Methods for producing mesenchymal stem cells

Abstract

The present invention produces a large number of mesenchymal stem cells in a short time in a culture medium containing no heterologous components by including a step of culturing a cell population containing mesenchymal stem cells in the presence of a fibronectin fragment.

Description

Technical Field

[0001] This invention relates to a method for generating mesenchymal stem cells (MSCs), particularly a method for generating adipose-derived stem cells (ASCs or ADSCs). The invention also relates to mesenchymal stem cells obtained by this method and regenerative medicine compositions containing these cells as active ingredients. Background Technology

[0002] Mesenchymal stem cells (MSCs) are somatic stem cells derived from mesodermal tissue (mesenchyma). Because MSCs have the ability to differentiate into mesenchymal cells, they are expected to be used in regenerative medicine, including the reconstruction of bone, blood vessels, and myocardium. Furthermore, MSCs can serve as research tools in embryology, molecular biology, and pharmacology. MSCs are known to be derived from various tissues, including bone marrow, adipose tissue, blood, placenta, umbilical cord, and dental pulp. Because MSCs possess different properties depending on the tissue from which they are obtained, they are referred to as bone marrow-derived stem cells, adipose-derived stem cells, etc., depending on the tissue from which they are obtained.

[0003] Mesenchymal stem cells are routinely derived from bone marrow. However, only a small number of mesenchymal stem cells are found in bone marrow, and their number decreases with age. Therefore, from a clinical perspective, it is necessary to collect hundreds of milliliters of bone marrow to obtain a sufficient number of mesenchymal stem cells, which is a significant burden on patients. On the other hand, mesenchymal stem cells are abundant in subcutaneous fat. Since subcutaneous fat is easily obtained through fat aspiration under local anesthesia, it is less of a burden on patients. In recent years, clinical research using fat-derived stem cells has been increasing.

[0004] A common method for obtaining adipose-derived stem cells involves enzymatically treating a small amount of fat slices taken from a human to obtain a cell population, separating the stromal vascular fraction (SVF) deposited from the cell population by centrifugation, and continuously culturing the fraction in a medium containing bovine serum to proliferate (Non-Patent Literature 1). However, from a clinical application perspective, the use of bovine serum containing animal-derived fractions presents significant problems.

[0005] List of cited references

[0006] Non-patent literature

[0007] Non-patent literature 1: Tissue Engineering, 2001, Vol. 7, No. 2, pp. 211-218. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] This invention addresses the aforementioned problems inherent in conventional methods for generating mesenchymal stem cells. One objective of this invention is to provide a method for generating a large number of mesenchymal stem cells in a short period of time.

[0010] Solution to the problem

[0011] The inventors of this invention conducted in-depth research to achieve this objective, and as a result, they discovered that by culturing a cell population containing mesenchymal stem cells in the presence of fibronectin fragments, a large number of mesenchymal stem cells were generated in a heterologous culture medium within a short period of time. Therefore, this invention was completed.

[0012] The summary of the present invention includes:

[0013] [1] A method for generating mesenchymal stem cells, the method comprising the step of culturing a cell population containing mesenchymal stem cells in the presence of fibronectin fragments.

[0014] [2] [1] method, wherein the fibronectin fragment is a fragment having at least one domain selected from a cell adhesion domain and a heparin binding domain,

[0015] [3] [1]'s method, wherein the culture step in the presence of fibronectin fragments is a step of culturing a cell population in contact with a solid phase coated with fibronectin fragments.

[0016] [4] [1] method, wherein the culture step is the step of culturing cell populations in a foreign-free culture medium.

[0017] [5] [4] The method wherein the heterologous culture medium is a serum-free culture medium or a culture medium containing allogeneic serum,

[0018] [6] [1] method, wherein the cell population is derived from human cells,

[0019] [7] [6] The method, wherein the cell population is a cell population directly isolated from body tissue,

[0020] [8] [7] method, wherein the body tissue is selected from adipose tissue, bone marrow, blood, placenta, umbilical cord and dental pulp,

[0021] [9] [6] method, wherein the cell population is derived from embryonic stem cells or induced pluripotent stem cells differentiated from or induced mesenchymal stem cells,

[0022]

[10] A mesenchymal stem cell obtained by any one of the methods in [1]-[9],

[0023]

[11] A therapeutic composition containing

[10] cells as an active ingredient,

[0024]

[12] A method for producing cells belonging to the mesenchyme, the method comprising the step of differentiating mesenchymal stem cells obtained by any one of the methods in [1]-[9], and

[0025]

[13]

[12] 's method, wherein the differentiation step is a culture step in a culture medium containing a differentiation inducer, the differentiation inducer inducing mesenchymal stem cells to differentiate into another type of cell.

[0026] Invention Effects

[0027] This invention enables the generation of a large number of mesenchymal stem cells in a foreign-free culture medium in a short period of time. Attached Figure Description

[0028] [ Figure 1 ] Figure 1 The images shown are micrographs taken on day 8 after the start of culturing in Example 1. Left: Condition A, Middle: Condition B, Right: Condition C.

[0029] [ Figure 2 ] Figure 2 This shows the number of culture days and the number of cells collected in Example 2.

[0030] [ Figure 3 ] Figure 3 This demonstrates the differentiation of mesenchymal stem cells into various cell types in Example 3. Detailed Implementation

[0031] Definitions, etc.:

[0032] The term “mesenchymal stem cells (MSCs)” as used in this article refers to somatic stem cells derived from mesodermal tissue (mesenchyma).

[0033] Mesenchymal stem cells are known to express specific cell surface markers, such as CD73, CD90, and CD105. Mesenchymal stem cells present in a sample as starting material or as the final cell population can be detected or quantified by immunological methods (using antibodies to detect them) to test the expression of these markers.

