Patent Description:
Mesenchymal stem cells, also called mesenchymal stromal cells, are somatic stem cells reported to exist in the bone marrow, adipose tissues, tooth pulp and the like. Recently, it has been revealed that these cells also exist in fetal appendages including the placenta, umbilical cord, and fetal membrane. Since mesenchymal stem cells are capable of differentiating into bones, cartilage, and fats, etc., mesenchymal stem cells have been gaining attention as a promising cell source in regenerative medicine.

In addition, since mesenchymal stem cells have immunosuppressive capacity as well as differentiation capacity, it has been reported that intravenous administration thereof enables treatment of immune-related diseases, inflammatory diseases and the like.

Patent Literature <NUM> describes a method for producing an amniotic mesenchymal cell composition, a method for cryopreserving the composition, and a therapeutic agent. Particularly, this literature discloses that cryopreserved amniotic mesenchymal cells can be produced as a cell preparation optimized for transplantation, by cryopreserving a mixture comprising amniotic mesenchymal cells in a solution containing <NUM> to <NUM>% by mass of dimethyl sulfoxide and containing <NUM> to <NUM>% by mass of hydroxyethyl starch or <NUM> to <NUM>% by mass of dextran.

Patent Literature <NUM> describes a method for preparing an amniotic mesenchymal stem cell population, comprising the steps of: (D) collecting a cell population of mesenchymal cells from the amnion of a mammal; (E) inoculating the collected cell population at a cell concentration of <NUM> to <NUM> cells/cm<NUM>, followed by initial culture for <NUM> to <NUM> days; (F) inoculating the cultured cells at <NUM>/<NUM> or more and less than <NUM>/<NUM> of the cell concentration of the initial culture, and repeating subculture three to four times with medium replacement twice a week; and (G) maintaining the culture of the cells in the same culture dish until confluence when a colony of cells having a fusiform shape is formed in the subculture.

In Patent Literature <NUM>, proteome analysis of human mesenchymal stem cells has confirmed that human mesenchymal stem cells secrete COL11A1 and COL16A1. <CIT> discloses a method for producing amniotic mesenchymal stromal cell composition, method for cryopreserving the same and a therapeutic agent.

In recent years, it has been found that mesenchymal stem cells derived from a fetal appendage are a heterogeneous cell population comprising various cells having different differentiation capacities, proliferative capacities, and cytokine producing capacities. For producing a cell preparation with a stable quality, it is necessary to prepare a purified and highly homogenous cell population. In addition, it has been pointed out that when a cell suspension comprising mesenchymal stem cells is intravenously administered, there is a risk that a cell aggregate consisting of the administered mesenchymal stem cells clogs a capillary vessel or a vessel thicker than that and thereby causes an embolus. Thus, from the viewpoint of improving the safety as a cell therapy agent, it is necessary to obtain mesenchymal stem cells having lower cell aggregability.

Patent Literature <NUM> discloses that cryopreserved amniotic mesenchymal cells can be produced as a cell preparation optimized for transplantation, by cryopreserving a mixture comprising amniotic mesenchymal cells in a particular cryopreservation solution to prevent a decrease in the survival rate of amniotic mesenchymal cells after thawing. However, this literature makes no mention about the selective preparation of mesenchymal stem cells having a particular excellent feature from among mesenchymal stem cells, specifically, the selective preparation of a cell population rich in mesenchymal stem cells having low cell aggregability, which is useful for intravenous administration of a cell preparation by utilizing the characteristics of mesenchymal stem cells as indices. Further, in Patent Literature <NUM>, a mesenchymal stem cell population having high proliferative capacity and differentiation capacity is prepared by inoculating cells at a low density. However, this literature neither describes nor suggests a cell population rich in mesenchymal stem cells having low cell aggregability by utilizing characteristics of mesenchymal stem cells comprised in a mesenchymal stem cell population as indices.

Patent Literature <NUM> describes that mesenchymal stem cells secrete COL11A1 and COL16A1. However, Patent Literature <NUM> does not describe expression intensities of COL11A1 and COL16A1, and it neither describes nor suggests selectively preparing a cell population rich in mesenchymal stem cells having low cell aggregability by using characteristics of mesenchymal stem cells as indices.

The present invention provides a method for producing a cell population comprising mesenchymal stem cells, the method comprising obtaining a cell population having the following cell characteristics:.

As a result of intensive studies in order to solve the above object, the present inventors have found that a cell population comprising cells collected from fetal appendages comprises mesenchymal stem cells having low expression levels of COL11A1 gene and COL16A1 gene, and further found that cell aggregation is reduced in a cell population comprising mesenchymal stem cells having the above cell characteristics. The present invention as defined by the claims has been completed on the basis of these findings.

According to the present invention as defined by the claims, a cell population comprising mesenchymal stem cells having low cell aggregability can be obtained. In addition, according to the present invention as defined by the claims, relative expression levels of various genes to housekeeping genes can be used as indices for formation of a cell population comprising mesenchymal stem cells having low cell aggregability. This enables an efficient production of a cell preparation (pharmaceutical composition) useful for intravenous administration.

as defined by the claims Embodiments of the present invention will be specifically described below. The descriptions below are intended to facilitate understanding of the present invention as defined by the claims.

The term "fetal appendage" used herein refers to a fetal membrane, a placenta, an umbilical cord, and amniotic fluid. In addition, the term "fetal membrane" refers to a fetal sac containing fetal amniotic fluid, which consists of an amnion, a chorion, and a decidua in that order from the inside. Among them, the amnion and the chorion are originated from the fetus. The term "amnion" refers to a transparent thin membrane with few blood vessels, which is located in the most inner layer of the fetal membrane. The inner layer (also called epithelial cell layer) of the amnion is covered with a layer of epithelial cells having a secretory function and secretes amniotic fluid. The outer layer (also called extracellular matrix layer, which corresponds to the stroma) of the amnion comprises mesenchymal stem cells.

The term "mesenchymal stem cells (MSCs)" used herein refers to stem cells that satisfy definitions described below, and are used interchangeably with "mesenchymal stromal cells. " The term "mesenchymal stem cells" is also referred to as "MSCs" in the present specification.

The term "mesenchymal stem cell population" used herein means a cell population comprising mesenchymal stem cells. Examples of the form thereof include, but are not particularly limited to, cell pellets, cell aggregates, cell-floated liquids and cell suspensions.

The term "amniotic mesenchymal stem cells" used herein refers to mesenchymal stem cells derived from the amnion, and are used interchangeably with "amniotic mesenchymal stromal cells. " The term "amniotic mesenchymal stem cells" used herein is also referred to as "amniotic MSCs.

The term "cell aggregation" used herein means that a plurality of single cells are adhered to one another to form a group. Cell aggregation can be evaluated, for example, by observation using a microscope, or by cell counting using a cell counter, aggregation rate measurement, size distribution measurement or the like.

The phrase "proportion of CDH6-positive mesenchymal stem cells" used herein refers to the proportion of cells positive for the surface antigen analyzed by flow cytometry as described in Examples mentioned later. The phrase "proportion of CDH6-positive mesenchymal stem cells" used herein is also referred to as "positive rate.

The cell population comprising mesenchymal stem cells provided by the present invention as defined by the claims is a cell population having the following cell characteristics:
the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less.

Satisfying the above conditions allows formation of a cell population comprising mesenchymal cells having low cell aggregability. Thus, in the present invention as defined by the claims, the conditions can be used as indices for the formation of a cell population having low cell aggregability.

The upper limit of the relative expression level of COL11A1 gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. Also, the lower limit of the relative expression level of COL11A1 gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

The upper limit of the relative expression level of COL16A1 gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. Also, the lower limit of the relative expression level of COL16A1 gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

According to one aspect of the present invention as defined by the claims, the cell population comprising mesenchymal stem cells may satisfy the relative expression level of COL4A5 gene to the expression level of SDHA gene of <NUM> or less.

