Method for producing mesenchymal stem cell culture

Abstract

Provided are a method for producing a mesenchymal stem cell culture, a mesenchymal stem cell culture, a pharmaceutical composition containing a mesenchymal stem cell culture, and a method for producing a population of mesenchymal cells. This method for producing a mesenchymal stem cell culture includes a step (a) for culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum starvation conditions or 2-deoxy-D-glucose (2-DG)-containing culture medium conditions to thereby separate a culture of mesenchymal stem cells that survived the serum starvation conditions or 2-DG-containing culture medium conditions. The population of mesenchymal stem cells is not exposed to growth conditions from after collection of the tissue-derived sample from an individual until culturing is performed under serum starvation conditions or 2-DG-containing culture medium conditions.

Description

Method for producing mesenchymal stem cell culture

[0001] The present disclosure relates to methods for producing mesenchymal stem cell cultures, mesenchymal stem cell cultures, and pharmaceutical compositions comprising mesenchymal stem cell cultures. The present disclosure further relates to methods for producing populations of mesenchymal cells.

[0002] Mesenchymal stem cells (MSCs) are non-blood cell stem cells that can differentiate into a variety of mesenchymal cells, including fat, bone, and cartilage. Because mesenchymal stem cells have the ability to proliferate in vitro and differentiate into a variety of cells, they are expected to have a wide range of applications in regenerative medicine. Research is being actively conducted worldwide toward the realization of regenerative medicine using mesenchymal stem cells, and mesenchymal stem cells are already being used in actual treatments.

[0003] Mesenchymal stem cells are known to undergo cellular senescence during extended culture. Cell senescence is associated with cellular senescence-related genes, including the p53 pathway. Through cellular senescence, mesenchymal stem cells gradually lose their proliferation and differentiation capabilities, properties crucial for therapeutic use. The issue of cellular senescence is a major obstacle to the clinical application of mesenchymal stem cells and is one of the major factors limiting their therapeutic potential. Furthermore, conventional mesenchymal stem cell samples typically contain undifferentiated stem cells as well as adherent fibroblasts, resulting in heterogeneous cell populations. This is thought to cause a decline in the functionality of stem cell preparations and promote aging.

[0004] Previous attempts have been made to place mesenchymal stem cells under low-serum conditions. For example, in Patent Document 1, mesenchymal stem cells prepared as components of mononuclear cells isolated from human bone marrow or umbilical cord blood are cultured in a standard medium containing 20% ​​FBS (fetal bovine serum) for two weeks to form culture dish-adherent cells, which are then subcultured in the same standard medium for three to five generations. The cells are then placed in α-modified MEM (referred to as a "starvation medium" in Patent Document 1) lacking b-FGF and containing 0.5% FBS for five to seven days. Patent Document 1 describes the production of pluripotent cells (cells capable of forming teratomas), which are comparable to mesenchymal stem cells, by a method including a culture step under such "starvation conditions."

[0005] Patent Document 2 describes that dental pulp stem cells were pre-cultured in a culture medium containing 5%, 10%, or 15% FBS, and then cultured for one week without any medium changes, resulting in a "low serum state." Patent Document 3 describes a method for isolating pluripotent stem cells or pluripotent cell fractions, which involves exposing cells derived from biological tissues to cell stress and recovering surviving cells. One of the various cell stresses listed is "culture under serum starvation." In the examples of Patent Document 3, a bone marrow stromal cell line purchased from a distributor was passaged for 4 to 10 generations in a 10% FBS-containing medium and then cultured for two days in a serum-free medium. Patent Document 3 teaches the production of pluripotent stem cells (cells capable of forming embryoid body-like cell clusters), which are comparable to mesenchymal cells, using a method including a step of exposing cells to such cell stress.

[0006] In all of the above-mentioned prior art examples, cells isolated from tissues are first grown in a growth condition using a medium containing a high concentration of serum, and then cultured in a low-serum condition. In other words, the methods disclosed in these examples do not include a step of immediately culturing cells isolated from tissues in a serum-starved condition for a certain period of time without exposing them to a growth condition.

[0007] JP 2004-057198 A International Publication No. 2018 / 143378 International Publication No. 2011 / 007900

[0008] Various embodiments of the present disclosure are directed to providing methods for producing mesenchymal stem cell cultures, mesenchymal stem cell cultures, and pharmaceutical compositions comprising mesenchymal stem cell cultures. Embodiments of the present disclosure are further directed to providing methods for producing populations of mesenchymal cells.

[0009] The present inventors have discovered that by directly transferring a tissue-derived sample containing a population of mesenchymal stem cells collected from an individual to culture under serum-starved conditions without exposing it to growth conditions, and then isolating the surviving mesenchymal stem cell culture, it is possible to produce a highly pure culture of mesenchymal stem cells (referred to herein as STAR MSCs) with a distinctive gene expression profile and metabolic properties, and have thus completed the present invention.

[0010] One aspect of the present disclosure provides a method for producing a mesenchymal stem cell culture, the method comprising the steps of: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starvation conditions or in a 2-deoxy-D-glucose (2-DG)-containing medium condition, thereby isolating a culture of mesenchymal stem cells that survive said conditions, wherein the population of mesenchymal stem cells is not exposed to proliferation conditions between collection of the tissue-derived sample from an individual and culturing under said conditions.

[0011] One aspect of the present disclosure provides a mesenchymal stem cell culture, the mesenchymal stem cell culture being produced by a method comprising the steps of: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starvation conditions or 2-DG-containing medium conditions, thereby isolating a culture of mesenchymal stem cells that survive said conditions, wherein the population of mesenchymal stem cells is not exposed to growth conditions between collection of the tissue-derived sample from an individual and culturing under said conditions.