[0034] As used in this article, the term "adipose-derived stem cells (ADSC or ASC)" refers to stem cells that exist in adipose tissue. Adipose-derived stem cells are included within mesenchymal stem cells and retain the same differentiation capacity as mesenchymal stem cells.

[0035] The term "alien" as used in this article refers to an organism or the genes of an organism that originate from the same species as the standard species. The term "alien" includes both those with the same genes and those that are self-generated.

[0036] The term “heterogeneous” as used in this article refers to an organism or the genes it possesses that originate from a species different from the standard species.

[0037] The term “heterogeneous” as used in this article means that it does not contain components derived from species different from the standard species.

[0038] The invention will now be described in detail.

[0039] (1) The method for generating mesenchymal stem cells of the present invention

[0040] <Cell population containing mesenchymal stem cells>

[0041] The method for generating mesenchymal stem cells of the present invention includes the step of culturing a cell population containing mesenchymal stem cells in the presence of fibronectin fragments. Hereinafter, "cell population containing mesenchymal stem cells" is described in detail.

[0042] "A cell population containing mesenchymal stem cells" is not particularly limited and can be any cell population containing mesenchymal stem cells.

[0043] The organism from which the cells are used in this invention are not particularly limited. The cells used in this invention can be derived from any organism, preferably a mammal. The age and sex of the organism are not particularly limited. In one embodiment, cells derived from primates (e.g., chimpanzees, Japanese macaques, humans) are used in this invention. Human cells are most preferably used, but the invention is not limited to the use of human cells. In cases where mesenchymal stem cells are generated by this invention for administration to humans, a cell population obtained from a donor having the same or similar histocompatibility antigen type as the recipient is preferably used as the raw material. More preferably, a cell population derived from the recipient is used to generate mesenchymal stem cells.

[0044] "A cell population containing mesenchymal stem cells" can be primary cells directly isolated from living tissue containing mesenchymal stem cells, mesenchymal stem cells differentiated from embryonic stem cells or induced pluripotent stem cells, or cryopreserved cells containing the above. The term "direct" as used herein means without in vitro culture / proliferation steps.

[0045] Examples of living tissues containing mesenchymal stem cells include bone marrow, adipose tissue, blood, placenta, umbilical cord, and dental pulp. Adipose tissue can be obtained through liposuction or adipectomy, which carries a low risk of individual functional impairment and is therefore suitable as a source of the aforementioned cell populations.

[0046] Adipose tissue is composed of adipocytes and is a living tissue. Examples of sites from which adipose tissue is derived for use in this invention include (but are not particularly limited to) subcutaneous fat, visceral fat, intramuscular fat, and intramuscular fat. Subcutaneous fat is a preferred source of adipose tissue because it is readily available under local anesthesia.

[0047] There are no particular restrictions on the methods for preparing adipose tissue. Adipose tissue can be prepared from tissue fragments aspirated during cosmetic surgery or from excised adipose tissue contained in tissue removed during surgery. Since adipose-derived stem cells are located around large blood vessels, a larger quantity can be obtained from excised adipose tissue than from liposuction. On the other hand, preparing stem cells from liposuction leaves smaller surgical scars, which places less burden on the donor.

[0048] Although one type of adipose tissue is typically used, two or more types can be used in combination. Adipose tissue can be collected multiple times, and the collected adipose tissue can be mixed before use.

[0049] The amount of adipose tissue collected can be determined depending on the type of donor, the type of donor tissue, or the required amount of mesenchymal stem cells. While the invention should not be particularly limited, mesenchymal stem cells can be obtained from 0.3–20 g of adipose tissue. Larger amounts of adipose tissue can be used as starting material by scaling up or repeating the process.

[0050] If necessary, the collected adipose tissue is subjected to a process to remove any attached blood components and then cut into fragments. The collected adipose tissue is then subjected to an enzymatic treatment (protease treatment) as described below. Blood components can be removed by washing the adipose tissue with a suitable buffer or culture medium.

[0051] Enzymatic treatment is carried out by digesting adipose tissue with proteases such as collagenase, trypsin, or dispersant enzymes. This enzymatic treatment can be carried out according to methods and conditions known to those skilled in the art (see, for example, RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). Preferably, the enzymatic treatment is carried out according to the methods and conditions described in the following examples.

[0052] Enzyme-treated adipose tissue contains two main cell populations: the stromal vascular component and mature adipocytes. The stromal vascular component is a mixture of cells including preadipocytes, mature endothelial cells, endothelial progenitor cells, vascular smooth muscle cells, pericytes, parietal cells, macrophages, fibroblasts, and adipose-derived stem cells. Adipose-derived stem cells are mesenchymal stem cells capable of readily differentiating into adipocytes, osteoblasts, chondrocytes, etc. The types and ratios of cells constituting the cell populations depend on the source and type of adipose tissue used.

[0053] The enzyme-treated adipose tissue is then centrifuged to separate the two cell populations. After centrifugation, the precipitate (containing the stromal vascular component) is collected as a sediment. Centrifugation conditions vary depending on the cell type and number. For example, centrifugation is performed at 300-2000 xg for 1-15 minutes. Before centrifugation, the enzyme-treated cell population can be subjected to filtration or the like to remove undigested tissue. For filtration, a filter with a pore size of 50-200 μm, preferably 100 μm, can be used, for example. In this invention, the stromal vascular component can be used as "a cell population containing mesenchymal stem cells".

