Method for improving ability to differentiate into osteoblasts and / or adipocytes

Abstract

The present invention addresses the problem of providing a method for supplying a large amount of high-quality mesenchymal stem cells by conferring differentiation ability again to mesenchymal stem cells that have partially lost differentiation ability in the process of isolation.　Provided is a method for preparing a cell population containing genetically modified mesenchymal stem cells that are able to differentiate into osteoblasts and / or adipocytes, the method comprising: a genetic modification step for genetically modifying mesenchymal stem cells in a cell population by introducing a gene encoding a telomerase reverse transcriptase or an active fragment thereof and a gene encoding a polycomb complex protein Bmi-1 or an active fragment thereof into the mesenchymal stem cells, wherein the mesenchymal stem cells are derived from amnion and are unable to differentiate into osteoblasts, unable to differentiate into adipocytes, or unable to differentiate into either osteoblasts or adipocytes; and an expression culture step for culturing the genetically modified mesenchymal stem cells and expressing the genes.

Description

Method for improving the differentiation ability into osteoblasts and / or adipocytes

[0001] The present invention relates to a method for preparing a cell population containing genetically modified mesenchymal stem cells having the ability to differentiate into osteoblasts and / or adipocytes, a method for improving the differentiation ability into osteoblasts and / or adipocytes, and a composition containing a nucleic acid molecule for use in these methods.

[0002] Pluripotent stem cells are cells that have the ability to differentiate into various somatic cells. Examples of pluripotent stem cells include iPS cells obtained by introducing reprogramming factors into somatic cells, ES cells derived from the inner cell mass, and mesenchymal stem cells which are somatic stem cells.

[0003] Among these, mesenchymal stem cells are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, etc., and have been found to exist in various tissues such as bone marrow, adipose tissue, dental pulp, fetal appendages (placenta, umbilical cord, amnion, etc.). We have developed a method for easily isolating mesenchymal stem cells from amnion and have been conducting research and development for clinical application as a cell preparation (Patent Document 1). In fact, clinical applications for Duchenne muscular dystrophy and spinal cord injury are progressing by utilizing the immunosuppressive ability of amnion-derived mesenchymal stem cells. In addition, amnion mesenchymal stem cells may be clinically applicable to a wide range of diseases such as acute graft-versus-host disease (GVHD), Crohn's disease, cardiomyopathy, and osteoarthritis deformans. Furthermore, since a large number of cells can be collected from a single amnion and large-scale culture is also possible, amnion-derived mesenchymal stem cells are also useful as a regenerative medical material for ex vivo gene therapy.

[0004] While some reports (Non-Patent Documents 1 and 2) indicate that amniotic mesenchymal stem cells (MSCs) possess the ability to differentiate into osteoblasts, chondrocytes, and adipocytes, similar to MSCs derived from other tissues, others (Non-Patent Document 3) suggest they do not differentiate into adipocytes and hardly differentiate into osteoblasts. The two reports differ in their isolation methods and culture medium compositions, which are thought to have led to the differences in MSC differentiation potential. Despite various methods being investigated for MSC isolation and culture medium composition, standardization remains difficult due to individual differences and other factors. Therefore, there has been a need for the development of a method to stably and abundantly supply high-quality MSCs with the ability to differentiate into osteoblasts, adipocytes, and other cells.

[0005] WO2015 / 025810

[0006] Mu, Y. et al BMC Cell Biol. , 13;19(1):27 (2018) Diaz-Prado, S. et al Tissue Eng Part C Methods. , 17(1):49-59 (2011) Bilic, G. et al Cell Transplant. , 17(8):955-968 (2008)

[0007] Because amniotic mesenchymal stem cells are cells whose characteristics are easily altered by the environment, even slight differences in isolation conditions can lead to a partial loss of differentiation potential, and identifying the cause is often difficult. This has been one of the challenges in ensuring a stable and large-scale supply of high-quality mesenchymal stem cells.

[0008] However, there has been very little research to date on restoring differentiation ability to mesenchymal stem cells that have partially lost their differentiation potential.

[0009] Therefore, the object of the present invention is to provide a method for restoring differentiation ability to mesenchymal stem cells that have partially lost their differentiation ability during the isolation process, thereby providing a stable and large supply of high-quality mesenchymal stem cells.

[0010] To solve the above problems, the inventors conducted intensive research and found that by introducing a combination of the human telomerase reverse transcriptase gene (hTERT gene) and the Polycomb complex protein gene (BMI1 gene) into amniotic mesenchymal stem cells, even mesenchymal stem cells that have lost the ability to differentiate into osteoblasts and adipocytes can acquire the ability to differentiate into these cells.

[0011] The present invention is based on the aforementioned novel findings and provides the following: [1] A method for preparing a cell population including genetically modified mesenchymal stem cells having the ability to differentiate into osteoblasts and / or adipocytes, comprising: a gene modification step of genetically modifying mesenchymal stem cells by introducing a gene encoding telomerase reverse transcriptase or an active fragment thereof, and a gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof into a cell population including mesenchymal stem cells derived from amniotic membrane that do not have the ability to differentiate into osteoblasts and / or adipocytes; and an expression culture step of culturing the genetically modified mesenchymal stem cells to express the genes. [2] The method according to [1], further comprising a single-cell formation step of single-cell formation of the genetically modified mesenchymal stem cells. [3] The method according to [1] or [2], further comprising a large-scale culture step of large-scale culture of the genetically modified mesenchymal stem cells. [4] The method according to any one of [1] to [3], wherein the mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes are mesenchymal stem cells isolated by treatment of the amnion, including the epithelial cell layer, with a mixture of dispase and collagenase. [5] The method according to any one of [1] to [4], wherein the mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes are mesenchymal stem cells that have not been treated with any enzymes including DNase I, trypsin, and hyaluronidase. [6] The method according to [5], wherein the mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes are mesenchymal stem cells isolated by treatment with a mixture containing substantially only dispase and collagenase as enzymes. [7] The method according to any one of [1] to [6], further comprising a selection step of selecting the genetically modified mesenchymal stem cells. [8] The method according to any one of [1] to [7], further comprising a first culture step of culturing the mesenchymal stem cells in a first culture medium before the genetic modification step. [9] The method according to [8], wherein the first culture medium comprises 3 v / v% to 20 v / v% human platelet lysate.

[10] A method for improving the differentiation ability to osteoblasts and / or adipocytes, comprising a gene modification step of genetically modifying the mesenchymal stem cells in a cell population including mesenchymal stem cells derived from amnion and lacking the differentiation ability to osteoblasts and / or adipocytes, by introducing a gene encoding telomerase reverse transcriptase or an active fragment thereof, and a gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof into the mesenchymal stem cells.

[11] A composition for use in any of the methods of [1] to

[10] , comprising a nucleic acid molecule containing an exogenous gene encoding telomerase reverse transcriptase or an active fragment thereof, and / or a nucleic acid molecule containing an exogenous gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof. This specification includes the disclosures of Japanese Patent Application No. 2025-013826, which forms the basis of the priority of this application.

[0012] The present invention provides a method for preparing a cell population containing genetically modified mesenchymal stem cells that have the ability to differentiate into osteoblasts and / or adipocytes from a cell population containing mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes, and a method for improving the differentiation ability to osteoblasts and / or adipocytes in a cell population containing mesenchymal stem cells that lack the ability to differentiate into osteoblasts and / or adipocytes.

[0013] Furthermore, the present invention provides compositions comprising nucleic acid molecules containing exogenous genes for use in those methods.

[0014] 1. Method for Preparing Cell Populations 1-1. Overview The first aspect of the present invention is a method for preparing a cell population including genetically modified mesenchymal stem cells (hereinafter sometimes abbreviated as "preparation method"). The preparation method of the present invention is characterized by introducing the TERT gene and the BMI1 gene. According to the preparation method of the present invention, genetically modified mesenchymal stem cells having the ability to differentiate into osteoblasts and / or adipocytes can be prepared from amniotic mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes.

[0015] 1-2. Definitions of Terms The following terms, which are frequently used in this specification, are defined below.

[0016] In this specification, "fetal appendages" refers to tissues or organs other than the fetal body that support fetal development within the uterus, specifically the amniotic membrane, placenta, umbilical cord, and amniotic fluid. The "amniotic membrane" is the gestational sac containing the amniotic fluid of the fetus, and consists of the amnion, chorion, and decidua from the inside out. The "amnion" refers to the innermost layer of the amniotic membrane, a transparent membrane with few blood vessels. The inner layer of the amnion (also called the epithelial cell layer) is covered with a single layer of epithelial cells with secretory function that secrete amniotic fluid, and the outer layer of the amnion (also called the extracellular matrix layer, corresponding to the stroma) contains mesenchymal stem cells.

[0017] In this specification, “mesenchymal stem cells” refers to stem cells that (i) exhibit adhesion to the surface of a plastic culture vessel under culture conditions in standard medium (basal medium supplemented with serum, serum substitute reagent, or growth factor), and (ii) are positive for surface antigens CD105, CD73, and CD90, and negative for CD45. In this specification, “mesenchymal stem cells” is also written as “MSC” and is synonymous with “mesenchymal stromal cells.” The species from which mesenchymal stem cells are derived is not particularly limited as long as it is a species that has mesenchymal stem cells, for example, fish, birds, or mammals. Mesenchymal stem cells may be, for example, mouse cells, chimpanzee cells, or human cells. Furthermore, while mesenchymal stem cells can be derived from bone marrow, hematopoietic stem cells, umbilical cord blood, umbilical cord, amniotic membrane, amniotic fluid, placental villi, nerves, adipose tissue, pancreas, synovial membrane, dental pulp, deciduous teeth, sperm, testes, cornea, etc., this invention focuses on mesenchymal stem cells derived from amniotic membrane.

[0018] In this specification, "cell population containing mesenchymal stem cells" refers to a cell population containing mesenchymal stem cells derived from the amniotic membrane. The form of the cell population containing mesenchymal stem cells is not particularly limited and may be, for example, a cell pellet, cell aggregate, cell suspension, or cell suspension. The cell population may further contain cells other than mesenchymal stem cells or cells derived from mesenchymal stem cells, or it may be a cell population containing only cells derived from mesenchymal stem cells, such as mesenchymal stem cells or cells differentiated from mesenchymal stem cells. The cell population containing mesenchymal stem cells may be derived from a single or multiple mesenchymal stem cells, or it may be a mixture of mesenchymal stem cells obtained from multiple individuals. For example, it may be a cell population containing only cells derived from a single mesenchymal stem cell. The proportion of mesenchymal stem cells derived from the amniotic membrane when cells other than mesenchymal stem cells derived from the amniotic membrane are not particularly limited, but may be, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%. The proportion of mesenchymal stem cells can be determined, for example, based on positive and / or negative markers for mesenchymal stem cells.