[0012] One aspect of the present disclosure provides a pharmaceutical composition comprising a mesenchymal stem cell culture produced by a method comprising the steps of: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starvation conditions or 2-DG-containing medium conditions, thereby isolating a culture of mesenchymal stem cells that survive said conditions, wherein the population of mesenchymal stem cells is not exposed to growth conditions between the time of collection of the tissue-derived sample from an individual and the time of culturing under said conditions.

[0013] One aspect of the present disclosure provides a method for producing a population of mesenchymal cells, the method comprising: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starvation conditions or 2-DG-containing medium conditions, thereby isolating a culture of mesenchymal stem cells that survive said conditions, wherein the population of mesenchymal stem cells is not exposed to growth conditions between the time of collection of the tissue-derived sample from an individual and the time of culturing under said conditions; and further differentiating the mesenchymal stem cell culture into mesenchymal cells in vitro.

[0014] (A) An example of a procedure for isolating CFU-F and STAR MSCs. Mouse mesenchymal stem cells (MSCs) can be purified by isolating Pdgfrα and Sca-1 co-positive cells from the CD45-, CD31-, and Ter119-negative fraction of bone marrow samples. Mouse mesenchymal stem cells were isolated using a flow cytometer and cultured in DMEM medium containing 20% ​​or 0.2% FBS for one week to obtain CFU-F or STAR MSC cells, respectively. (B) Micrographs of mouse mesenchymal stem cell cultures cultured in medium containing 20% ​​FBS (left) or 0.2% FBS (center) for one week. STAR MSCs, mesenchymal stem cells obtained by serum-starved culture (0.2% FBS), were switched to medium containing 20% ​​serum and cultured for an additional week to confirm that the cells retained their proliferative capacity (right). (A) Quantification of STAR MSC numbers. The number of colonies present on the flasks was counted after culture. Cell clusters consisting of 50 or more cells were counted as mesenchymal stem cell colonies. (B) Mesenchymal cells differentiated from STAR MSCs (adipocytes (left), chondrocytes (middle), and osteoblasts (right)) are shown. (C) Microarray analysis shows a comparison of gene expression between CFU-Fs (cultured in 20% FBS) and STAR MSCs (cultured in 0.2% FBS). In the heatmap, red clusters (top right and bottom left) indicate genes with over 2-fold higher expression (199 genes in STAR MSCs), while blue clusters (top left and bottom right) indicate genes with over 2-fold lower expression (492 genes in STAR MSCs). Representative genes in each cluster are shown on the right. The results of gene ontology (GO) analysis of genes with significant changes in expression levels in STAR MSCs compared to CFU-Fs are shown. (A) shows a list of increased GO terms, and (B) shows a list of decreased GO terms. (C) shows an expanded view of the GO terms listed in (A). (D) shows an expanded view of the GO terms listed in (B). Gene set enrichment analysis compared CFU-F and STAR MSCs.The enrichment scores for the cell cycle gene set (A, left) and the citric acid (TCA) cycle gene set (A, right) are shown. The enrichment scores for the cellular senescence gene set (B, left) and the p53 signaling pathway gene set (B, right) are shown. (A) The colony formation ability of STAR MSCs cultured in medium supplemented with 2-DG (a glucose metabolism inhibitor, 1 mM) and / or oligomycin (OligoM) (an oxidative phosphorylation pathway inhibitor, 1.5 μM) is shown. (B) The top 10 genes with increased expression among genes involved in cell adhesion that were highly expressed in STAR MSCs are shown. Figure 6 demonstrates the establishment of a STAR MSC culture method using 2-DG-supplemented medium. CFU-F cells (top row) were cultured in 20% FBS medium without 2-DG, and STAR MSCs (bottom row) were cultured in 20% FBS medium with 2-DG. Photographs were taken on days 7, 14, and 21.

[0015] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0016] [Method for Producing Mesenchymal Stem Cell Culture] In one embodiment, a method for producing a mesenchymal stem cell culture is provided. This method comprises the steps of (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starved conditions or in a 2-DG-containing medium, thereby isolating a culture of mesenchymal stem cells that survive the serum-starved conditions or the 2-DG-containing medium. In this method, the population of mesenchymal stem cells is not exposed to growth conditions between collection of the tissue-derived sample from an individual and culturing under serum-starved conditions or in a 2-DG-containing medium.

[0017] In the present disclosure, "mesenchymal stem cells" refers to non-blood cell stem cells or stromal cells that have the ability to self-renew and differentiate into mesenchymal cells, including at least adipocytes, osteocytes, and chondrocytes, as understood by those skilled in the art. Furthermore, a "culture" of cells refers to cells cultured in vitro. For example, a mesenchymal stem cell culture may contain mesenchymal stem cells, a medium composition for maintaining the mesenchymal stem cells, and secretions secreted from the mesenchymal stem cells. The medium composition may optionally contain components for cell maintenance known to those skilled in the art, such as a buffer system, inorganic salts, amino acids, nucleic acid bases, sugars, lipids, vitamins, trace elements, serum, growth factors, serum, serum replacements, growth factors, hormones, etc. Optionally, the medium composition may not contain any of these components. The serum used in the present disclosure may be any mammalian serum, as known to those skilled in the art. Preferably, fetal bovine serum (also known as fetal bovine serum (FBS) or fetal calf serum (FCS)) can be used.