[0054] <Fibronectin fragment>

[0055] Fibronectin is a large glycoprotein with a molecular weight of 250,000, found in animal blood, the surface of cultured cells, and the extracellular matrix of tissues, and is known to have various functions. Fibronectin has seven domains. The amino acid sequences of these domains include three similar sequences, and the whole is composed of repetitive sequences of these sequences. These three similar sequences are called type I, type II, and type III. Type III consists of 71-96 amino acid residues, with 17-40% identity among these residues. There are 14 type III sequences in fibronectin, with sequences 8, 9, and 10 (hereinafter referred to as III-8, III-9, and III-10, respectively) contained in the cell adhesion domain, and sequences 12, 13, and 14 (hereinafter referred to as III-12, III-13, and III-14, respectively) contained in the heparin-binding domain. III-10 contains a region with binding activity to integrin α5β1 (called VLA-5), and its core sequence is RGD. Additionally, there is a region called IIICS on the C-terminus of fibronectin. Within IIICS is a sequence called CS-1, which consists of 25 amino acid residues and exhibits binding activity to integrin α4β1 (called VLA-4).

[0056] The amino acid sequences of III-8 to III-14 and CS-1 are shown in the sequence listing as SEQ ID NO: 1-7 and 8, which is part of the specification. In this invention, any fibronectin fragment may be used, provided it possesses characteristics suitable for culturing mesenchymal stem cells. For example, a fibronectin fragment containing one or more amino acid sequences selected from the above-described amino acid sequences may be used.

[0057] As fibronectin fragments, many fragments containing intramolecular cell adhesion domains or heparin-binding domains are known (see, for example, J. Biochem., Vol. 110, pp. 284-291, 1991).

[0058] Examples of fibronectin fragments used in this invention include (but are not particularly limited to) fragments containing at least one domain selected from cell adhesion domains (III-8, III-9 and III-10) and heparin binding domains (III-12, III-13 and III-14).

[0059] The fibronectin fragments used herein may be fragments containing the fibronectin functional domains described above, namely cell adhesion domains or heparin-binding domains, and need not necessarily consist of a portion of the continuous amino acid sequence of fibronectin. Fibronectin fragments can be generated by fragmenting naturally occurring fibronectin via enzymatic digestion or recombinant DNA technology. Recombinant fibronectin fragments are suitable for this invention due to the less effort required for impurity removal and for designing and obtaining the desired fragments, regardless of enzyme recognition sites, etc. For the production or processing of recombinants, the fibronectin fragments used in this invention preferably have a molecular weight of no more than 100 kDa.

[0060] An example of a fibronectin fragment containing both a cell adhesion domain and a heparin-binding domain is RetroNectin (registered trademark, manufactured by TAKARA BIO INC.). RetroNectin is described as CH-296 in the aforementioned J. Biochem. RetroNectin is a recombinant protein with a molecular weight of approximately 63,000 (574 amino acid residues) containing cell adhesion domains (III-8, III-9, and III-10), heparin-binding domains (III-12, III-13, and III-14), and CS-1. The amino acid sequence of RetroNectin is shown in the sequence listing as SEQ ID NO: 9, which is part of the specification.

[0061] The fibronectin fragments used herein include proteins that have been chemically modified, such as by acetylation. To generate mesenchymal stem cells in this invention, single-molecule fibronectin fragments or mixtures of two or more fibronectin fragments may be used.

[0062] Substances functionally equivalent to fibronectin fragments may also be used, such as those possessing cell adhesion domains and / or heparin-binding domains. Derivatives of fibronectin fragments may also be used.

[0063] Culture in the presence of the fibronectin fragments of the present invention can be carried out by any method in which a cell population containing mesenchymal stem cells is contacted with the fibronectin fragments. For example, a culture medium in which the fibronectin fragments are dissolved can be used for culture, or a solid phase immobilized with the fibronectin fragments can be used for culture. A preferred aspect of the invention includes the step of culturing a cell population containing mesenchymal stem cells in contact with a solid phase coated with fibronectin fragments. Hereinafter, "solid phase coated with fibronectin fragments" is described in detail.

[0064] In a preferred aspect of the invention, the fibronectin fragments are used in a state of being coated with a suitable solid phase, such as a container or carrier (microbeads, etc.) for cell culture. The culture container used can be of any material and any shape, as long as the material or shape does not inhibit the maintenance, survival, differentiation, maturation, and self-renewal of cells. Examples of materials for culture containers include glass, synthetic and natural resins (including nonwoven fabrics), and metals. Examples of shapes for culture containers include any shape of polygonal prisms such as triangular prisms, cubes and cuboids, cylinders, polygonal pyramids such as triangular and square pyramids, cones, gourds, etc., as well as spheres, hemispheres, circles, ellipses, and semicircles. Commercially available culture flasks, culture dishes, culture bags, hollow fiber culture instruments, etc., can also be used.

[0065] The culture bag is preferably a breathable culture bag. Larger culture vessels can be used when a large number of cells are required. Although cell culture can be performed in open or closed systems, it is preferred to perform cell culture in a closed system for the purpose of distributing the obtained mesenchymal stem cells to humans.

[0066] Coating a solid surface with fibronectin fragments can be carried out by known methods. For example, solutions of fibronectin fragments in sterile water, buffer solutions, saline, etc., can be used for coating. Fibronectin fragments are preferably dissolved in phosphate-buffered saline (PBS).

[0067] The amount of fibronectin fragment used in this invention is not particularly limited and can be determined by those skilled in the art. For example, when RetroNectin is used as the fibronectin fragment, coating can be performed by adding PBS containing a final concentration of 20 μg / mL RetroNectin to a culture vessel and incubating the vessel at room temperature for 1 hour.

[0068] Containers coated with fibronectin fragments can be stored at low temperatures, such as 4°C, until use. Just before use, the solution containing the fibronectin fragments is removed from the culture instrument by aspiration. The culture instrument is washed once with PBS, then once with each cell culture medium, and then provided for cell culture.

