Mesenchymal stem cells and therapeutic agent for liver disease

Abstract

To provide a novel therapeutic agent for liver disease.SOLUTION: The present invention relates to a mesenchymal stem cell characterized by high expression of the Tissue Factor Pathway Inhibitor (TFPI). It is preferable that the mesenchymal stem cell is allogeneic and is derived from a fatty tissue. Moreover, the present invention includes a therapeutic agent for liver disease containing the mesenchymal stem cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a mesenchymal stem cell and a therapeutic agent for liver disease. [Background technology]

[0002] Liver cirrhosis is the terminal stage of various liver diseases characterized by significant fibrosis and regenerative nodule formation. It is characterized by the formation of pseudolobules due to remodeling of the liver lobule and abnormalities in intrahepatic and extrahepatic hemodynamics. As a result, liver cell function declines (see Non-Patent Document 1). In liver cirrhosis, chronic inflammation also occurs. This causes repeated degeneration and necrosis of hepatocytes and liver regeneration, during which excess extracellular matrix is ​​produced in the liver. It is known that the accumulation of inflammatory cytokines leads to fibrosis.

[0003] The causes of liver cirrhosis include persistent infection with hepatitis viruses, excessive alcohol intake, obesity, and insulin. Thrombocytopenic purpura resistance, autoimmunity (primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis) Among these, hepatitis caused by hepatitis virus is the most common. The most common cause is hepatitis C virus (HCV). There are 170 million CV carriers worldwide, and 1.5 to 2 million in Japan. It is estimated that approximately 70% of HCV-infected individuals will remain infected and develop chronic hepatitis. In cases where HCV infection has progressed, spontaneous clearance of the virus is rare (0.2% per year), and the persistence of inflammation due to HCV infection This prolongation induces liver fibrosis, which progresses to liver cirrhosis and hepatocellular carcinoma (see Non-Patent Document 3).

[0004] When cirrhosis progresses from the compensated stage to the decompensated stage accompanied by advanced fibrosis, existing drugs The current treatments are not sufficient to address this issue. Therefore, new treatments with different mechanisms of action are being developed. There is a need for the development of medicines.

[0005] Mesenchymal stem cells were first isolated from bone marrow by Friedenstein (1982). Mesenchymal stem cells are progenitor cells with multipotency that have been determined by the endothelial cell type (see Non-Patent Document 4). It has been revealed that mesenchymal stem cells are present in various tissues such as the umbilical cord and fat. It is expected to become a new treatment for various intractable diseases (see Patent Documents 1 and 2). Recently, it has been reported that cells with equivalent functions exist in interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc. Therefore, mesenchymal stem cells are called stromal cells. They are also called stromal cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2012-157263 A [Patent Document 2] Special Publication No. 2012-508733 [Non-patent literature]

[0007] [Non-Patent Document 1] Meiji Watanabe, Liver Cirrhosis: Pathogenesis and Treatment Handbook, Medical Review Co., Ltd., April 20, 2007, p.12 [Non-Patent Document 2] Edited by the Japanese Society of Gastroenterology, Guidelines for the Treatment of Liver Cirrhosis 2015, Nankodo Co., Ltd., published October 2015, p.2 [Non-Patent Document 3] Kiyosawa K. et. al., Hepatology,(1990), 12(4 Pt 1), pp.671-675 [Non-Patent Document 4] Pittenger FMet al.Science ,(1999),284,pp.143-147 Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above-mentioned circumstances, an object of the present invention is to provide a novel therapeutic agent for liver diseases. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the present inventors have discovered a tissue factor pathway inhibitor. -(Tissue factor pathway inhibitor; TFPI) Highly expressing mesenchymal stem (stromal) cells We have found that MSCs are effective in treating liver diseases and have completed the present invention. According to the present invention, it is possible to provide an effective therapeutic agent for treating liver diseases. , as follows.