The upper limit of the relative expression level of COL4A5 gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. Also, the lower limit of the relative expression level of COL4A5 gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

According to one aspect of the present invention as defined by the claims, the cell population comprising mesenchymal stem cells satisfies any one or more of: the relative expression level of VCAN gene to the expression level of SDHA gene of <NUM> or less; the relative expression level of DCN gene to the expression level of SDHA gene of <NUM> or less; and the relative expression level of LUM gene to the expression level of SDHA gene of <NUM> or less; preferably satisfies any two or more of the above; and more preferably satisfies all of the above.

The upper limit of the relative expression level of VCAN gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. The lower limit of the relative expression level of VCAN gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

The upper limit of the relative expression level of DCN gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. The lower limit of the relative expression level of DCN gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

The upper limit of the relative expression level of LUM gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. The lower limit of the relative expression level of LUM gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

According to one aspect of the present invention as defined by the claims, the cell population comprising mesenchymal stem cells may satisfy the relative expression level of GPC4 gene to the expression level of SDHA gene of <NUM> or less.

The upper limit of the relative expression level of GPC4 gene to the expression level of SDHA gene may be <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less or <NUM> or less. In addition, the lower limit of the relative expression level of GPC4 gene to the expression level of SDHA gene may be <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more.

As a method for measuring the relative expression level of each gene to the expression level of SDHA gene, measurement using a microarray can be used. Microarray can be performed specifically by the following procedures (<NUM>) to (<NUM>). The following procedures (<NUM>) to (<NUM>) can be entrusted to and performed by RIKEN GENESIS Co.

The sequence of SDHA (Succinate dehydrogenase complex, subunit A) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of VCAN (Versican) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of DCN (Decorin) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of LUM (Lumican) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of GPC4 (Glypican-<NUM>) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of COL11A1 (collagen type XI alpha <NUM> chain) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of COL16A1 (collagen type XVI alpha <NUM> chain) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The sequence of COL4A5 (Collagen Type IV Alpha <NUM> Chain) gene is registered as ID: <NUM> in the gene database of National Center for Biotechnology Information.

The timing for measuring the gene expression level described above is not particularly limited, and examples thereof include immediately after separation of cells from a biological sample, during a culture step, after purification in the culture step, immediately after the Nth passage (N represents an integer of <NUM> or more), during maintenance culture, before cryopreservation, after thawing, before formulation, or after formulation.

According to one aspect of the present invention as defined by the claims, the proportion of CDH6-positive mesenchymal stem cells in a cell population comprising mesenchymal stem cells is <NUM>% or more.

CDH6 refers to Cadherin6, and is one member of the cadherin superfamily. VCAN refers to Versican. DCN refers to Decorin. LUM refers tp Lumican.

The proportion of CDH6-positive mesenchymal stem cells in the cell population may be <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, or <NUM>% or more.

Each surface antigen marker can be detected by any detection method known in the art. Examples of the method for detecting a surface antigen marker include, but are not limited to, flow cytometry and cell staining. When cells that emit stronger fluorescence as compared with a negative control (isotype control) are detected in flow cytometry using a fluorescently labeled antibody, the cells are determined to be "positive" for the marker. Any antibody known in the art can be used as the fluorescently labeled antibody. Examples thereof include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), or the like. When cells that are stained or emit fluorescence are observed under a microscope in cell staining, the cells are determined to be "positive" for the marker. The cell staining may be immunostaining of cells using an antibody, or may be non-immunostaining of cells without using an antibody.

The proportion of cells positive for each surface antigen marker (positive rate) can be measured specifically using a flow cytometry dot-plot analysis by the following procedures (<NUM>) to (<NUM>).

The proportion of cells negative for each surface antigen (negative rate) is calculated by the following equation.

The timing for detecting the surface antigen marker described above is not particularly limited, and examples thereof include immediately after separation of cells from a biological sample, during a culture step, after purification in the culture step, immediately after the Nth passage (N represents an integer of <NUM> or more), during maintenance culture, before cryopreservation, after thawing, before formulation, or after formulation.

The cell aggregability of a cell population can be evaluated by calculating a cell aggregability through the following procedures (<NUM>) to (<NUM>).

The timing for evaluating the cell aggregability described above is not particularly limited, and examples thereof include immediately after separation of cells from a biological sample, during a culture step, after purification in the culture step, immediately after the Nth passage (N represents an integer of <NUM> or more), during maintenance culture, before cryopreservation, after thawing, before formulation, or after formulation.

The cell population comprising mesenchymal stem cells provided by the present invention as defined by the claims may be passaged. The lower limit of the number of passages is preferably <NUM> or more, more preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, and further preferably <NUM> or more. In addition, the upper limit of the number of passages is preferably <NUM> or less, further preferably <NUM> or less, further preferably <NUM> or less, and further preferably <NUM> or less.

Population doubling may be carried out on the cell population comprising mesenchymal stem cells provided by the present invention as defined by the claims. The lower limit of the number of population doubling is preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, and further preferably <NUM> or more. In addition, the upper limit of the number of population doubling is preferably <NUM> or less, further preferably <NUM> or less, and further preferably <NUM> or less.

The number of population doubling is a number of times of division of cell population in a certain culture period and is calculated according to an equation: [log<NUM>(cell number at the completion of culture) - log<NUM>(cell number at the start of culture)] / log<NUM> (<NUM>). In a case where subculture is performed, the number of population doubling for each passage is calculated according to the equation described above and then cumulated, and thereby a total number of population doubling is calculated.

The origin of mesenchymal stem cells is not particularly limited, and mesenchymal stem cells derived from, for example, a fetal appendage, bone marrow, adipose or tooth pulp can be used. The mesenchymal stem cells are preferably mesenchymal stem cells derived from a fetal appendage, and more preferably mesenchymal stem cells derived from the amnion. The mesenchymal stem cells are mesenchymal stem cells isolated from an autologous, allogeneic or heterologous biological sample, and preferably mesenchymal stem cells isolated from an allogeneic biological sample.

The mesenchymal stem cells are recombinant or non-recombinant mesenchymal stem cells, and preferably non-recombinant mesenchymal stem cells.

The cell population may contain any number of mesenchymal stem cells. The cell population can include, but not limited to, not less or not more than <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, <NUM> × <NUM><NUM>, or <NUM> × <NUM><NUM> mesenchymal stem cells.

The cell population may comprise any component in addition to mesenchymal stem cells. Examples of such a component can include, but are not limited to, salts, polysaccharides (e.g., hydroxyethyl starch (HES) and dextran), proteins (e.g., albumin), DMSO, amino acids, and medium components (e.g., components contained in RPMI1640 medium).

The cell population may be preserved in a frozen state until immediately before use. The cell population described above may comprise cryopreservation solution in addition to mesenchymal stem cells. As the cryopreservation solution described above, a commercially available cryopreservation solution may be used. Examples thereof include, but are not limited to, CP-<NUM> (registered trademark) (manufactured by Kyokuto Pharmaceutical Industrial Co, Ltd. ), BAMBANKER (manufactured by Lymphotec Inc. ), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co. ), ReproCryo RM (manufactured by REPROCELL Inc. ), CryoNovo (manufactured by Akron Biotechnology, LLC. ), MSC Freezing Solution (manufactured by Biological Industries Inc. ), and CryoStor (manufactured by HemaCare Inc.

The cell population may be provided as a composition in combination with a vehicle. As the vehicle, preferably a liquid vehicle (e.g., media, DMSO, cryopreservation solutions or pharmaceutically acceptable vehicles described below) can be used.

The composition comprising the cell population and the vehicle may be in any cell concentration. Exemplary cell concentrations of the composition comprising the cell population and a vehicle include, but are not limited to, not less or not more than <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, <NUM> × <NUM><NUM> cells/mL, or <NUM> × <NUM><NUM> cells/mL.

The method for producing a cell population comprising mesenchymal stem cells according to the present invention as defined by the claims is a method comprising obtaining a cell population having the following cell characteristics:
the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less.

