzymatically treating amniotic membrane collected from a donor to obtain a cell population containing adherent stem cells; (b) culturing the cell population; and (c) obtaining a cell population having the following cell characteristics (i) to (iv) in the cell population: (i) The ratio of adherent stem cells showing positive for KCNAB1 is 85% or more; (ii) The relative expression level of the SULT1E1 gene with respect to the expression level of the SDHA gene is 0.1 or more; (iii) The relative expression level of the MN1 gene with respect to the expression level of the SDHA gene is 0.7 or more; and (iv) The relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene is 0.4 or less; A production method comprising the above.

9. The method according to claim 8, wherein the culture in (b) includes one or more subcultures in a medium containing platelet lysate.

10. The method according to claim 8 or 9, wherein in the culture in (b), the seeding density is 100 to 20,000 cells / cm2.

11. The method according to any one of claims 8 to 10, wherein the culture in (b) includes culturing until the confluence rate is 95% or less.

Images