[0010] [1] Mesenchymal stem cells characterized by high expression of tissue factor pathway inhibitor (TFPI) Stem cells. [2] The mesenchymal stem cell described in [1], which is of allogeneic origin. [3] The mesenchymal stem cell according to [1] or [2], which is derived from adipose tissue. [4] A therapeutic agent for liver disease, comprising the mesenchymal stem cells described in any one of [1] to [3]. [5] The therapeutic agent for liver disease according to [4], wherein the liver disease is a liver disease accompanied by fibrosis of liver tissue. . [6] By using mesenchymal stem cells with high expression of tissue factor pathway inhibitor (TFPI), A method for treating liver disease, comprising: [7] The method for treating a liver disease described in [6], wherein the mesenchymal stem cells are of allogeneic origin. [8] The treatment for liver disease according to [6] or [7], wherein the mesenchymal stem cells are derived from adipose tissue. method. Effect of the Invention

[0011] According to the present invention, a novel therapeutic agent for liver diseases can be provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the inhibitory effect of rTFPI on the expression of fibrosis-related gene ACTA2 in hepatic stellate cells. [Diagram 2] FIG. 2 shows the inhibitory effect of rTFPI on the expression of fibrosis-related gene COL1A1 in hepatic stellate cells. [Diagram 3] FIG. 3 shows the results of comparing TFPI mRNA expression in the mesenchymal stem cells of the present invention with other cells. [Figure 4] FIG. 4 shows the results of comparing TFPI mRNA expression in the mesenchymal stem cells of the present invention with other cells (mesenchymal stem cells under conventional culture conditions). [Diagram 5] FIG. 5 shows the results of comparing the expression level of TFPI protein in the mesenchymal stem cells of the present invention with other cells (mesenchymal stem cells under conventional culture conditions). [Figure 6] FIG. 6 shows the results of comparing the amount of TFPI protein secreted from the mesenchymal stem cells of the present invention with other cells (mesenchymal stem cells under conventional culture conditions). [Figure 7] FIG. 7 shows the results of comparing the effect of the mesenchymal stem cells of the present invention on suppressing the expression of fibrosis-related gene ACTA2 with other cells (mesenchymal stem cells under conventional culture conditions). [Figure 8] FIG. 8 shows the results of comparing the effect of the mesenchymal stem cells of the present invention on suppressing the expression of fibrosis-related gene COLIA1 with other cells (mesenchymal stem cells under conventional culture conditions). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The mesenchymal stem cells and the therapeutic agent for liver disease of the present invention are described in detail below.

[0014] [Mesenchymal stem cells] The mesenchymal stem cells of the present invention are These tumors are characterized by high expression of tissue factor pathway inhibitor (TFA) and IFN-γ receptor agonist (IFN-γ receptor agonist).

[0015] TFPI is a glycoprotein with a molecular weight of approximately 42,000 that converts activated factor X into activated TF. Kunitz-type protein is thought to bind to factor VII and inhibit its blood clotting activity. It is an ase inhibitor [Broze, GJ, Proc. Natl. Acad. Sci.,84, p1886(1987)]. TFPI is mainly expressed in vascular endothelial cells, It is bound to the cell membrane, but the bond is broken by thrombin, MMPs, heparin, etc., and it becomes mobile. It is known to leave.

[0016] High expression of TFPI means high expression of TFPI mRNA, or TFPI protein is highly expressed, or both are highly expressed. .

[0017] In addition, the mesenchymal stem cells of the present invention may be sufficient if they express TFPI at a higher level than other cells. Specifically, the mesenchymal stem cells of the present invention are cultured under conventional culture conditions (e.g., in a culture medium containing 10% FBS). Compared to mesenchymal stem cells obtained by culturing in DMEM medium, Preferably, the number of mesenchymal stem cells obtained under conventional culture conditions is at least 1.5 times as large as that obtained under conventional culture conditions. Preferably, the expression is at least 2-fold, more preferably at least 3-fold, and particularly preferably at least 5-fold. .

[0018] The mesenchymal stem cells of the present invention have a higher TFPI activity than dermal fibroblasts, smooth muscle cells, and epithelial cells. It is sufficient that the IL-1 gene is highly expressed in skin fibroblasts, smooth muscle cells, or epithelial cells. 1.1 times or more, more preferably 1.15 times or more, and even more preferably 1.2 times or more Particularly preferably, two or more of skin fibroblasts, smooth muscle cells, and epithelial cells are expressed. More preferably, the number of cells is 1.1 times or more, even more preferably, the number of cells is 1.15 times or more, and even more preferably, the number of cells is 1.1 times or more. is expressed 1.2 times or more. More preferably, the expression level is increased in skin fibroblasts, smooth muscle cells, epithelial cells, and the like. For three or more of the cells, the number of cells is preferably 1.1 times or more, and most preferably 1.15 times or more. and most preferably, the expression is 1.2-fold or more.