In other words, the method for producing a cell population comprising mesenchymal stem cells according to the present invention as defined by the claims is a method comprising a step of preparing a cell population comprising mesenchymal stem cells under such a condition that the cell characteristics described above (the condition that the cell population comprising mesenchymal stem cells satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less) are maintained. The condition described above serves as indices for formation of a cell population comprising mesenchymal stem cells having low cell aggregability; and the production method of the present invention as defined by the claims is not particularly limited as long as the indices are satisfied.

The production method of the present invention as defined by the claims comprises a cell population obtainment step of obtaining a cell population comprising mesenchymal stem cells by enzymatically treating a sample (for example, a fetal appendage such as the amnion) comprising mesenchymal stem cells. In addition, the cell population obtainment step may comprise a step of washing a sample comprising mesenchymal stem cells.

The amnion consists of an epithelial cell layer and an extracellular matrix layer, and the latter layer comprises amniotic mesenchymal stem cells. Like other epithelial cells, the amniotic epithelial cells are characterized by expression of epithelial cadherin (E-cadherin: CD324) and an epithelial adhesion factor (EpCAM: CD326). On the other hand, the amniotic MSCs do not express such epithelial-specific surface antigen markers and they can be easily distinguished by flow cytometry.

The cell population comprising mesenchymal stem cells according to the present invention as defined by the claims is preferably a cell population obtained by treating a sample comprising an epithelial cell layer and an extracellular matrix layer collected from a fetal appendage with at least collagenase.

The enzymatic treatment of the sample collected from a fetal appendage (preferably a sample comprising an epithelial cell layer and an extracellular matrix layer) is preferably a treatment with an enzyme (or a combination of enzymes) that can release mesenchymal stem cells contained in the extracellular matrix layer of the fetal appendage, and does not degrade the epithelial cell layer. Examples of such an enzyme can include, but are not particularly limited to, collagenase and/or metalloproteinase. Examples of the metalloproteinase can include, but are not particularly limited to, thermolysin and/or dispase, which is metalloproteinase that cleaves nonpolar amino acids at their N-terminal sides.

The active concentration of the collagenase is preferably <NUM> PU/ml or higher, more preferably <NUM> PU/ml or higher, further preferably <NUM> PU/ml or higher, further preferably <NUM> PU/ml or higher, and further preferably <NUM> PU/ml or higher. The active concentration of the collagenase is, but is not particularly limited to, for example, <NUM> PU/ml or lower, <NUM> PU/ml or lower, <NUM> PU/ml or lower, <NUM> PU/ml or lower, <NUM> PU/ml or lower, or <NUM> PU/ml or lower. In this context, PU (Protease Unit) is defined as the amount of the enzyme that degrades <NUM>µg of FITC-collagen in <NUM> minute at <NUM> and pH <NUM>.

The active concentration of the metalloproteinase (e.g., thermolysin and/or dispase) is preferably <NUM> PU/ml or higher, more preferably <NUM> PU/ml or higher, further preferably <NUM> PU/ml or higher, further preferably <NUM> PU/ml or higher, and further preferably <NUM> PU/ml or higher. Also, the active concentration of the metalloproteinase is preferably <NUM> PU/ml or lower, more preferably <NUM> PU/ml or lower, further preferably <NUM> PU/ml or lower, further preferably <NUM> PU/ml or lower, further preferably <NUM> PU/ml or lower, and further preferably <NUM> PU/ml or lower. In this context, PU (Protease Unit) in an aspect of using dispase as the metalloproteinase is defined as the amount of the enzyme that releases an amino acid corresponding to <NUM>µg tyrosine from casein lactate in <NUM> minute at <NUM> and pH <NUM>. In the concentration range of the enzyme described above, mesenchymal stem cells contained in the extracellular matrix layer can be efficiently released while preventing contamination with epithelial cells contained in the epithelial cell layer of the fetal appendage. The preferred combination of the concentrations of the collagenase and/or the metalloproteinase can be determined by the microscopic observation of the fetal appendage after the enzymatic treatment, or the flow cytometry of the obtained cells.

It is preferred to treat the fetal appendage at the same time with collagenase and metalloproteinase in combination, from the viewpoint of efficiently collecting live cells. In this case, thermolysin and/or dispase can be used as the metalloproteinase, though the metalloproteinase is not limited thereto. Mesenchymal stem cells can be easily obtained by treating the fetal appendage only once with an enzyme solution containing collagenase and metalloproteinase. The treatment at the same time can reduce the risk of contamination by bacteria, viruses, and the like.

For the enzymatic treatment of the fetal appendage, it is preferred to immerse the amnion washed using a washing solution such as physiological saline or Hank's balanced salt solution in the enzyme solution, and perform the treatment with stirring using stirring means. A stirrer or a shaker can be used as such stirring means from the viewpoint of efficiently releasing mesenchymal stem cells contained in the extracellular matrix layer of the fetal appendage, though the stirring means is not limited thereto. The stirring rate is not particularly limited and is, for example, <NUM> rpm or more, <NUM> rpm or more, <NUM> rpm or more, <NUM> rpm or more, <NUM> rpm or more or <NUM> rpm or more when using a stirrer or a shaker. Also, the stirring rate is not particularly limited and is, for example, <NUM> rpm or less, <NUM> rpm or less, <NUM> rpm or less, <NUM> rpm or less or <NUM> rpm or less when using a stirrer or a shaker. The enzymatic treatment duration is not particularly limited and is, for example, <NUM> minutes or longer, <NUM> minutes or longer, <NUM> minutes or longer, <NUM> minutes or longer, <NUM> minutes or longer, <NUM> minutes or longer, <NUM> minutes or longer, <NUM> minutes or longer or <NUM> minutes or longer. Also, the enzymatic treatment duration is not particularly limited and is, for example, <NUM> hours or shorter, <NUM> hours or shorter, <NUM> hours or shorter, <NUM> hours or shorter, <NUM> hours or shorter, <NUM> minutes or shorter, <NUM> minutes or shorter. The enzymatic treatment temperature is not particularly limited and is, for example, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher, <NUM> or higher or <NUM> or higher. Also, the enzymatic treatment temperature is not particularly limited and is, for example, <NUM> or lower, <NUM> or lower, <NUM> or lower or <NUM> or lower.

In the production method of the present invention as defined by the claims, if desired, the released mesenchymal stem cells can be separated and/or collected from the enzyme solution containing the released mesenchymal stem cells by a known method such as a filter, centrifugation, a hollow fiber separation membrane, or a cell sorter. Preferably, the enzyme solution containing the released mesenchymal stem cells is filtered through a filter. In an aspect of filtering the enzyme solution through a filter, only the released cells pass through the filter, whereas an undegraded epithelial cell layer remains on the filter without passing through the filter. Therefore, not only can the released mesenchymal stem cells be easily separated and/or collected, but the risk of contamination by bacteria, viruses, and the like can be reduced. Examples of the filter can include, but are not particularly limited to, mesh filters. The pore size (mesh size) of the mesh filter is not particularly limited and is, for example, <NUM> or larger, <NUM> or larger, <NUM> or larger, <NUM> or larger, <NUM> or larger, or <NUM> or larger. Also, the pore size of the mesh filter is not particularly limited and is, for example, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, <NUM> or smaller, or <NUM> or smaller. The filtration rate is not particularly limited. By using the pore size of the mesh filter within the range described above, the enzyme solution containing the mesenchymal stem cells can be filtered by free fall. This can prevent decrease in cell survival rate.

Nylon is preferably used as a material for the mesh filter. A tube containing a <NUM>, <NUM>, <NUM> or <NUM> nylon mesh filter such as a Falcon cell strainer, which is widely used for research purposes, can be used. Alternatively, medical mesh cloth (nylon and polyester) used for hemodialysis and the like can be used. Further, an arterial filter used for extracorporeal circulation (polyester mesh filter, pore size: <NUM> or larger and <NUM> or smaller) can also be used. A mesh made of any other material, for example, a stainless-steel mesh filter, may also be used.