[0018] A "tissue-derived sample" refers to a sample derived from an individual's tissue and isolated from the individual. The tissue-derived sample used in step (a) of the disclosed method for producing a mesenchymal stem cell culture may include samples derived from bone marrow, fat, umbilical cord, placenta, or dental pulp tissue. These tissues are known to those skilled in the art to contain mesenchymal stem cells. A sample derived from bone marrow tissue is particularly preferred. These tissue-derived samples can be collected from an individual by methods known to those skilled in the art. For example, bone marrow fluid collected from an individual by bone marrow aspiration, digested material from the umbilical cord or placenta released from an individual during childbirth, or dental pulp collected along with a tooth during extraction may provide a tissue-derived sample.

[0019] The tissue-derived sample may be a tissue-derived sample that has undergone various processes after collection from an individual. Examples of such processes include enzymatic treatment (e.g., collagenase treatment, protease treatment, etc. to digest connective tissue and separate individual cells), removal of contaminants (e.g., removal of bone fragments that may be contained in an animal bone marrow sample), and removal of non-interest cells or purification or enrichment of interest cells (e.g., lysis of red blood cells with purified water, isolation of mononuclear cells by density gradient separation, etc.). In the context of mesenchymal stem cells, the terms "purification," "enrichment," and "isolation," as commonly understood by those skilled in the art, refer to the selective or preferential recovery of a population of mesenchymal stem cells from a tissue-derived sample consisting of various cell types to enrich the percentage of mesenchymal stem cells. Alternatively, in some embodiments, a tissue-derived sample that has not been purified, enriched, or isolated may be directly transferred to culture under serum-starvation conditions, as the ability to survive serum-starvation conditions is substantially specific to STAR MSC cells.

[0020] In some embodiments, the method may include purifying mesenchymal stem cells from a tissue-derived sample by flow cytometry after collection of the tissue-derived sample from the individual and before culturing the sample under serum-starved or 2-DG-containing medium conditions. Techniques for isolating a population of mesenchymal stem cells by flow cytometry are known to those skilled in the art. Typically, a fluorescently labeled antibody against a cell surface marker protein is incubated with the tissue-derived sample to specifically fluorescently label cells expressing the marker protein gene. The fluorescently labeled cells can be separated from other cells using a flow cytometer. Marker genes for such selection may be genes known to those skilled in the art that are specifically expressed in mesenchymal stem cells. Alternatively, marker genes not expressed in mesenchymal stem cells may be used for negative selection. Non-limiting examples of such markers include LNGFR in humans. + , THY-1 + , VCAM-1 + , Pdgfrα in mice + , Sca-1 + , CD45 - , CD31- , Ter119 - In one embodiment, the human tissue-derived sample contains LNGFR + , THY-1 + , VCAM-1 + In one embodiment, mesenchymal stem cells are purified based on a combination of markers including Pdgfrα (Stem Cell Reports, Vol. 1, 152-165). + , Sca -1 + , CD45 - , Ter119 - Mesenchymal stem cells are purified based on a combination of markers including (J. Exp. Med., Vol. 206, No. 11, 2483-2496; Nature Protocols, Vol. 7, No. 12, 2012, 2103). Cells may also be selected using some of the genes that are highly or lowly expressed in STAR MSCs, as described below. Examples of such genes include Bmp4, Fgfr2, Pparg, Alcam, CD44, and Fgf2.

[0021] As the tissue-derived sample, a mammalian tissue-derived sample can be used, and it is preferable to use a sample derived from human tissue.

[0022] In this technical field, mesenchymal stem cells are typically cultured in a medium containing serum such as fetal bovine serum or serum substitutes known to those skilled in the art. "Serum starvation" conditions refer to conditions in which cells are placed for a certain period of time in a medium environment in which the concentration of serum and serum substitutes has been reduced or removed to the point where cell growth is inhibited and cell death occurs, thereby causing the death of the majority (at least half) of the cells in the tissue-derived sample.

[0023] In the present disclosure, serum-starved conditions can refer to culture conditions in a medium that does not contain either serum or serum substitute, or whose total content does not exceed 0.002 wt%, 0.02 wt%, 0.1 wt%, 0.2 wt%, or 0.4 wt%. As known to those skilled in the art, a "serum substitute" is a composition that can replace serum in cell culture and can contain one or more components selected from hormones, growth factors, proteins, lipids, vitamins, amino acids, trace elements, and sugars necessary for maintaining cell culture. The serum substitute can be a composition that contains no or only trace amounts (<1 wt%) of animal-derived components.

[0024] Embodiments are also contemplated in which 2-deoxy-D-glucose (2-DG)-containing media conditions are used instead of or in addition to serum starvation conditions. As known to those skilled in the art, 2-DG is an inhibitor of glucose metabolism (glycolysis). While mesenchymal stem cell cultures are typically performed in a medium environment where cells can utilize glucose metabolism as their primary energy source, 2-DG-containing media conditions refer to conditions in which the medium contains 2-DG, such that the majority of cells in a tissue-derived sample die or their growth is inhibited, while 2-DG-resistant mesenchymal stem cells are selectively allowed to survive or proliferate. Culturing a tissue-derived sample in 2-DG-containing media conditions can yield a culture enriched or isolated for a population of mesenchymal stem cells that survive by utilizing the oxidative phosphorylation pathway, corresponding to a culture of mesenchymal stem cells that survive serum starvation conditions. The concentration of 2-DG in the 2-DG-containing media conditions can be, for example, within the range of 0.5 to 20 mM, or even within the range of 0.7 to 10 mM or 1 to 5 mM.