[0019] In this specification, "amniotic mesenchymal stem cells" refers to mesenchymal stem cells derived from the amniotic membrane. In this specification, amniotic mesenchymal stem cells are also referred to as "amniotic MSCs" or "AMSCs".

[0020] As mentioned above, various surface antigens are known for mesenchymal stem cells. In this specification, surface antigens that can characterize mesenchymal stem cells based on a high positive rate are sometimes referred to as "positive markers," and surface antigens that can characterize mesenchymal stem cells based on a low positive rate are sometimes referred to as "negative markers." Specific examples of surface antigens of mesenchymal stem cells include CD324 (differentiation cluster 324; also known as epithelial cadherin (E-cadherin)), CD90 (differentiation cluster 90; also known as Thy-1), CD326 (differentiation cluster 326; also known as EpCAM), CD73 (differentiation cluster 73; also known as 5-Nucleotidase or Ecto-5'-Nucleotidase), CD166 (differentiation cluster 166; also known as Activated leukocyte cell adhesion molecule (ALCAM)), CD105 (differentiation cluster 105; also known as Endoglin), and CD45 (differentiation cluster 45; PTPRC (Protein tyrosine phosphorase, receptor) Examples include type C) or LCA (also known as Leukocyte common antibody), CD34 (differentiation cluster 34; also known as Hematopoietic progenitor cell antibody), etc. In mesenchymal stem cells, CD73, CD90, CD105, and CD166 are classified as positive markers, while CD34, CD45, CD324, and CD326 can be classified as negative markers.

[0021] In this specification, the "positive rate" of a surface antigen refers to the proportion of cells in a cell population that are positive for a given surface antigen. For example, the positive rate can be determined by evaluating the expression of the surface antigen in a cell population using any expression analysis method such as flow cytometry or immunohistochemistry.

[0022] In this specification, "osteoblast" refers to a cell type that differentiates into osteocytes and secretes bone matrix molecules such as osteocalcin (OCN). Typically, osteoblasts differentiate from preosteoblasts, which are derived from osteoprogenitor cells. Known markers for osteoblasts include the expression of alkaline phosphatase (ALP), type I collagen, osteopontin (OPN), OCN, and bone sialoprotein (BSP).

[0023] In this specification, "adipocyte" refers to a cell characterized by carrying large lipid droplets in its cytoplasm. While types such as white adipocytes and brown adipocytes are known, the term "adipocyte" in this specification encompasses all of them, but typically refers to white adipocytes. Markers for adipocytes include perilipin, adipsin, leptin, adiponectin, and FABP4, as well as UCP1, PRDM16, TBX1, and TMEM26 expression in brown adipocytes.

[0024] In this specification, “differentiation” refers to the specialization and increased specificity of a cell’s morphology and / or function. In particular, differentiation refers to the restriction of a cell’s differentiation fate and a reduction in the number of cell types that can arise from it.

[0025] In this specification, "differentiation potential" refers to the possibility that a cell can differentiate into a specific cell type without undergoing dedifferentiation. In particular, in this specification, "differentiation potential into osteoblasts and / or adipocytes" refers to the possibility that a mesenchymal stem cell can differentiate into osteoblasts and / or adipocytes without undergoing dedifferentiation. Therefore, in this specification, "lacking differentiation potential" refers to the inability to differentiate into a specific cell type (e.g., osteoblasts and / or adipocytes) at all, or with little to no differentiation. On the other hand, in this specification, "having differentiation potential" refers to the ability to differentiate into a specific cell type (e.g., osteoblasts and / or adipocytes) with the efficiency normally expected for that cell type (e.g., amniotic mesenchymal stem cells) by applying appropriate stimulation.

[0026] In this specification, "lacking differentiation ability" means that a cell lacks the ability to differentiate into a specific cell type (e.g., osteoblasts and / or adipocytes), or that, while it may be able to differentiate into those cell types upon appropriate stimulation, the degree of differentiation is less than what would normally be expected in the source cell type (e.g., amniotic mesenchymal stem cells). Typically, a cell population containing mesenchymal stem cells lacking the ability to differentiate into osteoblasts and / or adipocytes (often abbreviated as "deficient population" in this specification) exhibits a certain level and / or significantly lower differentiation efficiency to the same cell type upon the same differentiation stimulation compared to a cell population containing mesenchymal stem cells that possess the ability to differentiate into osteoblasts and / or adipocytes.

[0027] In this specification, "improving differentiation ability" means improving the ability of a cell population to differentiate into a specific cell type (e.g., osteoblasts and / or adipocytes) that is deficient in that population. Typically, in a deficient population or a cell population containing mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes (often abbreviated as "undifferentiated population" in this specification), the differentiation efficiency to the same cell type given the same differentiation stimulus is increased to a certain level and / or significantly. The criteria for a certain level of increased differentiation efficiency are not particularly limited, but for example, the improved differentiation efficiency is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more higher than the differentiation efficiency in the deficient population.

[0028] In this specification, "cell population containing genetically modified mesenchymal stem cells" refers to a cell population containing mesenchymal stem cells, particularly including genetically modified mesenchymal stem cells. Such a cell population may also contain cells other than genetically modified mesenchymal stem cells, and the proportion of genetically modified mesenchymal stem cells in such cases is not particularly limited. For example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the cells constituting the cell population are genetically modified mesenchymal stem cells. The proportion of genetically modified mesenchymal stem cells can be determined, for example, based on various indicators described in the selection process. Such a cell population may originate from a single genetically modified mesenchymal stem cell or from multiple genetically modified mesenchymal stem cells.

[0029] In this specification, "telomerase reverse transcriptase (TERT)" refers to a protein that, together with telomerase RNA component (TERC), constitutes the telomerase complex. Telomerase binds to the ends of chromosomes and adds telomere repeat sequences to the 3' end of telomere DNA using TERC as a template. Telomerase is hardly expressed in normal somatic cells in humans, but is expressed in fetal cells, germ cells, etc. Cells lacking telomerase activity undergo telomere shortening with each cell division, leading to cellular senescence and thus limiting the number of cell divisions. However, cells with telomerase activity maintain telomere length through telomerase activity, potentially allowing for cell proliferation beyond the limit.

[0030] In this specification, the species from which TERT originates is not limited. Examples include TERT derived from mammals, specifically mice, rats, rabbits, cattle, cynomolgus monkeys, marmosets, or humans. In this specification, human-derived telomerase reverse transcriptase may be referred to as "hTERT". Furthermore, in this specification, TERT may be a mutant of TERT derived from any species, for example, a mutant of hTERT.

[0031] In this specification, the amino acid sequence of TERT is not particularly limited. Specific examples of the amino acid sequence of TERT include hTERT and its variants, which consist of the amino acid sequence shown in Sequence ID No. 1. The variant of hTERT is preferably one that possesses the enzymatic activity of TERT, such as reverse transcriptase activity. Examples of such variants include those consisting of amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequence shown in Sequence ID No. 1.

[0032] In this specification, "active fragment" of TERT means a fragment of any of the above-mentioned TERTs that has the enzymatic activity of TERT (e.g., reverse transcriptase activity), for example, a fragment having 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of hTERT consisting of the amino acid sequence shown in Sequence ID No. 1, or equivalent or greater activity. The amino acid length of the polypeptide constituting this fragment is not particularly limited, but for example, it may be a continuous region in any of the above-mentioned TERTs having an amino acid length of 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, or 1100 or more.

[0033] In this specification, "Polycomb complex protein Bmi-1" is also called BMI1 Proto-Oncogene, Polycomb Ring Finger, etc., and is a protein that constitutes Polycomb Group Complex 1 (PRC1). PRC1 functions as an essential epigenetic repressor of several regulatory genes involved in embryonic development and somatic stem cell self-renewal through chromatin remodeling. By inactivating p16, which arrests the cell cycle in the G1 phase, Bmi-1 can avoid cell cycle arrest and enable cell proliferation beyond its limits.

[0034] In this specification, the species from which Bmi-1 originates is not limited. Examples of Bmi-1 include Bmi-1 derived from mammals, such as rats, rabbits, cattle, cynomolgus monkeys, marmosets, humans, or mice. In this specification, Bmi-1 may also be a variant of Bmi-1 derived from any species, for example, a variant of Bmi-1 derived from a mouse.

[0035] In this specification, the amino acid sequence of Bmi-1 is not particularly limited. Examples include mouse Bmi-1 and its mutants, which consist of the amino acid sequence shown in Sequence ID No. 5. Mutants of mouse Bmi-1 are preferably those that have Bmi-1 activity, and examples of such mutants include those whose amino acid sequences have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequence shown in Sequence ID No. 5.

[0036] In this specification, the "active fragment" of Bmi-1 means a fragment of any of the above-mentioned Bmi-1 that has the activity of Bmi-1, for example, a fragment that has 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of Bmi-1 consisting of the amino acid sequence shown in Sequence ID No. 5, or an activity equivalent to or greater thereto. The amino acid length of the polypeptide constituting this fragment is not particularly limited, but for example, it may be a continuous region having an amino acid length of 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, or 300 or more in any of the above-mentioned Bmi-1.

[0037] In this specification, "a gene encoding telomerase reverse transcriptase or its active fragment" may be any gene encoding TERT or its active fragment as described above. In this specification, a gene encoding telomerase reverse transcriptase or its active fragment may be referred to as a "TERT gene." Specific examples of TERT genes include the hTERT gene consisting of the nucleotide sequence shown in Sequence ID No. 2, and mutant genes consisting of nucleotide sequences having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide identity with respect to the nucleotide sequence shown in Sequence ID No. 2. A gene encoding human-derived telomerase reverse transcriptase or its active fragment may be referred to as an "hTERT gene."

[0038] In this specification, "a gene encoding the Polycomb complex protein Bmi-1 or its active fragment" may be any gene encoding Bmi-1 or its active fragment as described above. In this specification, a gene encoding Bmi-1 or its active fragment may be referred to as the "BMI1 gene." Specific examples of the BMI1 gene include the Bmi-1 gene consisting of the nucleotide sequence shown in Sequence ID No. 6, and mutant genes consisting of nucleotide sequences having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide identity with respect to the nucleotide sequence shown in Sequence ID No. 6.

[0039] In this specification, "amino acid identity" refers to the percentage of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting gaps as needed into one or both of them to maximize the number of matching amino acid residues. Alignment of two amino acid sequences for calculating amino acid identity can be performed using known programs such as Blast, FASTA, and CrystalW. "Base identity" is calculated in the same manner.