Preferably, the mesenchymal stem cells are allowed to pass through a filter in natural drop (free fall). It is also possible to force the cells to pass through a filter by suction using a pump or the like. In this case, minimum necessary pressurization is desirable in order to avoid damage of the cells.

The mesenchymal stem cells that have passed through the filter can be collected by centrifugation after dilution of the filtrate with two times or more its volume of a medium or balanced salt buffer solution. Examples of the balanced salt buffer solution that can be used include, but are not limited to, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), and phosphate buffer (PBS).

The cell population obtained by the cell population obtainment step described above is prepared under the following conditions: the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less. The condition is useful as indices for obtaining a cell population comprising mesenchymal stem cells having low cell aggregability. The preparation method is not particularly limited as long as the indices are satisfied. Examples of such a method may include: separating a cell population satisfying the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less by a cell sorter, and then, selecting from the obtained cell population a cell population satisfying the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less; selecting a cell population satisfying the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less, and then separating from the obtained cell population a cell population satisfying the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less by a cell sorter. In addition, another method for preparing a cell population satisfying the indices may be to culture a cell population under a condition satisfying the two conditions described above (the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less).

Examples of a culture method satisfying the indices include a step of repeating a plurality of times the inoculation of the cell population into an uncoated plastic culture vessel at a density of <NUM> to <NUM>,<NUM> cells/cm<NUM> followed by culture. The density of the cell population for inoculation is further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, further preferably <NUM> cells/cm<NUM> or more, and further preferably <NUM> cells/cm<NUM> or more. The density of the cell population for inoculation is further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, further preferably <NUM> cells/cm<NUM> or less, and further preferably <NUM> cells/cm<NUM> or less.

Examples of the other culture methods that satisfy the indices include a step of repeating a plurality of times the inoculation of the cell population into a plastic culture vessel coated with a coating agent at a density of <NUM> to <NUM>,<NUM> cells/cm<NUM> followed by culture. Preferred density conditions for the inoculation of the cell population are similar to the conditions described above.

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

Examples of further other culture methods that satisfy the indices include culturing with addition of basic fibroblast growth factor (bFGF) to the basal medium for use in the culture. The concentration of the basic fibroblast growth factor is preferably <NUM> ng/mL or more, further preferably <NUM> ng/mL or more, further preferably <NUM> ng/mL or more, further preferably <NUM> ng/mL or more, and further preferably <NUM> ng/mL or more. The concentration of the basic fibroblast growth factor is preferably <NUM> ng/mL or less, further preferably <NUM> ng/mL or less, <NUM> ng/mL or less, further preferably <NUM> ng/mL or less, and further preferably <NUM> ng/mL or less. The timing for adding the basic fibroblast growth factor is not particularly limited, and examples thereof include the beginning of a culture step, during the culture step, after purification in the culture step, immediately after the Nth passage (N represents an integer of <NUM> or more), during maintenance culture, before cryopreservation, or after thawing.

Examples of the culture period of one culture process can include <NUM> to <NUM> days and can more specifically include <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days and <NUM> days.

The medium for use in the culture can be prepared by utilizing any liquid medium for animal cell culture as a basal medium and, if necessary, appropriately adding other components (serum, a serum replacement reagent, a growth factor, etc.) thereto.

Examples of the basal medium that can be used include, but are not particularly limited to, media such as BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, improved MEM zinc option medium, IMDM medium (Iscove's modified Dulbecco's medium), Medium <NUM> medium, Eagle MEM medium, αMEM (alpha modification of minimum essential medium eagle) medium, DMEM medium (Dulbecco's modified Eagle's medium), Ham's F10 medium, Ham's F12 medium, RPMI <NUM> medium, Fischer's medium, and mixed medium thereof (e.g., DMEM/F12 medium (Dulbecco's modified Eagle's medium/nutrient mixture F-<NUM> Ham)).

Alternatively, the medium for use in the culture may be a commercially available serum-free medium. Examples thereof include, but are not particularly limited to, STK1 and STK2 (DS Pharma Biomedical Co. ), EXPREP MSC Medium (BioMimetics Sympathies Inc. ), and Corning stemgro human mesenchymal stem cell medium (Corning Inc.

Examples of other components to be added to the basal medium include albumin, serum, serum replacement reagents and growth factors. In an aspect of adding albumin to the basal medium, the concentration of albumin is preferably higher than <NUM>% and <NUM>% or lower. In an aspect of adding serum to the basal medium, the concentration of serum is preferably <NUM>% or higher. In an aspect of adding a growth factor, a reagent (heparin, etc.) for stabilizing the growth factor in the medium may be further added in addition to the growth factor; or the growth factor may be stabilized with a gel, a polysaccharide or the like in advance, and then adding the stabilized growth factor to the basal medium.

The culture of mesenchymal stem cells can be performed by, for example, the following process. First, a cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Next, the cells are inoculated into an uncoated plastic culture vessel and cultured to <NUM>% or less confluence using a medium in an environment of a CO<NUM> concentration of <NUM>% or higher and <NUM>% or lower at <NUM>. Examples of the medium can include, but are not limited to, αMEM, M199, and media based thereon. The cells obtained by the culture as described above are cells cultured once.

The cells cultured once described above can be further passaged and cultured, for example, as follows: first, the cells cultured once are treated by cell dissociation means, then treated with trypsin, and thereby dissociated from the plastic culture vessel. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Finally, the cells are inoculated to an uncoated plastic culture vessel, and cultured to <NUM>% or less confluence using a medium in an environment of a CO<NUM> concentration of <NUM>% or higher and <NUM>% or lower at <NUM>. Examples of the medium can include, but are not limited to, αMEM, M199, and media based thereon. The cells obtained by the passage and the culture as described above are cells passaged once. Cells passaged N times can be obtained by similar passage and culture (N represents an integer of <NUM> or more). From the viewpoint of producing the cells at a large scale, the lower limit of passage number N is, for example, <NUM> or more, preferably <NUM> or more, more preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, further preferably <NUM> or more, and further preferably <NUM> or more. In addition, from the viewpoint of suppressing cell senescence, the upper limit of passage number N is, for example, preferably <NUM> or less, <NUM> or less, <NUM> or less, <NUM> or less, or <NUM> or less.

As the cell dissociation means, a cell dissociation agent, for example, may be used. As the cell dissociation agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA) or the like can be used, but the cell dissociation agent is not particularly limited. As the cell dissociation agent, a commercially available cell dissociation agent may be used. Examples thereof include, but are not limited to, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific Inc. ), TrypLE Select (manufactured by Thremo Fisher Scientific Inc. ), Accutase (manufactured by Stemcell Technologies Inc. ), and Accumax (manufactured by Stemcell Technologies Inc. In addition, as cell dissociation means, physical cell dissociation means may be used, and examples thereof to be used include, but are not limited to, a cell scraper (manufacture by Corning Inc. Cell dissociation means may be used alone or a plurality of cell dissociation means may be used in combination.

According to the production method of the present invention as defined by the claims, a cell population comprising mesenchymal stem cells having low cell aggregability can be obtained, and this enables production of a cell preparation (pharmaceutical composition) useful for intravenous administration. The lower limit of the obtained cell number per batch of culture (cell number obtained per unit surface area and per unit number of culture days) differs depending on an inoculated cell number, an inoculation density, etc. and is, for example, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more or <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or more. Also, the upper limit of the obtained cell number per batch of culture is not particularly limited and is, for example, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less, <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less or <NUM> × <NUM><NUM> (cells/cm<NUM>/day) or less.

According to the production method of the present invention as defined by the claims, a cell population comprising mesenchymal stem cells having low cell aggregability can be obtained. The mesenchymal stem cells obtained by the production method of the present invention as defined by the claims can be cultured preferably up to <NUM> days or later, more preferably up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, up to <NUM> days or later, or up to <NUM> days or later, after the start of in vitro culture.