[0069] <Cultivation>

[0070] In the method for generating mesenchymal stem cells of the present invention, the "step of culturing a cell population containing mesenchymal stem cells in the presence of fibronectin fragments" includes a step of isolating and culturing mesenchymal stem cells or a step of expanding mesenchymal stem cells, or both a step of isolating and culturing mesenchymal stem cells and a step of expanding mesenchymal stem cells. Therefore, the method for generating mesenchymal stem cells of the present invention can be a method of isolating and culturing mesenchymal stem cells (which includes the step of "culturing a cell population containing mesenchymal stem cells in the presence of fibronectin fragments") or a method of expanding mesenchymal stem cells (which includes the step of "culturing a cell population containing mesenchymal stem cells in the presence of fibronectin fragments"). A preferred embodiment of the present invention is a method for obtaining mesenchymal stem cells through two steps: isolating and culturing mesenchymal stem cells and expanding the isolated and cultured mesenchymal stem cells. These steps are described in detail below.

[0071] (i) Isolation and culture of mesenchymal stem cells

[0072] In this step, the desired mesenchymal stem cells are selectively proliferated by culturing a population of cells containing mesenchymal stem cells in the presence of fibronectin fragments.

[0073] The culture medium used in this step can be prepared based on media commonly used for animal cell culture. Preferred examples of media include those free of heterologous components, such as fetal bovine serum (FBS) or fetal bovine serum (FCS) or sheep serum. Although such heterologous-free media can be suitably prepared, known media or commercially available media can be used as is, or modified as needed. Examples of commercially available heterologous-free media include DEF-CS500 XF (manufactured by Cellartis) and DXF (manufactured by PromoCell).

[0074] In the case of generating mesenchymal stem cells from autologous tissue, the culture medium can be a medium containing allogeneic serum or a serum-free medium. Preferred examples of such media include media containing allogeneic serum but without heterologous components and serum-free media without heterologous components. The allogeneic serum is preferably autologous serum. As used herein, autologous serum and autologous plasma refer to serum and plasma obtained from blood taken from the same donor as the cell population to be cultured.

[0075] Because plasma contains serum components, culture media containing plasma can be used. Preferably, inactivated autologous plasma is added to the culture medium. For example, cells are cultured in a medium containing 10% (V / V) or less, preferably 5% (V / V) or less, more preferably 2% (V / V) or less of inactivated autologous plasma. The use of autologous plasma allows for the exclusion of foreign components from the production step, thereby providing a safe method for cell production.

[0076] Cell culture conditions are not particularly limited, and normal cell culture conditions can be used. Examples of cell culture conditions include (but are not limited to) cell culture at a temperature of 37°C, 95% humidity, and a CO2 concentration of 5%. For example, although cell culture can be carried out at a temperature of 30-40°C, 90-98% humidity, and a CO2 concentration of 3-7%, the temperature, humidity, and CO2 concentration do not need to fall within the above ranges, as long as the desired cell proliferation can be obtained. It is preferable to replace the culture medium containing the active ingredient with fresh culture medium at appropriate time intervals during the culture period.

[0077] In a preferred aspect of the invention, the stromal vascular component is cultured in a container coated with fibronectin fragments in a heterologous-free medium containing 5% autologous plasma for, for example, 4-14 days, preferably 7-10 days, with the medium replaced with fresh medium every 3 or 4 days. This culture results in selective proliferation of mesenchymal stem cells. In other words, mesenchymal stem cells constitute 80% or more, preferably 90% or more, of the cell population obtained after cell culture.

[0078] The cell concentration at the start of culture during the isolation and culture steps is not particularly limited, and examples include 0.1–10 x 10⁻⁶ cells / mL. 5 Cells / mL, preferably 0.3-5 x 10⁻⁵. 5 Cells / mL, and more preferably 0.5-2 x 10⁻⁶ cells / mL. 5 Cells / mL.

[0079] (ii) Expansion of mesenchymal stem cells

[0080] In this step, mesenchymal stem cells are subjected to expansion culture in the presence of fibronectin fragments to enable them to proliferate in large quantities. Examples of mesenchymal stem cells to be cultured in this step include mesenchymal stem cells isolated by any method or cell populations containing mesenchymal stem cells, such as mesenchymal stem cells isolated by step (i) above, established mesenchymal stem cell lines, and mesenchymal stem cells differentiated from embryonic stem cells or induced pluripotent stem cells.

[0081] The mesenchymal stem cells isolated by step (i) above can be isolated from the culture vessel by methods commonly used in the art, such as physical methods, methods including the use of chelating agents, enzymatic methods (including the use of separation solutions with protease activity and / or collagenase activity (e.g., Accutase (registered trademark) and Accumax (registered trademark) etc.)) or any combination thereof. A preferred method involves enzymatically dissociating the mesenchymal stem cells from the sheet and then physically finely dispersing the cells. The cells to be used are preferably cells cultured to 70-95% confluence in the culture vessel in use, more preferably cells cultured to 80-90% confluence in the culture vessel in use.

[0082] The isolated cell populations can be used as is or after cryopreservation. Subsets of cells isolated based on specific cell surface markers from the aforementioned cell populations, such as isolated mesenchymal stem cells, can also be used in this step.

[0083] The culture medium and culture conditions used in this step are the same as those described in step (i) above. This step can be carried out using the same culture media, containers, and culture conditions as those used in step (i) above, or using different culture media, containers, and culture conditions than those used in step (i) above. In the same manner as the culture in step (i) above, a culture medium free of heterologous components is preferably used, and any heterologous-free culture medium can be used as is, such as known or commercially available media, or after modification.

[0084] In a preferred aspect of the invention, isolated and cultured mesenchymal stem cells are expanded, for example, for 4-14 days, preferably 6-12 days, in a container coated with fibronectin fragments and in a serum-free, heterologous culture medium, with the culture medium replaced with fresh medium every 3 or 4 days, and the cells are passaged as appropriate. This culture results in a large proliferation of mesenchymal stem cells. In other words, through this culture, mesenchymal stem cells proliferate by 100-fold or more, preferably 300-fold or more.