[0040] In this specification, "culture medium" is not limited to any medium capable of maintaining cells such as mesenchymal stem cells as living cells. For example, the culture medium may be any medium known in the field that is commonly used in cell culture. The culture medium may be a basic medium, a serum-free medium, a low-serum medium, or a serum-supplemented medium, but usually a basic medium (e.g., a standard cell culture medium) or a serum-free medium is sufficient and can be prepared by adding other components as needed. "Standard cell culture medium" as used herein refers to a highly versatile basic medium mainly used for culturing various types of cells of mammalian origin. Specifically, for example, 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, MEM-α (Minimum Essential Medium α) medium, DMEM medium (Dulbecco's Modified Eagle's) Medium, Advanced DMEM 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 are not particularly limited. Various commercially available serum-free media can also be used. Other components to be added to the basic medium include, for example, albumin, blood-derived components, growth factors, etc.

[0041] In this specification, "significant" refers to being statistically significant. Statistical significance means that when the difference between the measured value of the test subject and the control value is statistically analyzed, there is a significant difference between the two. For example, when the significance level of the obtained value is small, specifically less than 5% (p < 0.05), less than 1% (p < 0.01), or less than 0.1% (p < 0.001). The "p (value)" shown here represents the probability that the test statistic happens to be that value within the distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p," the lower the probability that the test statistic is that value, and the more likely the null hypothesis is to be rejected. The statistical testing method may be any known test method that can determine the presence or absence of significance, and is not particularly limited. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank-sum test, analysis of variance, Tukey post-hoc test, etc., can be used, but are not particularly limited.

[0042] 1-3. Process The preparation method of the present invention includes a gene modification step and an expression culture step as essential steps, and may include a mesenchymal stem cell preparation step, a first culture step, a harvesting step, a single-cell formation step, an expanded culture step, and / or a selection step as optional steps. Each step in this embodiment will be described in detail below.

[0043] (Mesenchymal stem cell preparation step) The mesenchymal stem cell preparation step is an optional step in the preparation method of this embodiment, and is a step of preparing mesenchymal stem cells from the amniotic membrane for use in the method of this embodiment.

[0044] Methods for preparing amniotic mesenchymal stem cells (MSCs) are widely known in the art and are not particularly limited. In particular, when used in the preparation method of this embodiment, the prepared amniotic mesenchymal MSCs may have lost their ability to differentiate into osteoblasts and / or adipocytes.

[0045] Typically, amniotic mesenchymal stem cells (MSCs) are prepared from fetal appendages, but the specific processing method is not particularly limited. For example, the obtained amniotic tissue can be prepared by enzymatic treatment with a mixture of dispase and collagenase, while retaining all or part of the epithelial cell layer (e.g., while still bound to the epithelial cell layer). The case where a portion of the epithelial cell layer is retained includes cases where the epithelial cell layer has been almost completely removed by mechanical removal, but a portion remains, or where enzymatic removal of the epithelial cell layer (especially enzymatic removal with trypsin) has not been performed. Alternatively, the MSCs may be prepared without enzymatic treatment with any of DNase I, trypsin, or hyaluronidase. Specifically, for example, they can be isolated by treatment with a mixture containing substantially only dispase and collagenase as enzymes. Alternatively, for example, enzymatic treatment may be performed using high concentrations of dispase or collagenase.

[0046] "Dispase" refers to a neutral protease that primarily breaks down fibronectin and type IV collagen, and is an enzyme used for separating tissue from the basement membrane. The dispase used herein may be either dispase I or dispase II, or they may be used in combination. Alternatively, it may be either a natural dispase or an artificial dispase such as a recombinant protein, or they may be used in combination.

[0047] "Collagenase" is a type of collagen-degrading enzyme that has the activity to break down the peptide bonds of collagen. Types I, II, III, and IV of collagenase are known, and in this specification, any of them may be used, or they may be used in combination. Furthermore, either natural collagenase (including both animal and microbial origins) or artificial collagenase such as recombinant proteins may be used, or they may be used in combination.

[0048] The enzymes used may or may not contain animal-derived components.

[0049] The concentrations of dispase and collagenase are not particularly limited. For example, they can be independently set to concentrations of 20 PU / mL or higher, 50 PU / mL or higher, 60 PU / mL or higher, 100 PU / mL or higher, 125 PU / mL or higher, 150 PU / mL or higher, 200 PU / mL or higher, 300 PU / mL or higher, 350 PU / mL or higher, 370 PU / mL or higher, 390 PU / mL or higher, 400 PU / mL or higher, 450 PU / mL or higher, 460 PU / mL or higher, 470 PU / mL or higher, or 480 PU / mL or higher. In this context, PU (performance unit) refers to the amount of enzyme that releases an amino acid equivalent to 1 μg of tyrosine per minute from lactate casein at pH 7.5 and 30°C in the case of dispase, and the amount of enzyme that decomposes 1 μg of FITC-collagen per minute at pH 7.5 and 30°C in the case of collagenase.

[0050] The duration of the enzyme treatment is not particularly limited, but for example, it can be 40 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, 90 minutes or more, 120 minutes or more, or 180 minutes or more. Alternatively, for example, the duration of the enzyme treatment can be 6 hours or less, 3 hours or less, 2 hours or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, etc. Specifically, for example, if each treatment is performed separately, the dispase treatment and collagenase treatment can each be set to the above durations, and their total duration can be set to the above durations.

[0051] The solvent used in the enzymatic treatment is not particularly limited, as long as it is a solution capable of maintaining amniotic membrane MSCs. Examples include physiological saline, buffer solutions, and culture media. As for the culture media, for example, those described above in the definition section can be used.

[0052] The amniotic tissue from which amniotic MSCs are isolated may or may not undergo lithotripsy and / or fragmentation, but for example, it can be subjected to enzymatic treatment without either lithotripsy or fragmentation. There are no particular limitations on the size of the amniotic tissue subjected to enzymatic treatment, but for example, the size of the largest surface (base) is 0.1 mm. ２ Above 1 mm ２ More than 1cm ２ Above, 10cm ２Above, 20 cm ２ Above, 25 cm ２ Above, 40 cm ２ Above, 50 cm ２ Above, 65 cm ２ Above, 70 cm ２ Above, 80 cm ２ Above, 85 cm ２ Above, 90 cm ２ Above, 100 cm ２ Those above, or intact amniotic tissue that has not been fragmented can be used as it is.

[0053] In this specification, the "mixture containing substantially only dispase and collagenase as enzymes" refers to a mixture that does not contain other enzymes in an effective amount. Specifically, it means that the concentration of enzymes other than dispase and collagenase is less than 1 U / mL, less than 0.1 U / mL, less than 0.01 U / mL, less than 0.001 U / mL, or less than 0.0001 U / mL, etc.

[0054] (First culture step) The first culture step is an optional step in the preparation method of this embodiment, and is a step of culturing mesenchymal stem cells in a first medium before the gene modification step. When performing the mesenchymal stem cell preparation step, this step can be performed simultaneously with or after that step.

[0055] The composition of the medium, culture conditions, passage, etc. in this step shall conform to the description of the expression culture step. The first medium used in this step may have the same composition as the medium used in the expression culture step, or may have a different composition.

[0056] This step can be performed once or multiple times. For example, this step can be performed as the primary culture of mesenchymal stem cells prepared in the mesenchymal stem cell preparation step and the maintenance culture of the primary culture mesenchymal stem cells.

[0057] (Recovery Step) The recovery step is an optional step in the preparation method of this embodiment, and is a step in which a cell population including mesenchymal stem cells is recovered. The recovery method in this step can be appropriately selected according to the type of culture method in the first culture step. For example, if the culture method in the first culture step is adherent culture, mesenchymal stem cells can be recovered by detaching the cells from the culture vessel after adherent culture. Also, if the culture method in the first culture step uses microcarriers, the cells can be detached from the microcarriers, and in the case of spheroid culture, the spheroids can be dispersed to recover the mesenchymal stem cells.

[0058] In this specification, "detachment" means the physical separation of cells from the surface of a culture substrate such as a culture vessel or microcarrier. Cells may be detached in cell aggregate units or single cell units. Examples of detachment include physical detachment (e.g., mechanical detachment by scraping the surface of the container with a cell scraper, etc., splashing, methods using shaking or vibration, and ultrasonic treatment), enzymatic detachment using enzymes such as proteases (e.g., trypsin), chemical detachment using compounds such as chelating agents, and any combination thereof.

[0059] The method for recovering detached mesenchymal stem cells from culture vessels or microcarriers is not limited; for example, the cell suspension containing the detached mesenchymal stem cells may be recovered by aspiration, decantation, filtration, etc.

[0060] The number of mesenchymal stem cells recovered in this process is not limited, but for example, 1 x 10⁶ １ pcs or more, 1×10 ２ pcs or more, 1×10 ３ pcs or more, 1×10 ４ pcs or more, 1×10 ５ One or more, or 1 x 10 ６ The number of cells is 1 or more. Furthermore, the viability of the cells recovered in this process is not limited, but for example, it should be 50% or more, 70% or more, 80% or more, 90% or more, or 95% or more.

[0061] In one embodiment, this step involves recovering a cell population from the first culture medium used in the first culture step described above.

[0062] (Genetic Modification Step) The genetic modification step is an essential step in the preparation method of this embodiment. It is a step in which mesenchymal stem cells are genetically modified by introducing a gene encoding telomerase reverse transcriptase TERT or its active fragment, and a gene encoding Polycomb complex protein Bmi-1 or its active fragment, into a cell population (undifferentiated population) containing mesenchymal stem cells derived from amnion and lacking the ability to differentiate into osteoblasts and / or adipocytes. This step can be performed after the mesenchymal stem cell preparation step if one is performing it, and can be performed simultaneously with or after the first culture step and / or harvesting step if one is performing it.

[0063] The cell population used for gene transfer in this process is an undifferentiated population. The undifferentiated population can be any cell population that includes mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes, and is not particularly limited. For example, a cell population that includes mesenchymal stem cells that have undergone various treatments described in the mesenchymal stem cell preparation process can be used. Specifically, for example, mesenchymal stem cells prepared by subjecting the obtained amniotic tissue, with all or part of the epithelial cell layer still included (for example, while still bound to the epithelial cell layer), to enzymatic treatment with a mixture of dispase and collagenase; mesenchymal stem cells prepared without enzymatic treatment with any of DNase I, trypsin, and hyaluronidase; mesenchymal stem cells isolated by treatment with a mixture containing substantially only dispase and collagenase as enzymes; or mesenchymal stem cells that have been enzymatically treated with high concentrations of dispase and collagenase; or amniotic tissue without any disruption or fragmentation treatment (with a base size of 10 cm ２ You can use mesenchymal stem cells, etc., that have been subjected to enzyme treatment, such as amniotic tissue that has not been fragmented or other such tissue.

[0064] The undifferentiated population used in this process can be one that has been confirmed to contain mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes. Typically, if cells do not differentiate into osteoblasts and / or adipocytes even when induced using the method described later in the differentiation process, they can be treated as an undifferentiated population. If it is already known that an undifferentiated population can be obtained by preparing cells using a similar method, the cell population prepared by that method can be treated as an undifferentiated population without further confirmation.