[0025] Step (a) involves isolating the culture of mesenchymal stem cells that survive serum starvation or 2-DG-containing medium conditions. Isolating the surviving mesenchymal stem cells can include removing cells that died under serum starvation or 2-DG-containing medium conditions from the cell culture. One or more medium changes may be performed for this isolation. Typically, live mesenchymal stem cells remain attached to the culture substrate, while dead cells detach from the culture substrate and float in the medium, allowing for easy isolation of the surviving mesenchymal stem cells. Alternatively, isolation of the surviving mesenchymal stem cells may be performed using a cell sorter such as a flow cytometer.

[0026] The period for culturing a tissue-derived sample containing a mesenchymal stem cell population under serum-starved conditions is the period during which most cells die, based on the definition of serum-starved conditions, but the exact length is not particularly limited. The period can be 3 days or more, 1 week or more, or 1 month or more. The period can be, for example, 2 months or less, or 1 month or less. The period is preferably 3 days or more, and preferably 2 weeks or less. STAR MSCs have been found to be able to survive under serum-starved conditions for extended periods. During these periods, fresh medium (although still containing reduced or eliminated serum and serum substitutes) may be substituted. The period for culturing a tissue-derived sample under 2-DG-containing medium conditions is similar, and can be, for example, 1 week to 2 months.

[0027] The disclosed method for producing a mesenchymal stem cell culture can be characterized in that the mesenchymal stem cell population is not exposed to proliferation conditions between collection of a tissue-derived sample from an individual and culture in serum-starved or 2-DG-containing medium conditions (i.e., before the transition to culture in serum-starved or 2-DG-containing medium conditions). This facilitates the isolation of STAR MSC cultures with distinctive gene expression profiles and metabolic characteristics that contrast with conventional mesenchymal stem cell cultures, as described below. Compared to conventional mesenchymal stem cell cultures, STAR MSC cultures can offer one or more advantages selected from stem cell purity, the ability to maintain an undifferentiated state, suppression of senescence, etc. Without being bound by theory, repeated cell division due to active proliferation in nutrient-rich environments can cause the adverse effects of cellular senescence (e.g., telomere shortening) and can also amplify the adverse effects of contaminating cells in the culture. The disclosed method for producing a mesenchymal stem cell culture can establish a pure and unsenescent mesenchymal stem cell culture before such adverse effects occur. Furthermore, since the method does not include the pre-culture step that is performed in conventional methods for preparing mesenchymal stem cell cultures, such as those described in Patent Document 2, pure mesenchymal stem cells can be provided quickly and at low cost.

[0028] The method for producing a mesenchymal stem cell culture disclosed herein may include a step (b) of expanding the mesenchymal stem cell culture separated in step (a) by exposing it to growth conditions. In this disclosure, "growth conditions" with respect to a cell population refer to environmental and temporal requirements under which a cell population is placed in a culture medium environment that allows or promotes cell proliferation for a certain period of time, resulting in cell population doubling. "Exposing a cell population to growth conditions" means that the cell population satisfies the growth conditions. Such growth conditions may be, for example, culture conditions in a medium containing more than 2%, 5% or more, 10% or more, 15% or more, or 20% or more serum or serum substitute by weight. The concentration of serum or serum substitute is typically 30% by weight or less. These descriptions of "growth conditions" apply not only to the "growth conditions" in step (b) above, but also to the growth conditions in the case where a mesenchymal stem cell population is "not exposed to growth conditions" between collection of a tissue-derived sample from an individual and culture in serum-starved conditions or 2-DG-containing medium conditions, as described above.

[0029] In certain embodiments, for example, when serum starvation conditions are used in step (a), the medium used in the growth conditions in step (b) may contain 2-deoxy-D-glucose. For example, in addition to the serum or serum replacement described above, the medium may contain 0.1 to 10 mM or 0.5 to 2 mM 2-deoxy-D-glucose. Adding 2-deoxy-D-glucose to the medium can promote the maintenance of a population of surviving and proliferating mesenchymal stem cells by utilizing oxidative phosphorylation.

[0030] [Mesenchymal Stem Cell Culture] In another aspect of the present disclosure, there is provided a mesenchymal stem cell culture produced by the above-described method, i.e., comprising the steps of (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starved conditions or in a 2-DG-containing medium, thereby isolating a culture of mesenchymal stem cells that survive the conditions, wherein the population of mesenchymal stem cells is not exposed to growth conditions between the time of collection of the tissue-derived sample from an individual and the time of culturing under the conditions. The explanations provided in connection with the above-described embodiment of the production method can be applied to various elements of this mesenchymal stem cell culture (including the cells used for production and their origin, medium components, serum-starved conditions, growth conditions, culture period, etc.).

[0031] The mesenchymal stem cell cultures of this embodiment may exhibit (i) increased expression of one or more genes selected from Bmp4 and Fgfr2, (ii) decreased expression of one or more genes selected from Pparg, Alcam, CD44, and Fgf2, or both (i) and (ii) compared to corresponding cultures cultured under proliferation conditions instead of serum-starved or 2-DG-containing media conditions. The expression levels may refer to expression levels at the mRNA level or protein level, with expression levels at the mRNA level being preferred. Methods for determining a significant increase or decrease in expression levels are known to those skilled in the art, and may include, for example, a fold change of 1.4-fold or greater or a fold change of 2-fold or greater.