The mesenchymal stem cells obtained by the production method of the present as defined by the claims invention can also be cultured up to doubling number of preferably <NUM> or more, more preferably <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, or <NUM> or more, after the start of in vitro culture.

The production method of the present invention as defined by the claims comprises an identification step of identifying a cell population comprising mesenchymal stem cells having low cell aggregability by using, as indices, a condition that the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less.

Means for identifying the cell population comprising mesenchymal stem cells is preferably flow cytometry, microarray and/or quantitative RT-PCR.

The relative expression levels of COL11A1 gene and COL16A1 gene to the expression level of SDHA gene can be measured using microarray in accordance with the procedures described above in the present specification.

The timing to perform the above identification is not particularly limited, and examples thereof include immediately after separation of cells from a biological sample, during a culture step, after purification in the culture step, immediately after the Nth passage (N represents an integer of <NUM> or more), during maintenance culture, before cryopreservation, after thawing, before formulation, or after formulation.

In addition, the production method of the present invention as defined by the claims may include a step of selectively separating the identified cell population after identifying the cell population comprising the mesenchymal stem cells by using the above conditions as indices. Means for selectively separating the identified cell population is not particularly limited, but examples thereof include separation of a cell population by a cell sorter, and purification of a cell population by culture.

The production method of the present invention as defined by the claims may also comprise a step of cryopreserving the cell population comprising mesenchymal stem cells. In an aspect comprising the step of cryopreserving the cell population, the cell population may be thawed, and then, if necessary, identified, separated, collected and/or cultured. Alternatively, the cell population may be thawed and then directly used.

Examples of the means for cryopreserving the cell population comprising mesenchymal stem cells include, but are not particularly limited to, program freezers, deep freezers, and immersing in liquid nitrogen. In the case of using a program freezer, the temperature for freezing is preferably -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, or -<NUM> (liquid nitrogen temperature) or lower. In the case of using a program freezer, the freezing rate for freezing is, for example, preferably -<NUM>/min, - <NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min, -<NUM>/min or -<NUM>/min. In the case of using a program freezer as such freezing means, the temperature can be lowered to a temperature between - <NUM> or higher and -<NUM> or lower (e.g., -<NUM>) at a freezing rate of, for example, -<NUM>/min or more and -<NUM>/min or less, and further lowered to a temperature of -<NUM> or higher and - <NUM> or lower (e.g., -<NUM>) at a freezing rate of -<NUM>/min or more and -<NUM>/min or less (e.g., -<NUM>/min). In addition, when immersing in liquid nitrogen is used as such freezing means, the temperature can be rapidly lowered to, for example -<NUM> for freezing, and then, cryopreservation can be carried out in liquid nitrogen (gas phase).

For freezing by the freezing means, the cell population may be frozen in a state contained in any preservation container. Examples of such a preservation container include, but are not limited to, cryotubes, cryovials, freezing bags, and infusion bags.

For freezing by the freezing means, the cell population may be frozen in any cryopreservation solution. Examples of such a cryopreservation solution include, but are not limited to, BAMBANKER (manufactured by Lymphotec Inc. ), STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co. ), ReproCryo RM (manufactured by REPROCELL Inc. ), CryoNovo (Akron Biotechnology, LLC. ), MSC Freezing Solution (Biological Industries Inc. ), and CryoStor (HemaCare Inc.

The cryopreservation solution can contain polysaccharides at a defined concentration. The preferable concentration of polysaccharides is, for example, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher or <NUM>% by mass or higher. In addition, the preferable concentration of polysaccharides is, for example, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower or <NUM>% by mass or lower. Examples of the polysaccharide include, but are not limited to, hydroxyethyl starch (HES) and dextran (Dextran40 or the like).

The cryopreservation solution can contain dimethyl sulphoxide (DMSO) at a defined concentration. The preferable concentration of DMSO is, for example, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher or <NUM>% by mass or higher. In addition, the preferable concentration of DMSO is, for example, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower or <NUM>% by mass or lower.

The cryopreservation solution may be a solution containing albumin at a defined concentration higher than <NUM>% by mass. The preferable concentration of albumin is, for example, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher, <NUM>% by mass or higher or <NUM>% by mass or higher. In addition, the preferable concentration of albumin is, for example, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower, <NUM>% by mass or lower or <NUM>% by mass or lower. Examples of albumin can include, but are not limited to, bovine serum albumin, mouse albumin, and human albumin.

The production method of the present invention as defined by the claims can comprise a step of washing the cell population comprising mesenchymal stem cells. Examples of a washing solution to be used in the step of washing the cell population comprising mesenchymal stem cells include, but are not limited to, physiological saline, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS) and phosphate-buffer (PBS). Washing a cell population can reduce or remove allergen, endotoxin or the like. Examples of the allergen include, but are not limited to, bovine serum albumin (BSA), swine trypsin and swine heparin.

The production method of the present invention as defined by the claims can comprise a step of filling the cell population comprising mesenchymal stem cells into a preservation container. Examples of such a preservation container include, but are not limited to, cryotubes, cryovials, freezing bags, and infusion bags.

In the present invention as defined by the claims, in a cell population comprising mesenchymal stem cells, the cell aggregability of mesenchymal stem cells can be evaluated by performing measurement (preferably by performing measurement over time) utilizing, as indices, a condition that the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less. Examples of the step that requires the evaluation include a culture step, a cryopreservation step and/or a formulation step.

In the culture step, for example, measurement of the indices over time allows changes in the cell aggregability of mesenchymal stem cells to be quickly and easily known and predicted. It can be known that in a cell population comprising mesenchymal stem cells satisfying the indices, the mesenchymal stem cells maintain low aggregability. On the other hand, when a culture state continues with a value deviating from the indices, it can be predicted that the cell aggregability of the mesenchymal stem cells is increasing. When it is found from the indices that the cell aggregability is increasing, the cell aggregation can be reduced by properly changing culture conditions (change of an inoculation density, a medium, a growth factor, serum, etc.) as needed. In addition, when the indices are not satisfied, only a cell population comprising mesenchymal stem cells that satisfy the indices can be separated through the use of, for example, a cell sorting technique. Thereafter, mesenchymal stem cells in the cell population are inoculated again and subcultured, and thereby the cell aggregation can be reduced. At the early stage of culture, culture conditions (change of an inoculation density, a medium, a growth factor, serum, etc.) may be designed such that the indices are satisfied at the final stage of the step, and thus, the indices may be satisfied at least at the final stage.

The quality of a donor itself and/or a sample collected from the donor can be evaluated by obtaining a cell population comprising mesenchymal stem cells from the donor, measuring the relative expression level of COL11A1 gene to the expression level of SDHA gene and the relative expression level of COL16A1 gene to the expression level of SDHA gene, performing the measurement using as indices a condition that the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less. When a cell population comprising mesenchymal stem cells that satisfy the indices is obtained (preferably, easily obtained), the quality of the donor and/or the sample collected from the donor can be confirmed to be good. On the other hand, when the relative expression levels in the cell population comprising mesenchymal stem cells deviate from the indices, the cell population contained in the sample collected from the donor is highly likely to cause cell aggregation, and thus, the cell aggregation can be reduced by properly changing culture conditions (change of an inoculation density, a medium, a growth factor, serum, etc.). In addition, when the relative expression levels in the cell population comprising mesenchymal stem cells deviate from the indices, cell aggregation can be reduced by separating a cell population comprising mesenchymal stem cells satisfying the indices through the use of, for example, a cell sorting technique, and inoculating and culturing the mesenchymal stem cells in the cell population. At the early stage of culture, culture conditions (change of an inoculation density, a medium, a growth factor, serum, etc.) may be designed such that the indices are satisfied at the final stage of the step, and thus, the indices may be satisfied at least at the final stage. In confirming the quality of a sample collected from a donor, a method for preparing and treating the sample, and a method for culturing a cell population are not particularly limited, and any method can be employed.