[0065] In this process, the TERT gene and the BMI1 gene are introduced. The vectors used to carry each gene are as described in the second embodiment.

[0066] In the method of this embodiment, genes other than these genes may be introduced, and the introduced exogenous genes may be limited to only the TERT gene and the BMI1 gene. The additional exogenous genes that can be introduced are not particularly limited, but examples include immortalization genes. Examples of additionally introduced immortalization genes include one or more genes encoding one or more polypeptides selected from the group consisting of Simian virus 40 large T antigen (SV40 Large T Antigen), adenovirus early gene region 1A (E1A), adenovirus early gene region 1B (E1B), human papillomavirus type 16 early gene region E6 (HPV16E6), human papillomavirus type 16 early gene region E7 (HPV16E7), and cyclin-dependent kinase 4 (CDK4). Alternatively, as a specific example, exogenous genes other than immortalization genes may be introduced, such as IL-10 (Interleukin-10), HGF (Hepatocyte Growth Factor), VEGF (Vascular Endothelial Growth Factor), BDNF (Brain-Derived Neurotropic Factor), or gene sequences encoding miRNA (microRNA), for the purpose of enhancing function.

[0067] In this process, multiple genes are introduced, but the timing of their introduction is not particularly limited. For example, all genes may be introduced simultaneously, or one or more genes may be introduced at different times. When introducing two or more genes simultaneously, they may be included in a single vector, or they may be included in separate vectors and introduced simultaneously using those vectors. When using multiple vectors, genes may be introduced using a mixture of each vector, or one or more vectors may be used separately from the others for gene introduction. Preferably, the TERT gene and the BMI1 gene are introduced simultaneously, for example, by mixing the vectors containing each gene.

[0068] The ratio of the amounts of TERT gene and BMI1 gene introduced can be set as appropriate and is not particularly limited. For example, the copy numbers of TERT gene and BMI1 gene used for introduction can be 0.2:1 to 5:1, 0.25:1 to 4:1, 0.3:1 to 3:1, 0.5:1 to 3:1, 0.5:1 to 2:1, 0.75:1 to 1.5:1, 0.8:1 to 1.3:1, 0.9:1 to 1.1:1, or 1:1 for the same number of cells.

[0069] The method for introducing a gene expression vector into a cell population in this process is not particularly limited. For example, any known gene introduction method (transformation method) in the relevant field, as described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc., may be used. Specifically, examples include viral infection, heat shock method, lipofection method, electroporation method, microinjection method, calcium phosphate method, DEAE dextran method, introduction by cationic lipids, introduction by cationic polymers (e.g., polyethyleneimine (PEI)), introduction by nanoparticles, particle impact, etc.

[0070] The composition of the solution used for gene transfer can be selected as appropriate and is not particularly limited. For example, the solvent used can be a culture medium as described in the definition section, such as a basic medium (e.g., a standard cell culture medium such as Advanced DMEM medium). In this case, the culture medium may be the same as the culture medium used in the first culture step and / or expression culture step, or it may be a different culture medium.

[0071] The solution may contain additives such as serum or serum substitutes. The additives in this case are not particularly limited, but examples include those exemplified in the expression culture process. The concentration is not particularly limited, but if it is serum such as FBS, it may be included at concentrations of 0.1 v / v% or more, 0.5 v / v% or more, 1 v / v% or more, 1.5 v / v% or more, 2 v / v% or more, 3 v / v% or more, and / or 30 v / v% or less, 25 v / v% or less, 20 v / v% or less, 15 v / v% or less, 10 v / v% or less, 7 v / v% or less, and 5 v / v% or less, for example, at concentrations of 1 v / v% to 10 v / v%.

[0072] The duration of this process (exposure time to the vector) is not particularly limited, as long as it is sufficient time for gene transfer to occur. For example, it may be 1 hour or more, 2 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 16 hours or more, and / or 48 hours or less, 36 hours or less, 24 hours or less, 20 hours or less, or 18 hours or less, for example, 12 to 24 hours.

[0073] The composition of the solution can be changed during the process. The method of change is not particularly limited. For example, it can be changed by changing the culture medium, or by adding additives. For example, platelet lysates such as hPL can be added. The concentration in this case is not particularly limited. For example, it can be added at the concentrations exemplified in the expression culture process (e.g., 3 v / v% to 20 v / v%).

[0074] When changing the composition of the solution, there are no particular limitations on the timing. For example, it can be done 10 minutes or more after the start of the process, 20 minutes or more after, or 30 minutes or more after. Alternatively, it can be done before 3 hours, 2 hours or more after, 110 minutes or more after, 100 minutes or more after, 90 minutes or more after, 60 minutes or more after, 40 minutes or more after, or 30 minutes or more after, etc.

[0075] This process can be performed once or multiple times. If performed multiple times, the conditions may all be the same, or one or more conditions may differ. For example, the same vector may be introduced multiple times, the same gene may be introduced using different vectors, or a different gene may be introduced each time.

[0076] (Expression Culture Step) The expression culture step is an essential step in the preparation method of this embodiment, and is a step of culturing genetically modified mesenchymal stem cells and expressing the gene. This step can be performed simultaneously with or after the gene modification step.

[0077] The culture method used in this process is not particularly limited and may include, for example, adherent culture, suspension culture, or spheroid culture. Since mesenchymal stem cells are adherent cells, adherent culture is preferred as the culture method in this process, but suspension culture, in which microcarriers to which mesenchymal stem cells are attached are cultured in suspension, or spheroid culture, in which mesenchymal stem cells are cultured after being converted into spheroids, can also be used.

[0078] The composition of the culture medium used in this process is not particularly limited. For example, it is the same as the "culture medium" described above in the definition section. For example, the culture medium may be a basic medium, a serum-free medium, a low-serum medium, or a serum-supplemented medium. Usually, it is a basic medium (e.g., a standard cell culture medium such as αMEM) or a serum-free medium, and may be a basic medium to which other components (additives) such as human platelet lysate, albumin, blood-derived components, growth factors, or combinations thereof have been added.

[0079] In this specification, "platelet lysate" refers to a lysate of blood-derived platelets, which is a cell culture additive containing large amounts of multiple growth factors and chemokines as its components. In this specification, the animal species from which the platelet lysate is derived is not particularly limited, but it is preferably human. In this specification, human-derived platelet lysate (human platelet lysate) is often abbreviated as "hPL". Examples of growth factors that may be present in human platelet lysates include platelet-derived growth factor isoforms (PDGF-AA, -AB, -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain-derived neurotropic factor (BDNF), vascular endothermal growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth. Examples include factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connected tissue growth factor (CTGF), and morphogenic protein-2, -4, -6 (BMP-2, -4, -6). Furthermore, examples of chemokines that may be contained in the above-mentioned human platelet lysates include Interleukin-8 (IL-8), neurophil-activating peptide-2 (NAP-2), regulated on activation, normal T cell expressed and secreted (TANTES), monocyte chemotactic protein-1,-3 (MCP-1,3), macrophage inflammation protein-1 alpha (MIP-1α), and beta-thromboglobulin.

[0080] The method for preparing human platelet lysates used in the present invention is not particularly limited. Platelet lysates are usually produced by repeatedly freezing and thawing platelets extracted from whole blood, and then purifying them by removing cell fragments, etc., but the preparation method is not particularly limited and any method may be used. For specific preparation methods, for example, refer to the following document: Schallmoser k et al., Methods Mol Biol. 2013;946:349-62.

[0081] In the present invention, it is preferable that the human platelet lysate used has been inactivated and / or sterilized for bacteria and viruses.

[0082] In the present invention, commercially available human platelet lysates can also be used. Examples of commercially available human platelet lysates include Stemulate (manufactured by Cook Regentec), PLTMax (manufactured by Mill Creek Life Science), UltraGRO-PURE (manufactured by AventaCell BioMedical), PLUS (manufactured by Compass BioMedical), PLTMax Human Platelet Lysate (manufactured by Merck), ELAREM Perform (manufactured by PL Bioscience GmbH), and the like.

[0083] In the present invention, clarified human platelet lysates can also be used. Clarification, as used herein, refers to a process of removing insoluble fractions generated during long-term storage or freeze-thawing from the human platelet lysates. Methods of clarification include centrifugation, filtration, precipitation, and sedimentation (e.g., spontaneous sedimentation), but are not limited to these as long as the insoluble fractions can be removed.

[0084] In one embodiment, the culture medium may contain human platelet lysate at any concentration. The concentration of human platelet lysate in the culture medium is not particularly limited. The culture medium may contain, for example, human platelet lysate at concentrations of 0.1 v / v% or more, 0.5 v / v% or more, 1 v / v% or more, 2 v / v% or more, 3 v / v% or more, or 5 v / v% or more, and / or 40 v / v% or less, 35 v / v% or less, 30 v / v% or less, 25 v / v% or less, 20 v / v% or less, 15 v / v% or less, or 10 v / v% or less, and for example, human platelet lysate at concentrations of 3 v / v% to 20 v / v%. In this specification, when the concentration of human platelet lysate is expressed in units of "v / v%", it indicates the volume ratio of dilution of human platelet lysate (volume of hPL before dilution / volume after dilution), more specifically, the volume ratio to which crude platelet lysate or commercially available platelet lysate is diluted by the culture medium used for cultivation. Furthermore, as a commercially usable platelet lysate, for example, a diluted human platelet lysate with a total protein concentration of 40 mg / mL to 80 mg / mL may be used.

[0085] The concentration of human platelet lysate in the culture medium used for the above-mentioned culture can also be expressed on a dry weight basis. The concentration of human platelet lysate may be, for example, 0.06 mg / mL or more, 0.3 mg / mL or more, 0.6 mg / mL or more, 1.2 mg / mL or more, 1.8 mg / mL or more, or 3 mg / mL or more, and / or 24 mg / mL or less, 21 mg / mL or less, 18 mg / mL or less, 15 mg / mL or less, 12 mg / mL or less, 9 mg / mL or less, or 6 mg / mL or less, on a dry weight basis.

[0086] Furthermore, the concentration of human platelet lysate in the culture medium used for the above-mentioned culture can also be specified by the concentration of human platelet lysate protein per 1 mL of medium. For example, the concentration of human platelet lysate protein per 1 mL of medium may be 0.02 mg / mL to 32 mg / mL, 0.04 mg / mL to 28 mg / mL, 0.2 mg / mL to 24 mg / mL, 0.4 mg / mL to 20 mg / mL, 0.8 mg / mL to 16 mg / mL, 1.2 mg / mL to 12 mg / mL, 1.6 mg / mL to 8 mg / mL, or 2 mg / mL to 4 mg / mL.