[0085] There are no particular restrictions on the cell concentration at the start of the amplification step, and examples include 0.1–10 x 10⁻⁶ cells / year. 5 Cells / mL, preferably 0.3-5 x 10⁻⁵. 5 Cells / mL, and more preferably 0.5-2 x 10⁻⁶ cells / mL. 5 Cells / mL.

[0086] (2) Mesenchymal stem cells obtained by the method of the present invention

[0087] Mesenchymal stem cells or cell populations containing mesenchymal stem cells can be obtained by the mesenchymal stem cell generation method of the present invention as described above.

[0088] Mesenchymal stem cells obtained by the method of this invention can be identified by detecting molecules characteristic of mesenchymal stem cells, such as enzymes, receptors, and low molecular weight compounds. Examples of molecules characteristic of mesenchymal stem cells include (but are not limited to) cell surface markers (positive markers) such as CD73, CD90, CD105, and CD166. CD19, CD34, CD45, HLA-DR, CD11b, and CD14 are also called negative markers, which are not expressed on mesenchymal stem cells and can be used to identify them. The above molecules can be detected by immunological methods or by quantifying the mRNA content of each molecule.

[0089] The aforementioned positive biomarkers are expressed in, for example, 80% or more, preferably 90% or more, and more preferably 95% or more of the cell populations containing mesenchymal stem cells obtained by the method of the present invention. The expression rate of the negative biomarkers in the cell populations containing mesenchymal stem cells obtained by the method of the present invention is, for example, 5% or less, preferably 1% or less, and more preferably below the limit of detection.

[0090] Antibodies that recognize molecules characteristic of mesenchymal stem cells can also be used to isolate and purify mesenchymal stem cells obtained by the method of this invention. This invention includes cell populations obtained by isolating, sorting, and purifying mesenchymal stem cells based on molecules characteristic of mesenchymal stem cells.

[0091] The mesenchymal stem cells obtained by the method of this invention can also be used in studies such as the differentiation of mesenchymal stem cells, drug screening for various diseases, and efficacy and safety evaluation of drug candidate compounds. According to this invention, a large number of mesenchymal stem cells can be obtained in one stage, and therefore reproducible experimental results can be obtained without being affected by batch-to-batch variations in cells.

[0092] (3) The therapeutic composition of the present invention

[0093] A therapeutic composition containing mesenchymal stem cells obtained by the method of the present invention as described above as an active ingredient can be prepared.

[0094] Mesenchymal stem cells obtained by the method of this invention and compositions containing such cells can be used to treat diseases such as bone diseases, osteomalacia, heart disease, spinal cord injury, and graft-versus-host disease. By using mesenchymal stem cells obtained by the method of this invention, pharmaceutical compositions for treating various diseases can be produced.

[0095] When mesenchymal stem cells obtained by the method of this invention are formulated into pharmaceutical compositions, the cells and pharmaceutically acceptable carriers can be mixed by conventional methods to formulate a suitable dosage form for individual administration. Examples of carriers include saline and distilled water for injection, which are made isotonic by adding glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). The pharmaceutical composition may further contain buffers (e.g., phosphate buffers, sodium acetate buffers), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0096] (4) This invention pertains to a method for producing mesenchymal cells.

[0097] It is known that various cells differentiated from mesenchymal stem cells, i.e., cells belonging to the mesenchymal system, can be obtained by culturing mesenchymal stem cells obtained by the method of the present invention in a culture medium containing a differentiation inducer that induces mesenchymal stem cells to differentiate into another type of cell.

[0098] Examples of mesenchymal cells obtained by the method of this invention include (but are not limited to) adipocytes (brown adipocytes, white adipocytes), chondrocytes, osteoblasts, and myocytes. Methods for inducing differentiation from mesenchymal stem cells are known for various mesenchymal cells. For example, known differentiation inducers for the corresponding cells include dexamethasone, insulin, 3-isobutyl-1-methylxanthine, etc., for adipocytes; dexamethasone, ascorbic acid or ascorbic acid-2-phosphate, β-glycerophosphate, etc., for osteoblasts; hydrocortisone, ascorbic acid, β-glycerophosphate, etc., for osteoblasts; and insulin, ascorbic acid-2-phosphate, hydrocortisone, etc., for chondrocytes. The desired cells can be obtained by contacting mesenchymal stem cells with these differentiation inducers.

[0099] Although the culture media for differentiation induction described above can be suitably prepared, known culture media or commercially available culture media can be used as is, or used after modification. Examples of commercially available culture media for differentiation induction include hMSC differentiation BulletKits-Adipogenesis Medium (manufactured by LONZA) for differentiation into adipocytes, hMSC differentiation BulletKits-Osteogenesis Medium (manufactured by LONZA) for differentiation into osteoblasts, and hMSC differentiation BulletKits-Chondroogenesis Medium (manufactured by LONZA) for differentiation into chondrocytes.

[0100] Mesenchymal cells obtained by the above methods can also be formulated into pharmaceutical compositions. In this case, the suitable composition or form is selected depending on the purpose or method of administration. These mesenchymal cells can also be used to screen pharmaceutical candidate compounds or evaluate the safety of compounds. Example

[0101] The present invention will be further described in detail below through embodiments, but the present invention is not limited to these embodiments.

[0102] Example 1: Isolation and culture of adipose-derived stem cells

[0103] (1) Separation of autologous blood plasma

[0104] Blood was drawn from a healthy donor with informed consent using a blood collection syringe. The blood was centrifuged at 500 xg for 20 minutes, and plasma was collected. The collected plasma was inactivated at 56°C for 30 minutes, cooled at 4°C for 30 minutes, and then centrifuged at 800 xg for 30 minutes. The supernatant was used as inactivated autologous plasma (hereinafter referred to as "autologous plasma").