An optimal enzymatic treatment condition can be determined and/or predicted by measuring the relative expression level of COL11A1 gene to the expression level of SDHA gene, and the relative expression level of COL16A1 gene to the expression level of SDHA gene in a cell population obtained by enzymatically treating a sample collected from a donor, and evaluating by using as indices a condition that the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less. When a cell population comprising mesenchymal stem cells that satisfy the indices is obtained (preferably, easily obtained), the enzymatic treatment method can be determined and/or predicted to be appropriate for the sample collected from a donor. On the other hand, when a culture state continues with a value deviating from the indices, the enzymatic treatment method can be determined and/or predicted to be inappropriate for the sample collected from a donor. In determining and/or predicting an optimal enzymatic treatment method, a method for preparing and treating the sample, and a method for culturing a cell population are not particularly limited, and any method can be employed.

The indices can be measured at a necessary timing. Examples of the timing include, but are not particularly limited to, immediately after separation of cells from a biological sample, during a culture step, after purification in the culture step, immediately after the Nth passage (N represents an integer of <NUM> or more), during maintenance culture, before cryopreservation, after thawing, before formulation, or after formulation.

The cell population comprising mesenchymal stem cells produced by the method according to the present invention as defined by the claims can be used as a pharmaceutical composition.

The pharmaceutical composition is preferably a liquid preparation, and more preferably an injectable liquid preparation.

The pharmaceutical composition can be used as a cell therapy agent, for example, a therapeutic agent for intractable diseases.

The pharmaceutical composition can be used as a therapeutic agent for a disease selected from immune-related disease, ischemic disease, lower-limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological disease, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes mellitus, mycosis fungoides, scleroderma, disease caused by the degeneration and/or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscal damage, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye disease, angiogenesis-related disease, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, cardiac failure, cardiomyopathy, valvular disease, wound, epithelial damage, fibrosis, lung disease and cancer.

The cell population comprising mesenchymal stem cells produced by the method according to the present invention as defined by the claims may be for use in a pharmaceutical composition.

The cell population comprising mesenchymal stem cells produced by the method according to the present invention as defined by the claims may be for use in a cell therapy agent.

The cell population comprising mesenchymal stem cells produced by the method according to the present invention as defined by the claims may be for use in the treatment of a disease selected from immune-related disease, ischemic disease, lower-limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, neurological disease, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, lupus erythematosus, collagen disease, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes mellitus, mycosis fungoides, scleroderma, disease caused by the degeneration and/or inflammation of connective tissues such as cartilage, articular cartilage defect, meniscal damage, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, eye disease, angiogenesis-related disease, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, cardiac failure, cardiomyopathy, valvular disease, wound, epithelial damage, fibrosis, lung disease and cancer.

The cell population comprising mesenchymal stem cells produced by the method according to the present invention as defined by the claims may be for use in the regeneration of cardiac muscle, the production of cardiac muscle cells, angiogenesis, the repair of a blood vessel, or the suppression of immune response by administration to a patient or a subject.

A pharmaceutical composition may be obtained by diluting a cell population comprising mesenchymal stem cells with a pharmaceutically acceptable vehicle. The pharmaceutically acceptable vehicle is not particularly limited as long as it is a solution that can be administered to a patient or a subject. The pharmaceutically acceptable vehicle may be an infusion preparation, and examples thereof include, but are not limited to, water for injection, physiological saline, <NUM>% glucose solution, Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonated Ringer's solution, amino acid solution, starter solution (Solution I), rehydration solution (Solution II), maintenance infusion (Solution III), postoperative recovery solution (Solution IV), and Plasma-Lyte A (registered trademark).

The "patient or subject" used herein is typically a human and may be other animals. Examples of other animals include, but are not limited to, mammals such as dogs, cats, cattle, horses, pigs, goats, sheep, monkeys (cynomolgus monkey, rhesus monkey, common marmoset and Japanese monkey), ferrets, rabbits and rodents (mouse, rat, Mongolian gerbil, guinea pig and hamster); birds such as chickens and quails.

The "immune-related disease" used herein is not particularly limited as long as it is a disease related to an immune responses of a patient or subject. Examples thereof include graft-versus-host disease (GVHD), inflammatory bowel diseases (IBD), Crohn's disease, ulcerative colitis, radiation enteritis, diabetes mellitus, systemic lupus erythematosus, collagen disease, mycosis fungoides, multiple sclerosis, psoriasis, autoimmune bullous disease, and rheumatoid arthritis.

The pharmaceutical composition may comprise any component for use in the treatment of a patient or subject. Examples of the component include, but are not limited to, salts, polysaccharides (e.g., hydroxyethyl starch (HES) and dextran), proteins (e.g., albumin), DMSO, amino acids, and medium components (e.g., components contained in RPMI1640 medium).

The pharmaceutical composition may comprise various additives for increasing the preservation stability, the isotonicity, the absorbability, and/or the viscosity, such as an emulsifier, a dispersant, a buffer, a preservative, a wetting agent, an antioxidant, a chelating agent, a thickener, a gelling agent and a pH adjuster. Examples of the thickener include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose and hydroxypropyl methylcellulose. The concentration of the thickener can be optionally set according to the selected thickener, within the range of concentration that is safe when administered to the patient or the subject and achieves the desired viscosity.

The pH of the pharmaceutical composition can be adjusted to around neutral pH, for example, pH <NUM> or more or pH <NUM> or more, and/or pH <NUM> or lower or pH <NUM> or lower, but not limited thereto.

The pharmaceutical composition is preferably a liquid preparation, and more preferably an injectable liquid preparation. As the injectable liquid preparation, liquid preparations suitable for injection are known in, for example, <CIT> and <CIT>. The pharmaceutical composition may also be an injectable liquid preparation described in the above literatures.

The dose of the pharmaceutical composition is the amount of cells that allows a patient or a subject to whom the pharmaceutical composition has been administered to obtain therapeutic effects, compared with a patient or a subject to whom the pharmaceutical composition has not been administered. A specific dose can be appropriately determined depending on the form of administration, the administration method, the intended use, the age, body weight, symptoms of a patient or subject, and the like. A single dose of mesenchymal stem cells to a human is not particularly limited and is, for example, <NUM><NUM> cells/kg body weight or more, <NUM><NUM> cells/kg body weight or more or <NUM><NUM> cells/kg body weight or more. Also, a single dose of mesenchymal stem cells to a human is not particularly limited and is, for example, <NUM><NUM> cells/kg body weight or less, <NUM><NUM> cells/kg body weight or less or <NUM><NUM> cells/kg body weight or less.

The pharmaceutical composition can be preserved in a frozen state until immediately before use. The temperature for cryopreservation is preferably -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, -<NUM> or lower, - <NUM> or lower, -<NUM> or lower or -<NUM> (liquid nitrogen temperature) or lower. When the pharmaceutical composition is administered to a patient or subject, it can be used after being quickly thawed at <NUM>.

The present invention will be specifically explained with reference to the Examples below.

A fetal membrane and a placenta, which are fetal appendages, were aseptically collected from a pregnant woman (donor) of an elective cesarean section case after obtaining informed consent. The obtained fetal membrane and placenta were contained in a sterile tray containing physiological saline. An amnion was manually separated from the stump of the fetal membrane. The amnion was washed with a Hank's balanced salt solution (free of Ca and Mg) to remove attached blood and clots.

The amnion was enzymatically treated by immersing the amnion comprising an epithelial cell layer and an extracellular matrix layer in a Hank's balanced salt solution (containing Ca and Mg) containing <NUM> PU/mL collagenase and <NUM> PU/mL dispase I, and shaking and stirring under conditions of <NUM>, <NUM> minutes, and <NUM> rpm. The solution after enzymatic treatment was filtered through a nylon mesh having openings of <NUM> to remove undigested products of the amnion so as to collect a cell suspension containing amniotic MSCs. The obtained cell suspension was analyzed for the proportion of cells positive for the expression of CD90, which is one of surface antigens known as a typical positive marker of MSCs, using a flow cytometer. It was then confirmed that amniotic MSCs were able to be separated with high purity from the amnion.