[0087] If the culture medium contains albumin, the albumin concentration may be, for example, 0.05% to 5% by mass. The origin of the albumin used is not particularly limited. For example, albumin derived from mammals, specifically mice, rats, rabbits, cattle, goats, cynomolgus monkeys, marmosets, or humans, can be used.

[0088] Furthermore, blood-derived components include various types of serum (animal-derived serum such as fetal bovine serum (FBS or FCS), human serum, and / or plasma). When the culture medium contains blood-derived components in addition to human platelet lysates, the concentration of the blood-derived components may be, for example, 2 v / v% to 40 v / v%, 3 v / v% to 30 v / v%, or 5 v / v% to 20 v / v%, or even 10 v / v%.

[0089] If the culture medium contains growth factors, reagents to stabilize the growth factors in the medium (such as anticoagulants like heparin, gels, or polysaccharides) may be added in addition to the growth factors. Examples of growth factors include, but are not limited to, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and factors belonging to these families.

[0090] In one embodiment, the culture medium further comprises heparin or a heparin substitute. For example, the human platelet lysate contained in the culture medium may contain heparin or a heparin substitute. Heparin has anticoagulant activity and can stabilize the human platelet lysate in the culture medium. In this specification, "heparin substitute" means any substance having anticoagulant activity similar to heparin. Specific types of heparin substitutes are known in the art, and specific examples include fucoidan, sulfated fucane, and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK). The concentration of heparin or heparin substitute in the first culture medium is, for example, 0.1 U / mL or more, 1 U / mL or more, or 2 U / mL or more, for example, 2 U / mL.

[0091] The culture temperature in this process is not limited as long as it is within the range at which mesenchymal stem cells can grow. For example, the culture in this process can be carried out at 30-42°C, 32-40°C, 35-39°C, or 36-38°C, for example, 37°C.

[0092] The culture time in this process is not particularly limited, and culture may be performed for a specific period, or until a specific criterion is met. The specific culture period is not particularly limited, but for example, it may be 1 hour or more, 2 hours or more, 4 hours or more, 12 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 24 hours or more, 2 days or more, or 3 days or more, and / or 2 weeks or less, 1 week or less, 6 days or less, 5 days or less, or 4 days or less. The specific criterion is not particularly limited, but for example, mesenchymal stem cells may be cultured until they reach a certain confluence. The specific confluence is not particularly limited. For example, they may be cultured until they reach a confluence of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%. For example, they may be cultured until they reach subconfluence (70% to 90% confluence).

[0093] Since amniotic membrane MSCs can adhere to plastic, coating the culture vessel is not necessary, but it may be done as appropriate. The type of coating agent used is not particularly limited in this case.

[0094] The seeding density is not particularly limited, as long as it allows amniotic MSCs to grow. For example, 100 cells / cm². ２ The above 10 ６ cells / cm ２ Below, 500cells / cm ２ The above 10 ５ cells / cm ２ Below, 1000cells / cm ２ The above 10 ４ cells / cm ２ Below, 1000cells / cm ２ More than 5000cells / cm ２ Below, 1000cells / cm ２ More than 4000cells / cm ２ Below, 1000cells / cm２ More than 3000cells / cm ２ Below, 1000cells / cm ２ More than 2000cells / cm ２ Below, 1000cells / cm ２ More than 1400cells / cm ２ Below, 1200cells / cm ２ More than 2000cells / cm ２ Below, 1300cells / cm ２ More than 1500cells / cm ２ Below, 1400cells / cm ２ More than 1500cells / cm ２ The following densities can be used for sowing.

[0095] The culture medium may be changed during the culture period. The culture medium may be changed multiple times, only once, or continuously (for example, by perfusion). If the culture medium is changed multiple times, there is no particular limit to the frequency of the changes. For example, it may be changed at predetermined intervals, or as appropriate in response to changes in the culture medium (for example, in response to changes in the pH of the culture medium). If the culture medium is changed at predetermined intervals, there is no particular limit to the frequency. For example, it may be every 1 to 7 days, and specifically, it can be every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, etc. There is no particular limit to the amount of culture medium changed in one change. For example, all or half of the culture medium may be changed. The composition of the culture medium may be changed before and after the change, or it may be changed to a culture medium of the same composition.

[0096] Subculturing can be performed in this process. Subculturing can be carried out by repeating this process and the recovery process described later. There is no particular limit to the number of subculturing cycles in this case. For example, subculturing can be performed one or more times, two or more times, three or more times, four or more times, or five or more times. The number of subculturing cycles may also be, for example, 25 or fewer times, 20 or fewer times, 15 or fewer times, or 10 or fewer times. The culture conditions and / or recovery conditions in each subculturing cycle may be the same each time, or they may differ in one or more subculturing cycles.

[0097] The peeling method is not particularly limited. For example, the peeling method exemplified in the recovery process can be used.

[0098] When subculturing, there are no particular limitations on the confluence at the end of each culture, but examples include 1% or more, 10% or more, 30% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, and 100%.

[0099] There are no particular restrictions on the seeding density when performing subgeneration. For example, the seeding densities exemplified above can be used.

[0100] The culture conditions used in this process may be the same as or different from the culture conditions in other processes.

[0101] (Single-cell formation step) The single-cell formation step is an optional step in the preparation method of this embodiment, and is a step in which genetically modified mesenchymal stem cells are formed into single cells. This step can be performed after the genetic modification step. This step can also be performed before or after the expression culture step.

[0102] In this specification, "single-cell formation" refers to the state in which one mesenchymal stem cell is isolated from other mesenchymal stem cells. The single-cell formation may or may not be isolated from other cell types other than mesenchymal stem cells, but it is more preferable that it be isolated from other living cells (e.g., cells with proliferative capacity). For example, a single cell isolated from other living cells can be used as the single-cell formation. In this specification, "single cell" means a single cell that does not contain any other cells. In this specification, "single cell" is also written as "single cell".

[0103] In this specification, "single-cell cloning" refers to the isolation and culture of a single cell (i.e., a single cell) with proliferative capacity from a cell population containing two or more cells. For example, when performing single-cell cloning on mesenchymal stem cells, the isolated mesenchymal stem cell is cultured in a culture medium that does not contain other cells, such as other mesenchymal stem cells.

[0104] The method of single-cell isolation is not limited as long as it is a method that can isolate mesenchymal stem cells into single cells. For example, by detaching cells using a detachment method as exemplified in the recovery process, and then diluting the dispersed cell suspension to a multi-well plate such as a 96-well plate to a concentration of 1 cell / well, one cell can be isolated into each well. When mesenchymal stem cells are labeled with a fluorescent substance or dye, they can be isolated one by one using a cell sorter or the like by flow cytometry using the fluorescence or dye as an indicator. Mesenchymal stem cells can be labeled, for example, by introducing a labeling gene (marker gene) into the mesenchymal stem cells using a gene expression vector, or by binding an antibody labeled with a fluorescent substance to the mesenchymal stem cells. Alternatively, one cell can be isolated using a micromanipulator or micromesh filter.

[0105] This process can also isolate multiple single mesenchymal stem cells. In this case, multiple single mesenchymal stem cells can be divided into multiple culture vessels or multiple wells of a multi-well plate. The shape of the culture vessel or multi-well plate is not limited; for example, multiple cells can be isolated as single cells by dividing a single cell into each well of a multi-well plate such as a 6-well plate, a 12-well plate, or a 96-well plate.

[0106] If necessary, specific cell types may be selectively separated during single-cell separation, or if a selection step is performed later, selection may be omitted in this step and the desired cell population may be selected in the selection step. The criteria for selection are not particularly limited, but for example, cells may be separated based on whether they are living cells, mesenchymal stem cells, genetically modified cells, or a combination thereof.

[0107] In one embodiment, by labeling dead or living cells after the gene modification step described above, this step can isolate only living cells. Examples of labeling methods include staining dead cells with 7-AAD (7-Amino-Actinomycin D) stain or PI (Propidine Iodide).

[0108] When selectively separating cells based on the criterion of being mesenchymal stem cells, for example, cells exhibiting a signal based on the binding molecule (such as an antibody) that can bind to the surface antigen of mesenchymal stem cells, as exemplified in the definition section, can be separated as mesenchymal stem cells.

[0109] When selectively separating cells based on whether they are genetically modified, if the exogenous gene transfer vector contains a labeling gene, cells exhibiting a signal based on that labeling can be separated as genetically modified cells. In this case, it is preferable that cells into which both the TERT gene and the BMI1 gene have been introduced are selectively separated.

[0110] (Expansion Culture Step) The expansion culture step is an optional step in the preparation method of this embodiment, and is a step of expanding the culture of genetically modified mesenchymal stem cells. This step can be performed simultaneously with or after the expression culture step. If the single-cell isolation step is performed, this step can be performed before and / or after it.

[0111] The culture process in this step can be carried out in accordance with the description in the expression culture step.

[0112] The specific composition of the culture medium used in this process is the same as that described above under "Culture Medium". For example, it may be a basic medium, serum-free medium, low-serum medium, or serum-supplemented medium. Typically, it is a basic medium (e.g., a standard cell culture medium such as αMEM) or a serum-free medium, and may be a basic medium to which other components such as albumin, blood-derived components, or growth factors have been added.

[0113] The culture medium in this process may contain serum or blood-derived components. The specific details of these components are as described in the expression culture process. The culture medium in this process may contain, for example, human platelet lysate as a blood-derived component. In this case, the concentration of human platelet lysate is not particularly limited and may be, for example, 0.1 v / v% or more, 0.5 v / v% or more, 1 v / v% or more, 2 v / v% or more, 3 v / v% or more, or 5 v / v% or more, and / or 40 v / v% or less, 35 v / v% or less, 30 v / v% or less, 25 v / v% or less, 20 v / v% or less, 15 v / v% or less, or 10 v / v% or less, for example, 3 v / v% to 20 v / v%. Alternatively, any of the above concentrations expressed on a dry weight basis or the above human platelet lysate protein concentration may be used.

[0114] The culture medium in this process may contain any additives, such as antibiotics, as exemplified in the expression culture process.

[0115] The composition of the culture medium and the culture conditions used in this process may be the same as or different from those in the first culture step and / or the expression culture step.

[0116] There are no particular restrictions on the time between the end of the expression culture step described above and the start of the culture in this step. However, the culture in this step can be carried out within, for example, 24 hours, 20 hours, 18 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour from the culture in the expression culture step.

[0117] If this step is performed after the single-cell isolation step, single-cell cloning can be performed using this step. In this case, typically, the cells used for culture in this step do not contain any cells other than one mesenchymal stem cell. Also, even if the single-cell isolation step is included, for example, multiple single-cell isolated mesenchymal stem cells may be cultured in the same culture environment (e.g., in the same well). In this case, the culture may be carried out in a manner that does not involve mixing of cells derived from each mesenchymal stem cell, or in a manner that involves mixing of them.