[0105] (2) Separation of stromal vascular components (SVF)

[0106] Adipose tissue was obtained from healthy human donors as described in Example 1-(1) and stored in saline. In a biosafety cabinet, the adipose tissue was placed in a 10 cm culture dish and then cut into cubes with sides of 1-2 mm using sterile scissors. A new 10 cm culture dish was placed on a balance, and 15 g of the cut adipose tissue was weighed. 2 mg / mL of type I collagenase / HBBSS solution was added to the adipose tissue at a rate of 3 mL per 1 g of adipose tissue to suspend it. The suspension was kept in a constant temperature bath at 37°C for 1 hour with shaking at 100 rpm / min. Centrifuge tubes were placed in a biosafety cabinet, and the suspension was filtered through a 100 μm sieve and collected in centrifuge tubes. The suspension was centrifuged at 25°C and 200 xg for 10 minutes. The supernatant was carefully removed using an electric pipette. 10-20 mL of ACK lysis buffer (manufactured by LONZA) was added to the precipitate and the precipitate was resuspended. The suspension was allowed to stand at room temperature for 5 minutes, and then centrifuged at 25°C and 400 xg for 5 minutes. The supernatant was carefully removed from the centrifuged sample using an electric pipette to obtain the precipitate. An appropriate amount of PBS was added to the precipitate and the sample was resuspended to obtain the stromal vascular component.

[0107] (3) Wrap with RetroNectin (RN)

[0108] The culture apparatus to be used in the following experiments was coated with RN. Specifically, 5 mL of phosphate-buffered saline (PBS) containing RetroNectin (registered trademark) (manufactured by TAKARA BIO INC.) (final concentration: 20 μg / mL) was added to a T-25 flask (manufactured by Corning). As a control, a flask containing PBS without RetroNectin (referred to as "RN") was prepared.

[0109] Incubate these culture instruments at room temperature for 1 hour and store at 4°C until use. Just before use, remove the PBS from the culture instruments by aspiration. Wash the culture instruments once with PBS, and then once with each cell culture medium, before providing them for cell culture. Hereinafter, flasks coated with RN will be referred to as RN flasks.

[0110] (3) Culture of stromal vascular components

[0111] The samples were obtained from the stromal vascular components obtained in Examples 1-(2), and the total nucleated cell concentration was measured using a Nucleo Counter (manufactured by Chemmetec). Cell suspensions were then prepared by adding culture medium (basal medium and additives shown in Table 1) to the samples, resulting in a total nucleated cell concentration of 1 x 10⁻⁶ cells / mL. 5 Total number of nucleated cells / mL. Then, 10 mL of cell suspension was added to the RN flask or control flask prepared by Example 1-(3). Cell culture was started at 37°C and 5% CO2 (day 0 of culture). One day after the start of culture, the culture medium was replaced with fresh culture medium, and thereafter the culture medium was replaced every 3-4 days until the cells reached 80-90% confluence.

[0112] The basal culture medium and additives used for cell culture, the presence or absence of RN coating, the number of culture days required to reach 80-90% confluence, and the number of cells collected are shown in Table 1. Microscopic images taken on day 8 after the start of culture are shown below. Figure 1 As shown.

[0113] [Table 1]

[0114]

[0115] From Table 1 and Figure 1As can be seen, when DEF-CS500 XF containing 5% autologous plasma (Condition B) was used instead of DMEM containing 10% FBS (Condition A, conventional method), the cells never reached confluence. On the other hand, when cells were cultured in DEF-CS500 XF containing 5% autologous plasma in RN flasks (Condition C), the cell growth rate was significantly higher than that of the conventional method (Condition A), and more cells were obtained in a shorter time.

[0116] Due to the initial cell number at the start of culture (1 x 10⁻⁶) 6 It contains all cells of the stromal vascular component, and most cells were excluded by medium replacement on day 1 after the start of culture, so the number of cells collected is less than 1 x 10⁻⁶. 6 The reason why the number of cells converging 80-90% differs greatly between conditions A and C is that the cells are different in size and shape.

[0117] Example 2: Expansion of adipose-derived stem cells

[0118] (1) Culture of stromal vascular components

[0119] In the same manner as in Examples 1-(4), the stromal vascular component obtained by Examples 1-(2) was suspended in the culture medium so that the total nucleated cell concentration became 1 x 10⁻⁶. 5 Total nucleated cell count / mL. Then, 10 mL of cell suspension was added to the RN flask or control flask prepared according to Example 1-(3). Cell culture was started at 37°C and 5% CO2 (day 0 of culture). One day after the start of culture, the medium was replaced with fresh medium, and thereafter the medium was replaced every 3-4 days until the cells reached 80-90% confluence. The basal medium and additives used for cell culture and the presence or absence of RN coating are shown in Table 2.

[0120] [Table 2]

[0121]

[0122] (2) Amplification culture

[0123] Cells were collected when they reached 80-90% confluence. A cell suspension was prepared by adding culture medium (basal medium and additives shown in Table 2) to achieve a cell density of 1 x 10⁻⁶ cells / mL. 5 Cells / mL. Then, 10 mL of cell suspension was added to the RN flask or control flask prepared by Example 1-(3). Amplification culture was started at 37°C and 5% CO2 (P0).

[0124] One day after the start of culture, the culture medium was replaced with fresh medium, and thereafter every 3-4 days, until the cells reached 70-95% confluence. At 70-95% confluence, cells were collected (P1) and stained with trypan blue to measure the total nucleated cell concentration. Then, a cell suspension was prepared by adding culture medium again (basal medium and additives shown in Table 2) to achieve a cell density of 1 x 10⁻⁶ cells / day. 5 Cells / mL. The cell suspension was added to a new RN flask or control flask and amplified further at 37°C and 5% CO2. Cells were collected (P2) at 80-90% confluence and stained with trypan blue to measure the total nucleated cell concentration. Culture days and cell numbers collected are shown in Table 3. Graphs of these results are shown below. Figure 2 As shown.