[0032] Generally, "colony-forming unit-fibroblast (CFU-F)" refers to a population of fibroblasts resulting from the differentiation of stem cells, such as mesenchymal stem cells, and the number of colonies is understood by those skilled in the art to reflect the number of mesenchymal stem cells contained in the original cell population. In contrast, except for this paragraph, the term "CFU-F" is used throughout the present disclosure to refer to a population of "mesenchymal stem cells capable of differentiating into CFU-F." Furthermore, the term CFU-F (20% FBS) refers to a population of mesenchymal stem cells capable of differentiating into CFU-F, which are cultured under growth conditions containing 20% ​​FBS after a tissue-derived sample is isolated from an individual, without being cultured under serum-starved conditions (or 2-DG-containing medium conditions). CFU-F (20% FBS) is used as a control in the following examples of the present application.

[0033] Furthermore, STAR MSCs refer to mesenchymal stem cells that can be obtained by the method for producing a mesenchymal stem cell culture in the present disclosure, and STAR MSCs (0.2% FBS) refer to mesenchymal stem cells obtained from STAR MSCs by culturing them in a medium containing 0.2% FBS as the serum-starved condition in step (a).

[0034] [Pharmaceutical Composition] In one aspect of the present disclosure, there is provided a pharmaceutical composition comprising a mesenchymal stem cell culture produced by the above-described method, i.e., the method comprising the steps of: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starvation conditions or 2-DG-containing medium conditions, thereby isolating a culture of mesenchymal stem cells that survive said conditions, wherein said population of mesenchymal stem cells is not exposed to growth conditions after collection of the tissue-derived sample from an individual until culturing under said conditions.

[0035] The components of this pharmaceutical composition are applicable to the explanations provided in relation to the above-mentioned manufacturing method and mesenchymal stem cell culture embodiments. The subject of administration of this embodiment is a mammal, preferably a human, monkey, dog, cat, cow, pig, sheep, or horse, and more preferably a human. In addition to mesenchymal stem cells, the pharmaceutical composition of this embodiment may contain a pharmaceutically acceptable carrier or excipient. Carriers and excipients capable of maintaining cells in a viable state are known to those skilled in the art. The pharmaceutical composition of this embodiment is typically provided in the form of an aqueous suspension.

[0036] [Method for Producing a Mesenchymal Cell Population] One embodiment of the present disclosure provides a method for producing a mesenchymal cell population, which method comprises the above-described method, i.e., (a) culturing a tissue-derived sample containing a mesenchymal stem cell population under serum-starved conditions or 2-DG-containing medium conditions, thereby isolating a culture of mesenchymal stem cells that survive the conditions, wherein the mesenchymal stem cell population is not exposed to growth conditions between the time of collection of the tissue-derived sample from an individual and the time of culturing under the conditions, and further differentiating the mesenchymal stem cell culture into mesenchymal cells in vitro.

[0037] For the elements of the method for producing this population of mesenchymal cells, the explanations provided in relation to the embodiments of the production method, mesenchymal stem cells and pharmaceutical composition above are applicable.

[0038] In the method for producing a mesenchymal cell population disclosed herein, differentiating a mesenchymal stem cell culture into mesenchymal cells in vitro comprises differentiating the mesenchymal stem cells obtained by the above-described production method into mesenchymal cells by a method known to those skilled in the art. The mesenchymal cells are preferably adipocytes, osteoblasts, or chondrocytes, but may also be other mesenchymal differentiated cells.

[0039] Techniques for differentiating mesenchymal stem cells into mesenchymal cells are known to those skilled in the art. In the method for producing a mesenchymal cell population disclosed herein, differentiation into adipocytes can be achieved by culturing mesenchymal stem cells in a differentiation medium such as an adipocyte differentiation-inducing medium (#PT-3004, Lonza). Differentiation into adipocytes can be confirmed by staining methods known to those skilled in the art using dyes such as Oil Red O. In the method for producing a mesenchymal cell population disclosed herein, differentiation into osteoblasts can be achieved by culturing mesenchymal stem cells in a differentiation medium such as an osteoblast differentiation-inducing medium (#PT-3002, Lonza). Differentiation into osteoblasts can be confirmed by staining methods known to those skilled in the art using dyes such as Alizarin Red. In the method for producing a mesenchymal cell population disclosed herein, differentiation into chondrocytes can be achieved by culturing mesenchymal stem cells in a differentiation medium such as a chondrocyte differentiation-inducing medium (#PT-3003, Lonza). Differentiation into chondrocytes can be confirmed by staining methods known to those skilled in the art with dyes such as Toluidine Blue.

[0040] In this specification, numerical ranges expressed using the expression "A to B" (or "from A to B" or "between A and B") indicate ranges that include the recited numerical values ​​A and B as the minimum and maximum values, respectively, and also encompass ranges excluding those minimum and / or maximum values. Furthermore, expressions such as "A to B" are understood to provide literal basis for ranges such as "greater than or equal to A" or "greater than A" and ranges such as "less than or equal to B" or "less than B." When multiple numerical ranges are described, the upper or lower limit of one recited numerical range can be combined with the upper or lower limit of another recited numerical range, and the resulting numerical ranges are also understood to be disclosed in this specification. In this specification, the expression "comprising A" or "having A" means, unless the context indicates otherwise, an object that includes both A and other elements, and also encompasses the literal basis for an object that includes only A and nothing other than A, i.e., an object consisting of A, and vice versa. Reference to a singular object is understood to also encompass disclosure of embodiments in which a plural object is used.

[0041] Examples of the present invention will be described below, but the embodiments of the present invention are not limited to the specific examples shown below.