[0118] The cultured cells may be separated and cultured individually as appropriate, or the separately cultured cells may be combined and cultured together as appropriate. For example, if the cells are cultured in a manner that prevents mixing, the cells derived from each mesenchymal stem cell may later be cultured in separate culture environments. Also, even if single-cell cloning is performed, multiple clones derived from different mesenchymal stem cells may be combined and cultured together later.

[0119] The culture period is not particularly limited, but in addition to the periods exemplified in the expression culture process, the culture may be extended to 1 week or more, 8 days or more, 9 days or more, 10 days or more, 2 weeks or more, 15 days or more, 20 days or more, 3 weeks or more, 22 days or more, 23 days or more, 4 weeks or more, 1 month or more, 2 months or more, 3 months or more, 100 days or more, 4 months or more, 150 days or more, 6 months or more, 200 days or more, 7 months or more, 8 months or more, 9 months or more, or for a long period of 1 year or more. If this process is performed multiple times, the total culture period for those multiple times can usually be extended to the above-mentioned period or longer.

[0120] The culture period may be determined based on indicators that reflect the properties of the cells, such as proliferative capacity and viability. In this specification, "proliferative capacity" refers to the ability of a cell to increase its number through cell division. For example, when mesenchymal stem cells are cultured as single cells, they may or may not exhibit proliferative capacity. When a single-cell mesenchymal stem cell exhibits proliferative capacity, it has the ability to produce two or more cells through cell division and to form two or more cells derived from those cells.

[0121] The indicator used to reflect the growth capacity is not particularly limited, but for example, the cumulative number of doublings can be used.

[0122] In this specification, "cumulative doubling count" means the cumulative number of doubling counts. Specifically, it refers to the cumulative number of times cells have doubled from the start of culture to the end of culture. When subculturing is performed during the culture period, the cumulative doubling count can be calculated by accumulating the number of doubling counts from immediately after subculturing to before the next subculturing for each subculturing. The population doubling level (PDL) from the start of culture to the end of culture can be calculated using the following formula.

[0123] Number of doublings = log ２ (A / B) (wherein A represents the number of cells at harvest and B represents the number of cells at seeding.) For example, this process can be carried out until the cumulative number of doublings reaches a certain level. The specific number of cumulative doublings in this case is not particularly limited, but for example it can be 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 65 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, or 130 or more.

[0124] In this specification, "specific growth rate (μ)" refers to the rate of increase in the number of cells per unit time. Specifically, it can be calculated using the following formula.

[0125] Specific growth rate (μ) = {ln(N ２ / N １ )} / (t ２ -t １ ) (in the formula, N １ is time t １ The number of cells in N ２ is time t ２ (This indicates the number of cells in the cell culture.) During this process, the culture medium may be changed and / or subcultured as appropriate. The specific details of these steps are the same as those for the expression culture process. For example, one or more subcultures can be performed for long-term culture (long-term subculture). Alternatively, the culture medium may not be changed and / or subcultured.

[0126] There are no particular limitations on the number of subculturing steps performed in this process, but for example, subculturing can be done 1 or more times, 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, 10 or more times, 11 or more times, 12 or more times, 13 or more times, 14 or more times, 15 or more times, 20 or more times, 21 or more times, 25 or more times, or 30 or more times. Alternatively, the number of subculturing steps may be 60 or less, 50 or less, 40 or less, 35 or less, or 30 or less.

[0127] Furthermore, for example, this process can be continued until the cell viability, doubling cycle, specific growth rate, or a combination thereof falls below a certain level.

[0128] While there are no specific limits on the values ​​used when using cell viability as an indicator, for example, subculturing can be carried out until the viability reaches 65% or less, 70% or less, 80% or less, 85% or less, 90% or less, 95% or less, 96% or less, etc.

[0129] While there are no specific limits on the values ​​when using the number of doubling cycles as an indicator, for example, subculturing can be carried out until the number of doubling cycles reaches 0.1 or less, 0.5 or less, 1 or less, 1.5 or less, 2 or less, 2.5 or less, 3 or less, etc.

[0130] There are no specific limits on the value when using the specific growth rate as an indicator, but for example, 0.05 days. －１ Less than 0.06 days －１ Less than 0.1 days －１ Less than 0.1 days －１ Less than 0.15 days －１ Less than 0.2 days －１ Subculturing can be carried out until the specific growth rate is less than or equal to the specified value. Furthermore, there are no particular limitations on the specific growth rate during this process, but for example, 0.05 days. －１ The above is 0.1 days －１ The above is 0.2 days. －１ The above is 0.3 days. －１ The above is 0.4 days. －１ More than or equal to 0.5 days －１ The above and / or 1.0 day －１ Below, 0.9 days －１ Below, 0.8 days －１ Below, 0.7 days －１ The following or 1.0 day －１ Any of the following is acceptable, for example, 0.4 days －１ ~0.7 days －１ These values ​​can be determined, for example, based on measurements taken before and after succession.

[0131] The culture conditions and / or harvesting conditions in each passage may be the same each time, or they may differ in one or more passages.

[0132] Additional processing, such as freezing, may be performed as appropriate during this process. The content of this processing is not particularly limited. For example, when freezing is performed, the conditions are not particularly limited, but examples include a programmable freezer, a deep freezer, or immersion in liquid nitrogen. When using a programmable freezer, the freezing rate (based on the absolute value of the decrease in temperature) may be, for example, 0.1°C / min or more, 0.5°C / min or more, or 1°C / min or more, and / or 15°C / min or less, 10°C / min or less, 5°C / min or less, 4°C / min or less, 3°C / min or less, or 2°C / min or less, for example, 0.5°C / min to 3°C / min. In this case, the final temperature reached is not particularly limited, but for example, it may be -30°C or below, -40°C or below, -50°C or below, -80°C or below, or -100°C or below. When using a deep freezer, for example, cells can be stored in a bicell (manufactured by Nippon Freezer Co., Ltd.), Mr. The food may be frozen in a freezing container such as a Frosty (manufactured by Thermo Fisher Scientific) or a Cool Cell (manufactured by Corning). The temperature at which this is frozen is not particularly limited, but for example, it can be -30°C or below, -50°C or below, -80°C or below, -100°C or below, -120°C or below, or -150°C or below. When using liquid nitrogen, for example, the food may be frozen by rapidly lowering the temperature to -196°C, and then stored frozen in the liquid or gas phase of liquid nitrogen.

[0133] When performing the freezing procedure under the above conditions, the cells may be in any storage container. Examples of storage containers include cryotubes, cryovials, freezing bags, infusion bags, etc.

[0134] When freezing is performed under the above conditions, cells may be stored in any cryopreservation solution. Examples of cryopreservation solutions include CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.), BAMBANKER (manufactured by Lymphotec), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), ReproCryo RM (manufactured by ReproCELL), CryoNovo (manufactured by Akron Biotechnology), MSC Freezing Solution (manufactured by Biological Industries), CryoStor (manufactured by HemaCare), etc.

[0135] (Selection Step) The selection step is an optional step in the preparation method of this embodiment, and is a step of selecting genetically modified mesenchymal stem cells. This step can be performed simultaneously with or after the expression culture step. If a single-cell formation step and / or expansion culture step are performed, this step can be performed before, simultaneously with, and / or after those steps.

[0136] The selection criteria used in this process are not particularly limited. For example, cells can be selected based on cell properties such as proliferative capacity, expression patterns such as the presence or absence of surface antigens and exogenous genes, differentiation capacity, or a combination thereof.

[0137] When selecting cells based on their properties, the specific criteria used are not particularly limited. For example, selection can be made based on conditions such as proliferation ability, as exemplified in the expansion culture process. The selection method in this case is not particularly limited. For example, cells exhibiting a predetermined specific growth rate or number of doubling cycles after a certain period of culture may be selected, or cells capable of being passaged a certain number of times or more may be selected.

[0138] In the case of selection based on expression patterns, there are no particular limitations on the specific criteria used. For example, selection can be made based on the presence or absence of expression of a labeling gene contained in an exogenous gene transfer vector, or the presence or absence of surface antigens in mesenchymal stem cells as exemplified in the definition section. There are no particular limitations on the selection method in this case. For example, the expression of surface antigens in a cell population can be evaluated by visual inspection of a portion of the cell population, or by subjecting it to any expression analysis method such as flow cytometry or immunostaining, and the positive rate of each of the above surface antigens can be determined.

[0139] In the case of selection based on differentiation potential, for example, selection can be made based on the presence or absence of the ability to differentiate into osteoblasts and / or adipocytes. The method of selection in this case is not particularly limited. For example, it can be done by inducing differentiation using the method exemplified in the differentiation process and then confirming the success or failure of the differentiation. The method of confirming the success or failure of differentiation is not particularly limited, but for example, the method exemplified in the differentiation process can be used.

[0140] In this process, a portion of cells may be selected from a cell population, or any of several cell populations may be selected. The selected cells may then be managed separately according to their originating cell population, or they may be managed in a mixed state containing cells that originally belonged to different cell populations. Furthermore, in this process, it is sufficient to increase the proportion of a specific cell; it is not necessary to select cells to the point where they contain absolutely no other cells.

[0141] This process can be performed multiple times. In this case, the selection criteria may be the same each time, or one or more criteria may differ.

[0142] (Differentiation Step) When a differentiation step is performed, the method of this embodiment can be carried out as a method for preparing osteoblasts or adipocytes. In this case, this step is an essential step in the method for preparing osteoblasts or adipocytes, in addition to the gene modification step and the expression culture step. This step can be performed simultaneously with or after the expression culture step. If a single-cell formation step, a wide-culture step, and / or a selection step are included, this step can be performed after those steps.

[0143] Furthermore, as described above, when selection based on differentiation potential is performed in the selection step of a cell population including genetically modified mesenchymal stem cells, differentiation induction and confirmation of its success or failure can be performed in the selection step as described in this step.

[0144] In this process, mesenchymal stem cells are differentiated into osteoblasts or adipocytes. The method of differentiation into each cell type is not particularly limited. For example, methods known in the art can be used.

[0145] For example, mesenchymal stem cells can be differentiated by inducing their culture in a medium and differentiation-inducing agent suitable for inducing differentiation of mesodermal stem cells.

[0146] The culture medium used in this process is not particularly limited, but examples include DMEM medium; αMEM medium; Mesenchymal stem cell growth medium; MSCGM. ＴＭ Examples include Mesenchymal stem cell growth medium or a combination thereof.