The surface antigen analysis employed BD Accuri™ C6 Flow Cytometer from Becton, Dickinson (BD) and Company, and the measurement conditions involved analyzed cell number: <NUM>,<NUM> cells and flow rate setting: Slow (<NUM>µL/min). FITC Mouse Anti-Human CD90 (BD/model number: <NUM>) was used as the antibody against the CD90 antigen, and FITC Mouse IgG1, κ Isotype Control (BD/model number: <NUM>) was used as the antibody for isotype control.

The cell population obtained in the above section "Step2: Enzymatic treatment of amnion and collection of amniotic MSCs" was suspended in BAMBANKER (LYMPHOTEC Inc. ) so as to be <NUM> × <NUM><NUM> cells/mL and then aliquoted into cryotubes. The cryotube was placed in a BICELL (freezing container) (NIHON FREEZER Co. ) and stored at -<NUM> for <NUM> hours, and thereafter, cryopreserved at a liquid nitrogen temperature.

The cell population obtained in the above section "Step <NUM>: Cryopreservation of amniotic MSCs" was inoculated to an uncoated plastic culture vessel, and adherent cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing <NUM>% fetal bovine serum (FBS) (inactivated) and <NUM>× Antibiotic-Antimycotic (manufactured by Thermo Fisher Scientific Inc. ) until subconfluence. Thereafter, cells were dissociated using TrypLE Select (<NUM>×) (manufactured by Thermo Fisher Scientific Inc. ), a <NUM>/<NUM> amount of cells was inoculated to an uncoated plastic culture vessel at the same scale as that of the preceding culture and thereby subcultured. Medium replacement was carried out with a frequency of twice a week. Thus the subculture was continued.

An amnion was obtained in the same manner as in Step <NUM> of Comparative Example <NUM> except that a fetal membrane and a placenta, which are fetal appendages, were aseptically collected from a donor different from the donor of Comparative Example <NUM>.

A cell suspension comprising amniotic MSCs was collected by the same method as in Step <NUM> of Comparative Example <NUM>. The obtained cell suspension was analyzed in the same manner as in Comparative Example <NUM> for the proportion of cells positive for the expression of CD90, which is one of surface antigens known as a typical positive marker of MSCs, using a flow cytometer. It was confirmed that amniotic MSCs were able to be separated with high purity from the amnion.

The cell population obtained in the above section "Step <NUM>: Enzymatic treatment of amnion and collection of amniotic MSCs" was cryopreserved by the same method as in Step <NUM> of Comparative Example <NUM>.

The cell population obtained in the above section "Step <NUM>: Cryopreservation of amniotic MSCs" was adherent cultured by the same method as in Step <NUM> of Comparative Example <NUM> until subconfluence. Subculture was performed by the same method as in Step <NUM> of Comparative Example <NUM>.

For the cell populations of the 6th passage cultured in Comparative Example <NUM> and Example <NUM>, the proportions of cells positive for CDH6 were measured using a flow cytometer.

In this measurement, used were Anti-CDH6-Mouse Mono IgG1 (R&D Systems Inc. /model number: MAB2715) as a primary antibody against CDH6 antigen; Mouse Mono IgG1 (R&D Systems Inc. /model number: MAB002) as an isotype control antibody; and Mouse F(ab)<NUM> IgG (H+L) APC-conjugated Antibody (R&D Systems Inc. /model number: F0101B) as a secondary antibody against the primary antibody against CDH6 antigen and the isotype control antibody thereof.

Analysis results are shown in <FIG> and <FIG>. In comparison with the cell population of Comparative Example <NUM>, the positive rate of CDH6 was improved in the cell population of Example <NUM>. In addition, the proportion of CDH6-positive mesenchymal stem cells was less than <NUM>% (specifically <NUM>%) in the cell population of Comparative Example <NUM>, while the proportion of CDH6-positive mesenchymal stem cells was <NUM>% or more (specifically <NUM>%) in the cell population of Example <NUM>.

The cell populations of the 6th passage cultured in Comparative Example <NUM> and Example <NUM> were analyzed by microarray analysis for the expression of VCAN gene, DCN gene, LUM gene, GPC4 gene, COL11A1 gene, COL16A1 gene, COL4A5 gene and SDHA gene.

The microarray analysis was carried out by the following procedures (<NUM>) to (<NUM>). The following procedures (<NUM>) to (<NUM>) were entrusted to and performed by RIKEN GENESIS CO.

The expression level of each gene was determined as the relative expression level to the expression level of the SDHA gene. Results are shown in the following table.

From Table <NUM>, it was found that the relative expression levels of COL11A1 gene and COL16A1 gene were lower in the cell population of Example <NUM> in comparison with the cell population of Comparative Example <NUM>. Specifically, it was found that in the cell population of Example <NUM> the relative expression level of COL11A1 gene to the expression level of SDHA gene was <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene was <NUM> or less. On the other hand, it was found that in the cell population of Comparative Example <NUM> the relative expression level of COL11A1 gene to the expression level of SDHA gene was larger than <NUM> and the relative expression level of COL16A1 gene to the expression level of SDHA gene was larger than <NUM>.

In addition, from Table <NUM>, it was found that the relative expression level of COL4A5 gene was lower in the cell population of Example <NUM> in comparison with the cell population of Comparative Example <NUM>. Specifically, it was found that in the cell population of Example <NUM> the relative expression level of COL4A5 gene to the expression level of SDHA gene was <NUM> or less. On the other hand, it was found that in the cell population of Comparative Example <NUM> the relative expression level of COL4A5 gene to the expression level of SDHA gene was larger than <NUM>.

In addition, from Table <NUM>, it was found that the relative expression levels of VCAN gene, DCN gene and LUM gene were lower in the cell population of Example <NUM> in comparison with the cell population of Comparative Example <NUM>. Specifically, it was found that in the cell population of Example <NUM> the relative expression level of VCAN gene to the expression level of SDHA gene was <NUM> or less; the relative expression level of DCN gene to the expression level of SDHA gene was <NUM> or less; and the relative expression level of LUM gene to the expression level of SDHA gene was <NUM> or less. On the other hand, it was found that in the cell population of Comparative Example <NUM> the relative expression level of VCAN gene to the expression level of SDHA gene was larger than <NUM>; the relative expression level of DCN gene to the expression level of SDHA gene was larger than <NUM>; and the relative expression level of LUM gene to the expression level of SDHA gene was larger than <NUM>.

In addition, from Table <NUM>, it was found that the relative expression level of GPC4 gene was lower in the cell population of Example <NUM> in comparison with the cell population of Comparative Example <NUM>. Specifically, it was found that in the cell population of Example <NUM> the relative expression level of GPC4 gene to the expression level of SDHA gene was <NUM> or less. On the other hand, it was found that in the cell population of Comparative Example <NUM> the relative expression level of GPC4 gene to the expression level of SDHA gene was larger than <NUM>.

The cell aggregability was evaluated by the following procedures (<NUM>) to (<NUM>).

Measurement results are shown in the following table.

From Table <NUM>, the cell aggregability of the cell population of Comparative Example <NUM> was <NUM>% and the cell aggregability of the cell population of Example <NUM> was <NUM>%. From the above, it was revealed that cell aggregability was lower in the cell population of Example <NUM> in comparison with the cell population of Comparative Example <NUM>.

Thus, it was found that cell aggregability is low in the cell population having the following cell characteristic.

The cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less.

Further, it was found that cell aggregability is low when a cell population satisfies the relative expression level of COL4A5 gene to the expression level of SDHA gene of <NUM> or less.

Further, it was found that cell aggregability is low when a cell population satisfies any one or more of: the relative expression level of VCAN gene to the expression level of SDHA gene of <NUM> or less; the relative expression level of DCN gene to the expression level of SDHA gene of <NUM> or less; and the relative expression level of LUM gene to the expression level of SDHA gene of <NUM> or less.