[0125] [Table 3]

[0126]

[0127] From Table 3 and Figure 2 As can be seen, when cells are cultured in DXF in RN flasks (conditions E and F), more cells are obtained in a shorter time compared to cells cultured in DMEM with 10% FBS added (condition D, conventional method). Even when the culture medium does not contain autologous plasma (condition E), the cell growth rate is significantly higher than that of the conventional method (condition D).

[0128] Example 3: Analysis of cell surface markers

[0129] The P2 cell population obtained by Example 2-(2) was continued to expand under each culture condition, and the cells were passaged when they reached 80-90% confluence. Around day 20 after the start of expansion culture, cells were collected and washed with PBS containing 0.1% bovine serum albumin (BSA, manufactured by SIGMA) (hereinafter referred to as 0.1% BSA / PBS). The cell population was then resuspended in 0.1% BSA / PBS. Add each antibody reaction solution containing PE-Cy7-labeled mouse anti-human CD73 antibody (manufactured by Becton, Dickinson and Company), APC-labeled mouse anti-human CD90 antibody (manufactured by Becton, Dickinson and Company), APC-labeled mouse anti-human CD105 antibody (manufactured by Becton, Dickinson and Company), PE-labeled mouse anti-human CD34 antibody (manufactured by Becton, Dickinson and Company), FITC-labeled mouse anti-human CD45 antibody (manufactured by Becton, Dickinson and Company), PE-Cy7-labeled mouse anti-human HLA-DR antibody (manufactured by Becton, Dickinson and Company), FITC-labeled mouse anti-human CD11b antibody (manufactured by Becton, Dickinson and Company), and PE-labeled mouse anti-human CD14 antibody (manufactured by Becton, Dickinson and Company) to the suspension, and let the suspension stand at room temperature for 10 minutes. The cell population was then washed twice with 0.1% BSA / PBS and resuspended in 0.1% BSA / PBS. The cell population was then subjected to flow cytometry (FACSCantoII, manufactured by Becton, Dickinson and Company) to calculate the proportion of each cell surface marker present in the cell population. In this study, CD73, CD90, and CD105 were cell surface markers of mesenchymal stem cells (positive markers), and CD34, CD45, HLA-DR, CD11b, and CD14 were negative markers not expressed on mesenchymal stem cells. The results are shown in Table 4.

[0130] [Table 4]

[0131]

[0132] In the table, P3 refers to the cell population that reaches 80-90% confluence after passage culture of P2. P5 refers to the cell population that reaches 80-90% confluence after two passage cultures of P3.

[0133] As shown in Table 4, the expression rate of positive markers for mesenchymal stem cells was 95% or higher under all conditions. In contrast, negative markers for mesenchymal stem cells were expressed at low levels under the conventional method (condition D), while the expression rates of all negative markers were below the detection limit when cells were cultured in DXF in RN flasks (conditions E and F). In other words, it was found that cell culture in DXF in RN flasks is a more effective way to obtain a cell population containing a high proportion of mesenchymal stem cells compared to the conventional method.

[0134] Example 4: Differentiation into mesenchymal cells

[0135] The mesenchymal stem cells obtained through Example 3 (condition EP5) were differentiated into various cell types.

[0136] To differentiate into adipocytes, mesenchymal stem cells (condition EP5) were cultured in hMSC differentiation BulletKits-Adipogenesis medium (manufactured by LONZA) according to the manufacturer's instructions. After removing the medium, 4% paraformaldehyde (manufactured by SIGMA) was added to the cells, and they were incubated at room temperature for 10 minutes to fix the cells. After washing the cells with 60% isopropanol (manufactured by SIGMA), Oil Red O was added to the cells, and they were incubated at room temperature for 15 minutes to make lipid droplets visible.

[0137] To differentiate into osteoblasts, mesenchymal stem cells (condition EP5) were cultured in hMSC differentiation BulletKits-Osteoblast medium (manufactured by LONZA) according to the manufacturer's instructions. After removing the medium, 4% paraformaldehyde (manufactured by SIGMA) was added to the cells, and they were incubated at room temperature for 10 minutes to fix the cells. After washing the cells with distilled water, alizarin red staining solution was added, and the cells were incubated at room temperature for 45 minutes to make calcium deposition visible.

[0138] To differentiate into chondrocytes, mesenchymal stem cells (condition EP5) were cultured in hMSC differentiation BulletKits-Chondrogenic Medium (manufactured by LONZA) according to the manufacturer's instructions. After removing the medium, 4% paraformaldehyde (manufactured by SIGMA) was added to the cells, and they were fixed by incubation at room temperature for 60 minutes. After washing the cells with distilled water, Alcian Blue staining solution was added, and the cells were incubated overnight at room temperature to make proteoglycans visible. These proteoglycans are the main components of the cellular matrix that forms cartilage.

[0139] The results are as follows Figure 3 As shown. From Figure 3As can be seen, cells are stained only when cultured in each differentiation-inducing medium. These results indicate that mesenchymal stem cells obtained by the method of this invention can differentiate into various cell types known to arise from mesenchymal stem cells, i.e., cells belonging to the mesenchyme, by culturing them in a medium containing differentiation inducers.

[0140] Industrial applicability

[0141] This invention provides a method for generating a large number of mesenchymal stem cells in a short period of time. This method for generating mesenchymal stem cells is particularly useful in regenerative medicine.

[0142] Sequence List Free Text

[0143] SEQ ID NO: 1; a partial region of fibronectin named III-8.