[0042] Materials and Methods 1. Isolation and Culture of Mesenchymal Stem Cells (MSCs) MSCs were isolated essentially as described in Nature Protocols, Vol. 7, No. 12, 2012, 2103. Tissue samples collected from the bone marrow of the femurs and tibias of 6-week-old male mice (C57BL / 6 J) were placed in 0.2% collagenase solution and treated with collagenase by shaking at 37°C, 110 rpm, and 60 minutes. Bone fragments were removed using a 70 μm filter (BD Falcon), and red blood cells were lysed and removed using sterile water. The resulting cells were suspended in HBSS+ and stained with antibodies against CD45, CD31, Ter119, Sca-1, and PDGFRα for 30 minutes on ice. CD45(-), CD31(-), Ter119(-), Sca-1(+), and Pdgfrα(+) cells (PαS cells) were isolated using a flow cytometer. The isolated samples were tissue-derived samples purified for MSCs. These tissue-derived samples were seeded into culture dishes filled with MSC medium containing 0.2% or 20% FBS. Culture in 0.2% FBS provided serum-starvation conditions. Between collection of bone marrow-derived samples from mice and seeding into the culture dishes, the MSC samples were not exposed to growth conditions. The MSC medium, containing the same FBS concentration as above, was replaced every 3–4 days. Similarly, 2,000 PαS cells were sorted and cultured under serum-starvation conditions, followed by colony-forming unit-fibroblast (CFU-F) analysis and differentiation potential analysis. The compositions of the 0.2% collagenase solution, HBSS+, and MSCs medium used in the isolation and culture of the above-mentioned mesenchymal stem cells (MSCs) were as follows.・0.2% collagenase solution: 20 mL DMEM (Dulbecco's modified Eagle's medium), 10 mM HEPES, 1% penicillin-streptomycin, 0.2% collagenase, 500 U DNase I, 4 ng / mL DISPASE II. ・HBSS+ (calcium- and magnesium-free Hank's-balanced salt solution): 500 mL DMEM, 2% FBS, 10 mM HEPES, 1% penicillin-streptomycin. ・MSCs medium: DMEM glutamax (#10569-010, Gibco), 0.2 or 20% FBS, 5 μg / mL bFGF (basic fibroblast growth factor), 1% penicillin-streptomycin.

[0043] 2. Colony Formation Analysis To analyze colony formation ability, cells were cultured in 100 mm dishes for 14 days in the standard mesenchymal stem cell medium with the following composition. The cultured cells were fixed with 4% paraformaldehyde for 10 minutes, stained with the crystal violet staining solution with the following composition for 20 minutes at room temperature, washed with PBS, and the number of colonies containing 50 or more cells was counted. Mesenchymal stem cell standard medium: DMEM glutamax (#10569-010, Gibco), 20% FBS, 5 ng / mL b-FGF (basic fibroblast growth factor), 1% penicillin-streptomycin, 1% HEPES. Crystal violet staining solution: 10 mg / mL crystal violet, 50% (v / v) methanol, 50% (v / v) 4% paraformaldehyde.

[0044] 3. Induction of adipocyte differentiation: 2.0 × 10 cells were cultured in a 24-well plate. 5 Cells were seeded at a density of 100 cells / well and cultured until confluent. Once 100% confluent, differentiation was induced using adipogenic induction medium (#PT-3004, Lonza). Differentiation was induced using two cycles of induction medium / maintenance medium. Each cycle consisted of 4 days of culture in adipogenic induction medium followed by 3 days of culture in maintenance medium. Culture was performed in a 37°C, 5% CO2 incubator. CD73-positive cells differentiated into adipocytes were stained with Oil Red O. Specifically, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed three times with PBS, and treated with 60% isopropyl alcohol for 30 seconds at room temperature. Staining was performed for 30 minutes at room temperature in the dark using a staining solution made by mixing Oil Red O staining stock solution for adipogenic staining with ultrapure water at a 3:2 ratio. After staining, cells were treated with 60% isopropyl alcohol for 30 seconds at room temperature and washed three times with PBS. Cells with red-stained lipid droplets were analyzed as adipocytes.

[0045] 4. Induction of osteoblast differentiation: 5.0 × 10 cells were cultured in a 24-well plate. 4 Cells were seeded at a density of 1000 cells / well. After 3 days of culture, they were cultured in osteoblast differentiation medium (#PT-3002, Lonza) (containing basal medium, dexamethasone, L-glutamine, ascorbate, penicillin / streptomycin, MCGS, and β-glycerophosphate) at 37°C in a 5% CO2 incubator for 14 days. The medium was replaced with osteoblast differentiation medium every 3–4 days. CD73-positive cells differentiated into osteoblasts were stained with Alizarin Red. Specifically, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and washed three times with PBS. Then, cells were stained with Alizarin Red Solution from the Osteogenesis Quantitation Kit (#ECM815, Merck Millipore Corporation) for 20 minutes at room temperature. After staining, the cells were washed four times with ultrapure water. Osteoblast differentiation was quantitatively analyzed using red-stained calcium deposits.

[0046] 5. Induction of chondrocyte differentiation: 1.0 x 10 cells were placed in a 15 mL polypropylene tube. 6 Cells were placed in a tube containing 1000 cells / tube. The tube was centrifuged at 309 g for 5 minutes at 4°C. After centrifugation, the supernatant was removed and the cells were resuspended in chondrocyte differentiation medium (#PT-3003, Lonza) (containing chondrocyte basal medium, dexamethasone, ascorbate, ITS supplement, GA-1000, sodium pyruvate, proline, L-glutamine, 10 ng / ml TGF-β3, and 500 ng / ml recombinant human BMP-6). The cells were then centrifuged at 150 g for 5 minutes at 4°C. After centrifugation, the tube cap was half-loosened to allow for CO2 gas exchange, and the cells were cultured in a 37°C, 5% CO2 incubator for 21 days. The chondrocyte differentiation medium was replaced every 3–4 days. The CD73-positive cells differentiated into chondrocytes were embedded in paraffin and stained with Toluidine Blue.