[0147] Examples of osteoblast differentiation inducers include compounds such as ascorbic acid, ascorbic acid-2-phosphate, β-glycerophosphate, dexamethasone, hydrocortisone hemisuccinate, statins, isoflavone derivatives, ipriflavone derivatives such as TAK-778, helioxanthine derivatives such as TH, phenamyl, harmine and its analogs, acerogenin and its analogs, and resveratrol; and signaling molecules involved in bone formation such as BMP-2, BMP-4, IGF-1, bFGF, TGF-β1, PTH (parathyroid hormone), and Wnt, or combinations thereof.

[0148] Examples of differentiation-inducing agents for adipocytes include insulin, dexamethasone, 3-isobutyl-1-methylxanthine, or combinations thereof.

[0149] Alternatively, commercially available culture media or kits for differentiation induction may be used. Such culture media or kits are not limited to those mentioned above, but examples include MSC Osteogenic Difference Medium (Promocell) and hMSC Osteogenic Difference Medium Bullet Kit. ＴＭ (Lonza), MSC Adipogenic Differentiation Medium2 (Promocell), hMSC Adipogenic Differentiation Medium BulletKit ＴＭ Examples include (Lonza).

[0150] The success or failure of differentiation can be determined based on the properties of the target cells (cell morphology, genetic markers, functional markers, etc.). Specifically, for example, it can be determined based on whether or not the cells exhibit the properties of osteoblasts or adipocytes as exemplified in the definition section, and / or whether or not they exhibit the properties of mesenchymal stem cells.

[0151] For example, differentiation into osteoblasts can be confirmed by staining the deposited calcium with alizarin red and / or by alkaline phosphatase staining.

[0152] Furthermore, for example, differentiation into adipocytes can be confirmed by staining lipid droplets with oil red O stain and / or by measuring triglycerides.

[0153] Furthermore, a decrease in the expression of undifferentiated marker genes may indicate that differentiation has been induced.

[0154] In this specification, "undifferentiated marker gene" refers to a gene whose gene expression changes (e.g., increases or decreases) during the differentiation process of cells such as mesenchymal stem cells, and whose change in expression level can serve as an indicator of the degree of differentiation / undifferentiation. Specific examples of undifferentiated marker genes include the OCT3 / 4 gene, the Nanog gene, and / or the Sox2 gene. The expression levels of these genes may decrease to less than approximately 100%, less than 80%, less than 60%, less than 40%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% when mesenchymal stem cells differentiate. Conversely, if the expression level of an undifferentiated marker gene is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more of the expression level in undifferentiated mesenchymal stem cells, it is considered that sufficient undifferentiation is maintained.

[0155] 1-4. Effects According to the preparation method of the present invention, it is possible to produce a cell population that includes mesenchymal stem cells that have regained the ability to differentiate into osteoblasts and / or adipocytes, in a cell population that includes mesenchymal stem cells that do not have the ability to differentiate into osteoblasts and / or adipocytes.

[0156] Furthermore, this produced cell population not only has restored differentiation ability but also possesses excellent proliferative capacity, making it possible to efficiently obtain large quantities of mesenchymal stem cells capable of differentiating into osteoblasts and / or adipocytes.

[0157] 1-5. Method for Improving Differentiation Ability The present invention further relates to a method for improving the differentiation ability to osteoblasts and / or adipocytes. The improvement method in this embodiment includes a gene modification step as an essential step, and may include an expression culture step, a first culture step, a harvesting step, a single-cell formation step, a wide-bloom culture step and / or a selection step as optional steps.

[0158] The specific details of each process are as described above in section 1-3. Therefore, only the differences will be explained here.

[0159] In the improved method of this embodiment, a cell population (deficiency population) including mesenchymal stem cells lacking the ability to differentiate into osteoblasts and / or adipocytes is used. The deficiency population may be an undifferentiated population as used in the preparation method, but as described in the definition section, it may also be a cell population including mesenchymal stem cells that have the ability to differentiate into osteoblasts and / or adipocytes, but whose differentiation efficiency is reduced.

[0160] The deficiency population exhibits a certain level of and / or significantly lower differentiation efficiency to the same cell type in response to the same differentiation stimulus compared to a cell population containing mesenchymal stem cells capable of differentiating into osteoblasts and / or adipocytes. While there is no particular limit to the criteria for a certain level of lower differentiation efficiency, for example, the differentiation efficiency of the deficiency population is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more lower than the differentiation efficiency of a cell population containing mesenchymal stem cells capable of differentiating into osteoblasts and / or adipocytes.

[0161] 1-6. Other Aspects According to the present invention, a cell population produced by the preparation method of the present invention, a culture medium containing the cell population, and a culture supernatant of the cell population are also provided.

[0162] 2. Composition 2-1. Overview A second aspect of the present invention is a composition comprising nucleic acid molecules containing exogenous genes (hereinafter sometimes abbreviated as "the composition of the present invention"). The composition of the present invention comprises, as nucleic acid molecules, a nucleic acid molecule containing an exogenous gene encoding telomerase reverse transcriptase or its active fragment, and / or a nucleic acid molecule containing an exogenous gene encoding Polycomb complex protein Bmi-1 or its active fragment, and is characterized by being used in the method of the first aspect. The composition of the present invention can improve the differentiation ability of mesenchymal stem cells that do not have and / or lack the ability to differentiate into osteoblasts and / or adipocytes by using them.

[0163] 2-2. Composition The composition of the present invention comprises, as nucleic acid molecules, a nucleic acid molecule containing an exogenous gene encoding telomerase reverse transcriptase or its active fragment, and / or a nucleic acid molecule containing an exogenous gene encoding Polycomb complex protein Bmi-1 or its active fragment.

[0164] A nucleic acid molecule may contain an exogenous gene encoding telomerase reverse transcriptase or its active fragment and an exogenous gene encoding Polycomb complex protein Bmi-1 or its active fragment within the same molecule. Alternatively, each exogenous gene may be contained in a separate molecule.

[0165] If the composition of the present invention includes a nucleic acid molecule containing one exogenous gene, the composition of the present invention can be used in combination with a nucleic acid molecule containing the other exogenous gene. Specifically, for example, the present invention relates to a composition containing a nucleic acid molecule containing an exogenous gene encoding telomerase reverse transcriptase or its active fragment, for use in combination with a nucleic acid molecule containing a Polycomb complex protein Bmi-1 or its active fragment, for use in the method of the first embodiment, and a composition containing a nucleic acid molecule containing a Polycomb complex protein Bmi-1 or its active fragment, for use in combination with a nucleic acid molecule containing a telomerase reverse transcriptase or its active fragment, for use in the method of the first embodiment.

[0166] Each exogenous gene is defined as described in the definition section. The following describes the composition of nucleic acid molecules.

[0167] Nucleic acid molecules can be in the form of vectors, such as recombinant vectors or gene expression vectors.

[0168] In this specification, "gene expression vector" refers to a vector that contains a target gene or gene fragment in an expressible state and includes expression units that can control the expression of that gene or gene. A gene expression vector may be a plasmid vector or a viral vector. A gene expression vector may contain a labeling gene (selection marker) as needed.

[0169] In this specification, "expression-ready state" means that the gene to be expressed is located in the downstream region of the promoter, which is under the control of the promoter.

[0170] The plasmid vector may be a commercially available expression vector for mammalian cells, such as Promega's pCI vector or pSI vector, or a shuttle vector that can replicate between mammalian cells and bacteria such as E. coli.

[0171] Viral vectors that can be used include, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, Simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors, but lentiviral vectors are preferred. Viral vectors that lack replication ability so as not to self-replicate within infected cells may also be used.

[0172] The nucleic acid molecule may contain additional genes. Examples of additional genes include, but are not limited to, exogenous genes that can be introduced in addition as exemplified in the gene modification process of the first embodiment, and labeling genes.

[0173] In this specification, "labeling gene" or "marker gene" refers to a gene that encodes a labeling protein (marker protein), also called a selection marker or reporter protein. A "labeling protein" or "marker protein" is not limited to those that can label and / or select cells into which a gene expression vector has been introduced based on their activity. Examples include drug (e.g., antibiotic) resistance proteins, pigment proteins, fluorescent proteins, and luminescent proteins.

[0174] 2-3. Other Aspects The present invention also relates to a kit for use in the method of the first aspect. The kit may include, for example, one or more elements selected from the group consisting of nucleic acid molecules containing an exogenous gene encoding telomerase reverse transcriptase or an active fragment thereof, nucleic acid molecules containing an exogenous gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof, a culture medium, a gene transfer means, a culture vessel, a selection agent or equipment, and a means for adding the agent. Each element may be housed in the same container or in different containers. Instructions for use may also be included as appropriate.

[0175] The present invention further relates to the use of nucleic acid molecules comprising an exogenous gene encoding telomerase reverse transcriptase or an active fragment thereof, and / or nucleic acid molecules comprising an exogenous gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof, in the method of the first embodiment.

[0176] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0177] <Example 1: Production of Genetically Modified Amniotic Mesenchymal Stem Cells> (Objective) The gene encoding human telomerase reverse transcriptase (hereinafter referred to as "hTERT") (hereinafter referred to as the "hTERT gene") and the gene encoding Polycomb complex protein Bmi-1 (hereinafter referred to as the "BMI1 gene") are introduced as exogenous genes into mesenchymal stem cells derived from the amniotic membrane (hereinafter referred to as "amniotic MSCs").

[0178] (Methods and Results) (1) Preparation of Plasmid Vector for Gene Transfer A plasmid vector was prepared to introduce the hTERT gene into MSC cell lines using a lentiviral vector. The plasmid vector contained the hTERT gene (SEQ ID NO: 2), which encodes hTERT (SEQ ID NO: 1), and the TagBFP2 gene (SEQ ID NO: 4), which encodes the fluorescent marker protein TagBFP2 (SEQ ID NO: 3). This plasmid is called the "hTERT expression plasmid".

[0179] Furthermore, a plasmid vector was constructed for introducing the BMI1 gene into MSC cell lines using a lentiviral vector. This plasmid vector contained the BMI1 gene (SEQ ID NO: 6), which encodes Bmi-1 (SEQ ID NO: 5), and the mCherry gene (SEQ ID NO: 8), which encodes the fluorescent marker protein mCherry (SEQ ID NO: 7). This plasmid is called the "BMI1 expression plasmid."

[0180] (2) Preparation of amniotic membrane MSCs (2-1) Acquisition of amniotic membrane MSCs The amniotic membrane and placenta, which are fetal appendages, were aseptically collected from pregnant women in elective cesarean section cases who had given informed consent. The obtained amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amniotic membrane was manually peeled off from the cut end of the amniotic membrane. The amniotic membrane was washed with Hanks equilibrium salt solution (Ca and Mg-free) to remove any attached blood and blood clots.