[0144] SEQ ID NO: 2; a partial region of fibronectin named III-9.

[0145] SEQ ID NO: 3; a partial region of fibronectin named III-10.

[0146] SEQ ID NO: 4; a partial region of fibronectin named III-11.

[0147] SEQ ID NO: 5; a partial region of fibronectin named III-12.

[0148] SEQ ID NO: 6; a partial region of fibronectin named III-13.

[0149] SEQ ID NO: 7; a partial region of fibronectin named III148.

[0150] SEQ ID NO: 8; a partial region of fibronectin named CS-1

[0151] SEQ ID NO: 9; A fibronectin fragment named RetroNectin (CH-296) <110> TAKARA BIO INC. <120> Methods for generating mesenchymal stem cells <130> 673183 <150> JP 2015-236442 <151> 2015-12-3 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 87 <212> PRT <213> Artificial sequence <220> <223> A portion of fibronectin called III-8 <400> 1 Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val 1 5 10 15 Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg 20 25 30 Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser 35 40 45 Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu 50 55 60 Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro 65 70 75 80 Leu Arg Gly Arg Gln Lys Thr 85 <210> 2 <211> 90 <212> PRT <213> Artificial sequence <220> <223> A portion of fibronectin called III-9 <400> 2 Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn 1 5 10 15 Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr 20 25 30 Arg Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp 35 40 45 Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro 50 55 60 Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu 65 70 75 80 Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr 85 90 <210> 3 <211> 94 <212> PRT <213> Synthetic Sequence <220> <223> Partial Region of Fibronectin Named III-10 <400> 3 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 <210> 4 <211> 84 <212> PRT <213> Artificial sequence <220> <223> A portion of fibronectin called III-11 <400> 4 Gln Met Gln Val Thr Asp Val Gln Asp Asn Ser Ile Ser Val Lys Trp 1 5 10 15 Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg Val Thr Thr Thr Pro 20 25 30 Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr Ala Gly Pro Asp Gln 35 40 45 Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr Val Glu Tyr Val Val 50 55 60 Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser Gln Pro Leu Val Gln 65 70 75 80 Thr Ala Val Thr <210> 5 <211> 92 <212> PRT <213> Artificial sequence <220> <223> Partial region of fibronectin III-12 <400> 5 Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr 1 5 10 15 Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr 20 25 30 Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile 35 40 45 Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val 50 55 60 Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr 65 70 75 80 Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu 85 90 <210> 6 <211> 89 <212> PRT <213> Artificial sequence <220> <223> A portion of fibronectin called III-13 <400> 6 Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr 1 5 10 15 Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe 20 25 30 Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr 35 40 45 Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly 50 55 60 Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser 65 70 75 80 Ser Pro Val Val Ile Asp Ala Ser Thr 85 <210> 7 <211> 90 <212> PRT <213> Artificial sequence <220> <223> A portion of fibronectin called III-14 <400> 7 Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn 1 5 10 15 Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr 20 25 30 Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro 35 40 45 Arg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro 50 55 60 Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys 65 70 75 80 Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr 85 90 <210> 8 <211> 25 <212> PRT <213> Artificial sequence <220> <223> A portion of fibronectin called CS-1 <400> 8 Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly 1 5 10 15 Pro Glu Ile Leu Asp Val Pro Ser Thr 20 25 <210> 9 <211> 574 <212> PRT <213> Artificial sequence <220> <223> A fibronectin fragment named RetroNectin (CH-296) <400> 9 Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val 1 5 10 15 Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg 20 25 30 Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser 35 40 45 Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu 50 55 60 Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro 65 70 75 80 Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp 85 90 95 Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro 100 105 110 Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe 115 120 125 Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile 130 135 140 Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val 145 150 155 160 Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser 165 170 175 Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro 180 185 190 Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr 195 200 205 Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu 210 215 220 Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys 225 230 235 240 Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly 245 250 255 Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu 260 265 270 Ile Asp Lys Pro Ser Met Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe 275 280 285 Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn 290 295 300 Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr 305 310 315 320 Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val 325 330 335 Val Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala 340 345 350 Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr 355 360 365 Leu Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr 370 375 380 Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr 385 390 395 400 Gly Phe Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln 405 410 415 Arg Thr Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln 420 425 430 Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala 435 440 445 Arg Ser Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro 450 455 460 Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser 465 470 475 480 Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu 485 490 495 Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly 500 505 510 Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr 515 520 525 Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile 530 535 540 Gly Arg Lys Lys Thr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His 545 550 555 560 Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr 565 570

Claims

1. A method for selectively proliferating mesenchymal stem cells, the method comprising culturing a population of cells containing mesenchymal stem cells in a heterologous culture medium in the presence of fibronectin fragments, thereby selectively proliferating the mesenchymal stem cells, wherein the cell population is a stromal vascular component isolated from adipose tissue, and the heterologous culture medium is a culture medium containing autologous plasma.

2. The method for selectively proliferating mesenchymal stem cells according to claim 1, wherein the fibronectin fragment is a fragment having at least one domain selected from a cell adhesion domain and a heparin binding domain.

3. The method for selectively proliferating mesenchymal stem cells according to claim 1, wherein the culture step in the presence of fibronectin fragments is a step of culturing the cell population in contact with a solid phase coated with the fibronectin fragments.

4. The method for selectively proliferating mesenchymal stem cells according to claim 1, wherein the cell population is a human-derived cell population.

5. A method for producing cells belonging to the mesenchyme, the method comprising: The steps of obtaining the mesenchymal stem cells by the selective proliferation method of mesenchymal stem cells according to any one of claims 1-4, and the steps of differentiating the mesenchymal stem cells.

6. The method of generating cells belonging to mesenchyme according to claim 5, wherein the differentiation step is a culture step in a culture medium containing a differentiation inducer, wherein the differentiation inducer induces mesenchymal stem cells to differentiate into another type of cell.

Images