[0047] 6. Analysis of STAR MSC Viability by Metabolic Inhibitors. Two thousand PαS cells were sorted from mouse femurs and cultured in 0.2% FBS medium in 100 mm dishes. Cells were treated with one of the following additives: (1) 2-DG (a glucose metabolism inhibitor, 1 mM); (2) oligomycin (an oxidative phosphorylation pathway inhibitor, 1.5 μM); (3) 2-DG (1 mM) and oligomycin (1.5 μM); or (4) no additive. After one week of culture under these conditions, cell proliferation was stimulated by adding 20% ​​FBS medium. The formed colonies were counted to estimate and compare cell viability after each treatment (1)–(4).

[0048] Example 1 Mesenchymal stem cells (MSCs) samples are generally cultured in a medium containing a high concentration (10%-20%) of serum, and cell proliferation is promoted under these culture conditions. However, the culture conditions with high serum concentrations are significantly different from the in vivo environment, which leads to differences in gene expression and cell properties. The present inventors isolated MSCs (PDGFRα) from the bone marrow (BM) of adult mice. + SCA-1 +We isolated MSCs (cells) and then cultured them for one week in normal serum (20%) or in a medium with a serum concentration 100-fold lower (0.2%) (serum-starved conditions) (Fig. 1A). While cells proliferated in the normal serum (20%) culture (Fig. 1B, "FBS 20%), the MSCs cultured in the low-serum (0.2%) medium showed virtually no proliferation and the majority of cells died. However, a small number of cells appeared to survive the low-serum conditions (Fig. 1B, "FBS 0.2%"; a magnified image of the boxed single cell is shown in the lower left).

[0049] After culturing in low serum, the medium was replaced with a normal high-serum medium (20%), which stimulated the proliferation of the few surviving MSCs (Figure 1B, "FBS 0.2% → 20%"). Quantification of colony-forming cells revealed that approximately 1 / 7th of the colony-forming units (CFU-F) survived under this specific low-serum condition compared with the colony-forming units (CFU-F) in normal culture (Figure 2A). Cells that survived under low-serum conditions (serum starvation conditions) were named STAR MSCs, after the CFU-Fs that could withstand starvation. STAR MSCs exhibited MSC characteristics, including the ability to self-renew and differentiate into adipocytes, chondrocytes, and osteoblasts (Figure 2B). Similar experiments confirmed that STAR MSCs could survive under serum-starvation conditions for more than one month and could be isolated from human tissues (data not shown).

[0050] Example 2: We extracted mRNA from CFU-F (20% serum) and STAR MSCs (0.2% serum) and performed comprehensive gene expression analysis using microarrays. Comparing the two cultures, we identified 199 genes that showed significantly higher expression (fold change of 2-fold or greater) in cells cultured at low serum concentrations and 492 genes that showed significantly higher expression (fold change of 2-fold or greater) in cells cultured at high serum concentrations (Figure 2C). STAR MSCs showed elevated expression of Bmp4 and Fgfr2, which are important for development, and decreased expression of genes related to the differentiation and maturation of MSCs (Pparg, Alcam, CD44, and Fgf2) (Figure 2C).

[0051] Gene ontology (GO) analysis was performed to examine the gene functions of genes whose expression levels were confirmed to change by more than two-fold between CFU-F (20% serum) and STAR MSCs (0.2% serum) (Fig. 3). As a result, metabolism-related GO terms were significantly increased in STAR MSCs (Fig. 3A, C), while GO terms related to the cell cycle were decreased (Fig. 3B, D).

[0052] Gene set enrichment analysis (GSEA analysis) using methods known to those skilled in the art showed that the expression correlation of genes related to the cell cycle (Figure 4A, left) and citric acid cycle (TCA cycle) (Figure 4A, right) was reduced in STAR MSCs compared to CFU-Fs. On the other hand, STAR MSCs showed lower expression levels of gene sets related to cellular senescence (Figure 4B, left) and the p53 signaling pathway (Figure 4B, right) than CFU-Fs. These results suggest that STAR MSCs maintain an undifferentiated state and suppress senescence compared to CFU-Fs.

[0053] We observed that STAR MSCs exhibited decreased expression of genes related to the citric acid cycle and increased expression of genes related to the oxidative phosphorylation pathway. Therefore, we cultured STAR MSCs with 2-deoxy-D-glucose (2-DG) (a glucose metabolism inhibitor) and / or oligomycin (an oxidative phosphorylation pathway inhibitor). The results showed that inhibition of glucose metabolism by 2-DG alone had little effect on STAR MSC survival (Figure 5A, left). However, inhibition of the oxidative phosphorylation pathway by oligomycin alone induced cell death and significantly reduced viability (Figure 5A, center). Simultaneous inhibition of both glucose metabolism and oxidative phosphorylation resulted in substantial loss of STAR MSC viability (Figure 5A, right). These findings suggest that STAR MSCs utilize the oxidative phosphorylation pathway to survive serum starvation.

[0054] Figure 5B shows an expression heatmap of the top 10 genes with increased expression among the cell adhesion-related genes that were upregulated in STAR MSCs. High expression of Icam1, Itgb8, and CD274 (PD-L1; involved in immune evasion) genes is shown.