Further, it was found that cell aggregability is low when a cell population satisfies the relative expression level of GPC4 gene to the expression level of SDHA gene of <NUM> or less.

Thus, it was revealed that it is effective to satisfy at least the above condition (the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less) as the indices for obtaining a cell population having low cell aggregability, and that a cell population comprising mesenchymal stem cells having low cell aggregability can be obtained by using the above condition as indices. Therefore, by producing a cell preparation with a cell population comprising mesenchymal stem cells having low cell aggregability as defined by the claims obtained by the present invention as defined by the claims, a cell preparation with increased safety and reduced risk of causing an embolus can be produced.

"Step <NUM>: Collection of amnion", "Step <NUM>: Enzymatic treatment of amnion and collection of amniotic MSCs ", "Step <NUM>: Cryopreservation of amniotic MSCs", and "Step <NUM>: Culture of amniotic MSCs" were carried out in the same manner as in Example <NUM>, from a pregnant woman of an elective cesarean section case after obtaining informed consent (a donor different from those of Comparative Example <NUM> and Example <NUM>). A portion of the cell population of each passage was collected during culturing of the amniotic MSCs, and then each of the collected cell populations was evaluated for the conditions (a) and (b) below. The evaluation of the conditions was carried out using the same procedures as those of the above sections "Analysis of CDH6 expression" and "Analysis of gene expression".

There were a cell population satisfying the conditions (a) and (b) and a cell population not satisfying them in collected cell populations. Therefore, these two types of cell populations were evaluated for cell aggregability by the same method as in "Evaluation of cell aggregability" described in paragraph <NUM>.

Evaluation results are shown in the following table.

From Table <NUM>, the cell aggregability of the cell population not satisfying the conditions (a) and (b) was <NUM>%, and the cell aggregability of the cell population satisfying the conditions (a) and (b) was <NUM>%. Since cell aggregability is lower in the cell population satisfying the conditions (a) and (b) in comparison with the cell population not satisfying the conditions (a) and (b), a cell population having low cell aggregability can be selectively obtained by using the conditions (a) and (b) as indices.

In addition, the cell population satisfying the conditions (a) and (b) and the cell population not satisfying them were also evaluated for the following conditions (c) and (d).

The cell population satisfying the conditions (a) and (b) satisfied the conditions (c) and (d) while the cell population not satisfying the conditions (a) and (b) did not satisfy the conditions (c) and (d).

From the above, in a culture step, cell aggregability is lower in a cell population that satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less, compared to a cell population not satisfying the above conditions, and thus, the cell aggregability of mesenchymal stem cells can be evaluated by using the conditions as indices. In addition, a cell population having low cell aggregability can be selectively obtained by using the conditions as indices. Further, according to the present as defined by the claims invention, changes in the cell aggregability of mesenchymal stem cells can be quickly known and predicted by measuring (or measuring over time) the indices. This enables production of a cell preparation useful for intravenous administration.

A portion of the cell population obtained in the above Example <NUM> is subjected to preparation of a pharmaceutical composition. A pharmaceutical composition (cell preparation) consisting of <NUM> of RPMI1640 medium containing <NUM> × <NUM><NUM> amniotic MSCs, <NUM> of HES, <NUM> of DMSO and <NUM> of human serum albumin is prepared. The pharmaceutical composition is enclosed in a freezing bag and stored in a frozen state. The pharmaceutical composition can be thawed upon use and applied to a patient.

An amnion was obtained by the same method as in step <NUM> of Comparative Example <NUM>, Example <NUM> and Example <NUM> except that a fetal membrane and a placenta, which are fetal appendages, were aseptically collected from two donors (donors X and Y), who are different from those of Comparative Example <NUM>, Example <NUM> and Example <NUM>. Amniotic MSCs obtained from fetal appendages of the two were referred to as #X and #Y, respectively.

Cell populations comprising amniotic MSCs of #X and #Y obtained in the above were cryopreserved by the same method as in step <NUM> of Comparative Example <NUM>, Example <NUM> and Example <NUM>, and cultured by the following method.

The cell population obtained in the above was inoculated to an uncoated plastic culture vessel, and adherent cultured in αMEM containing <NUM>% FBS and <NUM>× Antibiotic-Antimycotic until subconfluence. The subculture was carried out by the same method as in step <NUM> of Comparative Example <NUM>, Example <NUM> and Example <NUM>.

A portion of the cell population of the 6th passage obtained in the above "Step <NUM>: Culture of amniotic MSCs" was collected and subjected to microarray analysis to confirm whether the following condition is satisfied, and at the same time, the remaining of the cell population was cryopreserved until results of microarray analysis were obtained.

The cell population comprising mesenchymal stem cells satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less.

Analysis results on each gene in the cell population of the 6th passage are shown in Table <NUM>.

As shown in Table <NUM>, in the donor X-derived cell population, the relative expression level of COL11A1 gene to the expression level of SDHA gene was <NUM> and the relative expression level of COL16A1 gene to the expression level of SDHA gene was <NUM>. Also, in the donor Y-derived cell population, the relative expression level of COL11A1 gene to the expression level of SDHA gene was <NUM> and the relative expression level of COL16A1 gene to the expression level of SDHA gene was <NUM>.

Hence, it was found that in the cell populations derived from the donors X and Y the relative expression level of COL16A1 gene to the expression level of SDHA gene was <NUM> or less and the relative expression level of COL11A1 gene to the expression level of SDHA gene was <NUM> or less. Specifically, it was found that in the cell populations of the present Example the relative expression level of COL16A1 gene to the expression level of SDHA gene was <NUM> or less and the relative expression level of COL11A1 gene to the expression level of SDHA gene was <NUM> or less.

In addition, from Table <NUM>, in the donor X-derived cell population, the relative expression level of VCAN gene to the expression level of SDHA gene was <NUM>; the relative expression level of DCN gene to the expression level of SDHA gene was <NUM>; and the relative expression level of LUM gene to the expression level of SDHA gene was <NUM>. Further, in the donor Y-derived cell population, the relative expression level of VCAN gene to the expression level of SDHA gene was <NUM>; the relative expression level of DCN gene to the expression level of SDHA gene was <NUM>; and the relative expression level of LUM gene to the expression level of SDHA gene was <NUM>.

Hence, it was found that in the cell populations derived from the donors X and Y, the relative expression level of VCAN gene to the expression level of SDHA gene was <NUM> or less; the relative expression level of DCN gene to the expression level of SDHA gene was <NUM> or less; and the relative expression level of LUM gene to the expression level of SDHA gene was <NUM> or less. Specifically, it was found that in the cell populations of the present Example the relative expression level of VCAN gene to the expression level of SDHA gene was <NUM> or less; the relative expression level of DCN gene to the expression level of SDHA gene was <NUM> or less; and the relative expression level of LUM gene to the expression level of SDHA gene was <NUM> or less.

Further, from Table <NUM>, in the donor X-derived cell population, the relative expression level of GPC4 gene to the expression level of SDHA gene was <NUM>. Also, in the donor Y-derived cell population, the relative expression level of GPC4 gene to the expression level of SDHA gene was <NUM>.

Hence, it was found that in the cell populations derived from the donors X and Y the relative expression level of GPC4 gene to the expression level of SDHA gene was <NUM> or less. Specifically, it was found that in the cell population of the present Example the relative expression level of GPC4 gene was <NUM> or less.

Claim 1:
A method for producing a cell population comprising mesenchymal stem cells, the method comprising obtaining a cell population having the following cell characteristics:
the cell population satisfies the relative expression level of COL11A1 gene to the expression level of SDHA gene of <NUM> or less and the relative expression level of COL16A1 gene to the expression level of SDHA gene of <NUM> or less,
wherein the step of obtaining the cell population comprises
treating a sample comprising an extracellular matrix layer collected from a fetal appendage with an enzyme solution to release the mesenchymal stem cells contained in the extracellular matrix layer; and
identifying the cell population having the cell characteristics from the released mesenchymal stem cells.