[0181] The amniotic membrane, containing the epithelial cell layer and the mesenchymal stem cell layer, was immersed in a Hanks equilibrium salt solution (containing Ca and Mg) containing 480 PU / mL collagenase and 400 PU / mL dispase I. The amniotic membrane was enzymatically treated by rotating and stirring it at 10 rpm for 60 minutes at 37°C using a rotator. After enzymatic treatment, the solution was filtered through a nylon mesh with a mesh size of 95 μm to remove undigested material from the amniotic membrane, and a cell suspension containing amniotic membrane MSCs was recovered.

[0182] (2-2) Culture of amniotic membrane MSCs: The cell suspension containing the above amniotic membrane MSCs is cultured at 1,400 cells / cm³. ２ Cells were seeded in culture vessels at the seeding density. After seeding, the cells were adherently cultured in αMEM containing 5% human platelet lysate (hPL; AventaCell BioMedical, catalog number: HPCFDCGLI50) until subconfluence (approximately 80% confluence) was reached. After reaching subconfluence, amniotic membrane MSCs were cultured at 1,000 cells / cm³. ２ The cells were subcultured at the seeding density and cultured until subconfluent. Thereafter, amniotic MSCs were subcultured under the same conditions as needed. Hereafter, this amniotic MSC cell line will be referred to as the "AMSC cell line".

[0183] (3) Gene transfer into amniotic MSCs (3-1) Preparation of hTERT-loaded lentiviral vector solution 50,000 cells / cm³ in a 6-well plate coated with collagen containing Lenti-X 293T (Takara Bio) cells ２ Seeds were seeded at the specified density and cultured for 2 days in DMEM (Dulbecco's Modified Eagle Medium) containing fetal bovine serum (FBS; Moregate, catalog number: 599-04425) with a final concentration of 10%. Subsequently, the culture medium was changed to Advanced DMEM containing 2% final concentration FBS and 1×GlutaMAX, and 2.5 μg of hTERT expression plasmid, 2.5 μg of pLenti-P3A (Applied Biological Materials Inc.), 2.5 μg of pLenti-P3B (Applied Biological Materials Inc.), and 2.5 μg of pVSV-G (Clontech) were transfected using Lipofectamine 3000 Reagent. After 4 hours, the culture medium was changed to Advanced DMEM containing 2% final concentration FBS and 1×GlutaMAX, and the cells were incubated for 2 days. Subsequently, the culture supernatant was filtered through a 0.8 μm filter to prepare a lentiviral vector solution.

[0184] (3-2) Preparation of BMI1-carrying lentiviral vector solution A BMI-carrying lentiviral vector solution was prepared in the same manner as in (3-1) above. However, a BMI1 expression plasmid was used instead of an hTERT expression plasmid.

[0185] (3-3) Infection of amniotic membrane MSCs with hTERT-borne lentiviral vector and BMI1-borne lentiviral vector In the subconfluent amniotic membrane MSCs cultured and subculturised as described in (2) above, the culture medium was removed and a mixed lentiviral vector solution prepared by mixing the hTERT-borne lentiviral vector solution prepared in (3-1) above and the BMI-borne lentiviral vector solution prepared in (3-2) above in a 1:1 ratio was added. Thirty minutes after addition, hPL was added to a final concentration of 5%, and the culture was incubated for a further 18 hours. After incubation, the culture medium was replaced with αMEM containing hPL at a final concentration of 5%. After the medium change, the culture was expanded for 10 days. Subculturing in the expanded culture was performed in the same manner as described in (2-2) above.

[0186] (4) Single-cell cloning of amniotic MSCs Amniotic MSCs cultured in large quantities as described in (3-3) above are detached using TrypLE™ Select Enzyme (1X) (manufactured by Thermo Fisher Scientific, catalog number: 12563-029) and the cell concentration is 1 × 10⁶ ６ Cells were suspended in 0.5% bovine serum albumin-containing phosphate-buffered saline to a concentration of cells / mL to prepare a cell suspension. Dead cells were stained by adding 7-AAD (7-amino-actinomycin D; manufactured by IMMUNOSTEP, catalog number: 7AAD) staining solution to this cell suspension.

[0187] Using the stained cell suspension described above as the sample, only the fractions that were 7-AAD negative (live cells), TagBFP2 positive, and mCherry positive were separated using a cell sorter MA900 (Sony, catalog number: MA900). The separated cells were seeded in a 96-well plate at a seeding density of 1 cell / well. The cells in each well were cultured for 23 days in αMEM containing 5% hPL at a final concentration. The expansion culture was performed in the same manner as described in (3-3) above. As a result, two MSC cell lines exhibiting a spindle-shaped cell morphology and into which the hTERT gene and BMI1 gene were introduced were obtained (hereinafter, these two cell lines will be referred to as "AMSC-TB1 cell line" and "AMSC-TB2 cell line").

[0188] The AMMSC-TB1 cell line and the AMMSC-TB2 cell line were repeatedly passaged in αMEM containing 5% hPL at a final concentration in the same procedure as in (2-2) above and cultured for 200 days or more. For both cell lines, the cumulative doubling number exceeded 130 times, and the specific growth rate was always 0.2 －１ or more, and it was confirmed that they grew stably.

[0189] The cumulative doubling number was calculated by summing up the doubling numbers (PDL) for each passage calculated by the following formula based on the number of cells at seeding and the number of cells at recovery for each passage. Doubling number = log ２ (A / B) (In the formula, A represents the number of cells at recovery, and B represents the number of cells at seeding.)

[0190] The specific growth rate was calculated by the following formula. Specific growth rate (μ) = {ln(N ２ / N １ )} / (t ２ -t １ ) (In the formula, N １ represents the number of cells at time t １ , and N ２ represents the number of cells at time t ２ .)

[0191] Each cell line was stored at -80°C in a deep freezer with cells suspended in BAMBANKER (manufactured by Rinfotec).

[0192] <Example 2: Evaluation of Differentiation Ability> (Purpose) Evaluate whether the various amniotic MSC cell lines obtained in Example 1 have the ability to differentiate into osteoblasts and adipocytes.

[0193] (Method and Results) In this example, the AMMSC cell lines (AMMSC-TB1 cell line and AMMSC-TB2 cell line) obtained in Example 1 and the non-transfected AMMSC cells obtained in (2) above were used. All of these cells had undergone 21 passages and were cultured in αMEM containing 5% hPL at a final concentration until they became sub-confluent and then subjected to differentiation induction treatment.

[0194] (1) Evaluation of differentiation potential into osteoblasts (1-1) Differentiation induction of various amniotic membrane MSC cell lines into fibronectin-coated 6-well plates at 10,000 cells / cm² ２ Seeds were sown at the specified density and cultured until confluence. The culture medium was changed to MSC Osteogenic Difference Medium (Promocell) and cultured for 14 days. The medium was changed every 2-3 days.

[0195] (1-2) Evaluation of differentiation potential: Whether or not the cells differentiated into osteoblasts was determined based on staining with Alizarin Red S (SIGMA).

[0196] In AMSC cell lines that had not undergone gene transfer, no areas stained positive with Alizarin Red S were observed. On the other hand, in AMSC-TB1 and AMSC-TB2 cell lines, areas stained positive with Alizarin Red S were observed, confirming the presence of osteoblasts.

[0197] These results indicate that while the AMSC cell line obtained in (2) above does not possess the ability to differentiate into osteoblasts, the MSC cell lines into which the hTERT gene and BMI1 gene were introduced all possessed the ability to differentiate into osteoblasts. This suggests that the introduction of the hTERT gene and BMI1 gene restores the ability to differentiate into osteoblasts.

[0198] (2) Evaluation of differentiation potential into adipocytes (2-1) Differentiation induction of various amniotic membrane MSC cell lines into fibronectin-coated 6-well plates at 10,000 cells / cm² ２ Seeds were sown at the specified density and cultured until subconfluence. The culture medium was changed to MSC Adipogenic Difference Medium 2 (Promocell) and cultured for 14 days. The medium was changed every 2-3 days.

[0199] (2-2) Evaluation of differentiation potential The likelihood of differentiation into adipocytes was determined based on staining with Oil Red O stain (SIGMA Corporation).

[0200] In AMSC cell lines that had not undergone gene transfer, no positive areas stained with Oil Red O were observed. On the other hand, in AMSC-TB1 and AMSC-TB2 cell lines, positive areas stained with Oil Red O were observed, confirming the presence of adipocytes.

[0201] These results indicate that while the AMSC cell line obtained in (2) above does not possess the ability to differentiate into adipocytes, the MSC cell lines into which the hTERT gene and BMI1 gene were introduced all possessed the ability to differentiate into adipocytes. This suggests that the introduction of the hTERT gene and BMI1 gene restores the ability to differentiate into adipocytes.

[0202] Furthermore, it was confirmed that both the AMSC-TB1 and AMSC-TB2 cell lines were capable of differentiating into osteoblasts and adipocytes even after being cultured for more than 160 days.

[0203] The above results demonstrate that by introducing the hTERT gene and the BMI1 gene, even AMSC cell lines that have lost the ability to differentiate into osteoblasts and adipocytes can regain the ability to differentiate into these cells, and that a large quantity of mesenchymal stem cells with high differentiation potential can be obtained regardless of the isolation conditions for mesenchymal stem cells. All publications, patents and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for preparing a cell population including genetically modified mesenchymal stem cells having the ability to differentiate into osteoblasts and / or adipocytes, comprising: a gene modification step of genetically modifying mesenchymal stem cells by introducing a gene encoding telomerase reverse transcriptase or an active fragment thereof, and a gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof, into a cell population including mesenchymal stem cells derived from amnion and not having the ability to differentiate into osteoblasts and / or adipocytes; and an expression culture step of culturing the genetically modified mesenchymal stem cells to express the genes.

2. The method according to claim 1, further comprising a single-cell conversion step of converting the genetically modified mesenchymal stem cells into single cells.

3. The method according to claim 1, further comprising a step of expanding culture the genetically modified mesenchymal stem cells.

4. The method according to claim 1, further comprising a selection step of selecting the genetically modified mesenchymal stem cells.

5. The method according to claim 1, further comprising a first culture step of culturing the mesenchymal stem cells in a first culture medium prior to the gene modification step.

6. The method according to claim 5, wherein the first culture medium contains 3 v / v% to 20 v / v% human platelet lysate.

7. A method for improving the differentiation ability to osteoblasts and / or adipocytes, comprising a gene modification step of genetically modifying mesenchymal stem cells by introducing a gene encoding telomerase reverse transcriptase or an active fragment thereof, and a gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof, into a cell population including mesenchymal stem cells derived from amnion and lacking the differentiation ability to osteoblasts and / or adipocytes.

8. A composition for use in the method according to any one of claims 1 to 7, comprising a nucleic acid molecule containing an exogenous gene encoding telomerase reverse transcriptase or an active fragment thereof, and / or a nucleic acid molecule containing an exogenous gene encoding Polycomb complex protein Bmi-1 or an active fragment thereof.