[0055] Example 3 Next, the inventors conducted an experiment to verify whether STAR MSCs could be expanded while maintaining their properties. Mesenchymal stem cells were cultured for 3 weeks in a 20% FBS-containing medium supplemented with 2-DG (which inhibits the glycolytic pathway). The results showed that STAR MSCs cultured in the presence of 2-DG proliferated, albeit at a slower rate, than CFU-Fs cultured in the absence of 2-DG (Figure 6). The morphology of STAR MSCs was different from that of CFU-Fs, being uniform cells with small cell bodies (Figure 6). 2-DG could be used for both the selection and maintenance of STAR MSCs.

[0056] Embodiments of the present invention include the following: [Item 1] A method for producing a mesenchymal stem cell culture, comprising the step of: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starved conditions or in a 2-deoxy-D-glucose (2-DG)-containing medium condition, thereby isolating a culture of mesenchymal stem cells that survive the serum-starved conditions or the 2-DG-containing medium condition, wherein the population of mesenchymal stem cells has not been exposed to growth conditions between collection of the tissue-derived sample from an individual and culturing under the serum-starved conditions or the 2-DG-containing medium condition. [Item 2] The method of Item 1, wherein the serum-starved conditions are culture conditions in a medium that does not contain either serum or serum substitute, or whose total content does not exceed 0.4% by weight. [Item 3] The method of Item 1 or 2, wherein the culturing under the serum-starved conditions or the 2-DG-containing medium condition is carried out for three days or more, one week or more, or one month or more. [Item 4] The method of any one of Items 1 to 3, wherein the tissue-derived sample comprises a sample derived from bone marrow, adipose, umbilical cord, placenta, or dental pulp tissue. [Item 5] The method of any one of Items 1 to 4, wherein the tissue-derived sample is a sample derived from human tissue. [Item 6] The method of any one of Items 1 to 5, comprising purifying the tissue-derived sample for mesenchymal stem cells by flow cytometry after collection of the tissue-derived sample from the individual and before culturing under serum-starvation conditions or in a 2-DG-containing medium condition. [Item 7] The method of any one of Items 1 to 6, further comprising a step (b) of expanding the culture of mesenchymal stem cells separated in step (a) by exposing them to growth conditions. [Item 8] The method of Item 7, wherein the growth conditions in step (b) are culture conditions in a medium containing more than 2% by weight of serum or serum substitute. [Item 9] The method of Items 7 or 8, wherein the medium used in the growth conditions in step (b) contains 2-deoxy-D-glucose. [Item 10] A mesenchymal stem cell culture produced by the method of any one of Items 1 to 9. [Item 11] A pharmaceutical composition comprising the mesenchymal stem cell culture of Item 10. [Item 12] A method for producing a population of mesenchymal cells, the method comprising differentiating in vitro the mesenchymal stem cell culture produced by the method of any one of Items 1 to 9 into mesenchymal cells.[Item 13] The method of Item 12, wherein the mesenchymal cells include adipocytes, osteoblasts, or chondrocytes.

[0057] Although the present invention has been described with reference to the above several embodiments, the present invention is not limited to the above individual embodiments. Those skilled in the art who have read this disclosure will understand that various changes can be made to the configuration and details of the present invention within the spirit and scope of the present invention.

Claims

1. A method for producing a mesenchymal stem cell culture, comprising the steps of: (a) culturing a tissue-derived sample containing a population of mesenchymal stem cells under serum-starvation conditions or in a medium containing 2-deoxy-D-glucose (2-DG), thereby isolating a culture of mesenchymal stem cells that survive the serum-starvation conditions or the 2-DG-containing medium conditions; wherein the population of mesenchymal stem cells is not exposed to proliferation conditions between collection of the tissue-derived sample from an individual and culturing in the serum-starvation conditions or the 2-DG-containing medium conditions.

2. The method according to claim 1, wherein the serum-starved conditions are culture conditions in a medium that does not contain either serum or serum substitutes, or the total content of serum and serum substitutes does not exceed 0.4% by weight.

3. The method according to claim 1, wherein the culture under serum starvation conditions or 2-DG-containing medium conditions is carried out for 3 days or more, 1 week or more, or 1 month or more.

4. The method of claim 1, wherein the tissue-derived sample comprises a sample derived from bone marrow, adipose, umbilical cord, placenta, or dental pulp tissue.

5. The method of claim 1, wherein the tissue-derived sample is a sample derived from human tissue.

6. The method of claim 1, comprising purifying the tissue-derived sample for mesenchymal stem cells by flow cytometry after collection of the tissue-derived sample from the individual and prior to culturing in the serum-starved or 2-DG-containing medium conditions.

7. The method of claim 1, further comprising a step (b) of expanding the culture of mesenchymal stem cells isolated in step (a) by exposing them to growth conditions.

8. The method according to claim 7, wherein the growth conditions in step (b) are culture conditions in a medium containing more than 2% by weight of serum or serum substitute.

9. The method of claim 7, wherein the medium used in the growth conditions of step (b) contains 2-deoxy-D-glucose.

10. A mesenchymal stem cell culture produced by the method according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising the mesenchymal stem cell culture of claim 10.

12. A method for producing a population of mesenchymal cells, comprising differentiating in vitro a mesenchymal stem cell culture produced by the method of any one of claims 1 to 9 into mesenchymal cells.

13. The method of claim 12, wherein the mesenchymal cells comprise adipocytes, osteoblasts, or chondrocytes.

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