[0019] In the present invention, mesenchymal stem cells are one or more types of cells belonging to the mesenchymal system (such as bone cells, cardiac muscle cells, etc.). , chondrocytes, tendon cells, adipocytes, etc.), preferably two or more types of cells, more preferably three types of cells. The term "cells" refers to cells that have the ability to differentiate into the above-mentioned cells and can proliferate while maintaining said ability. The term mesenchymal stem cells as used herein means the same cells as stromal cells, and the two are not specifically distinguished from each other. They are not differentiated from mesenchymal stem cells. They are also sometimes simply referred to as mesenchymal cells. Examples of tissues that can be used include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, etc. Examples of the tissue include the membrane, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. Adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue. Among these, from the viewpoint of efficacy in the treatment of liver disease, availability, From the viewpoint of ease of use, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, and adipose tissue-derived mesenchymal stem cells are preferred. More preferred are umbilical cord-derived mesenchymal stem cells.

[0020] The mesenchymal stem cells in the present invention may be derived from the same species as the subject to be treated. The species of mesenchymal stem cells in the present invention may be human, horse, or mouse. Examples of the animals include sheep, pigs, dogs, cats, rabbits, mice, and rats, and preferably The mesenchymal stem cells of the present invention are derived from the same species as the subject (specimen) being treated. They may be derived from the subject (subject), i.e., autologous (syngeneic), or from another They may be derived from the subject, i.e. allogeneic cells. Preferably, the allogeneic cells are (different lineage).

[0021] Mesenchymal stem cells are unlikely to cause rejection in allogeneic subjects, so The prepared donor cells are expanded and cryopreserved, and are used as the disease treatment agent of the present invention. Therefore, autologous mesenchymal stem cells can be prepared and used as mesenchymal stem cells. Compared to using synthetic drugs, it is easier to commercialize and it is easier to obtain stable and consistent results. From this viewpoint, the mesenchymal stem cells of the present invention are more preferably allogeneic.

[0022] In the present invention, mesenchymal stem cells refer to any cell population containing mesenchymal stem cells. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% are mesenchymal stem cells.

[0023] In the present invention, adipose tissue includes adipocytes and stromal cells including microvascular cells. For example, the tissue is obtained by surgically removing or aspirating subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferable to obtain the antibody from the same species of animal as the subject. More preferably, the fat is human subcutaneous fat. The fat source may be a living or dead individual. However, the adipose tissue used in the present invention is preferably collected from a living individual. When harvesting from an individual, liposuction is performed using, for example, PAL (power-assisted) liposuction. Examples include liposuction, Erconia laser liposuction, and body jet liposuction. From the viewpoint of maintaining the state of the cells, it is preferable not to use ultrasound.

[0024] In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and the placenta, and includes the umbilical vein, umbilical artery, and Veins, gelatinous tissue (Wharton's jelly), and umbilical cord matrix itself The umbilical cord is composed of the above-mentioned tissues and contains a large amount of mesenchymal stem cells. It is preferable that the agent is obtained from the same species as the subject of administration, and the agent is preferably administered to humans. Considering administration, a human umbilical cord is more preferable.

[0025] In the present invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and the cells present in it are called bone marrow cells. In addition to red blood cells, granulocytes, megakaryocytes, lymphocytes, and adipocytes, mesenchymal stem cells, hematopoietic stem cells, Bone marrow cells include, for example, human iliac, long bone, or other bone marrow cells. It can be obtained from the bones of

[0026] In the present invention, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, Mesenchymal stem cells derived from various tissues, such as adipose tissue-derived mesenchymal stem cells and umbilical cord stem cells, respectively. Any tissue-derived mesenchymal stem cells, including mesenchymal stem cells derived from various tissues, such as human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, etc. The cell population is at least 20%, preferably 30%, 40%, or more. 0%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 9 7%, 98% or 99% are adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells These are mesenchymal stem cells derived from various tissues, such as human mesenchymal stem cells.

[0027] The mesenchymal stem cells of the present invention highly express TFPI as described above, and furthermore, long-term characteristics (e.g., population doubling capacity, doubling time from passage to senescence), karyotyping (e.g., positive normal karyotype, maternal or neonatal lineage), flow cytometry (e.g., FACS analysis) Surface marker expression by immunohistochemistry and / or immunocytochemistry (e.g., epitope detection) gene expression profiling (e.g., gene chip arrays; reverse transcription PCR, reverse transcription PCR, real-time PCR, conventional PCR, and other polymerase chain reactions), miRNA expression profiling Rings, protein arrays, protein secretion such as cytokines (e.g., plasma coagulation analysis, ELISA, cytokine array), metabolites (metabolome analysis), and other methods known in the field The characteristics may be characterized by other methods, such as those described above.

[0028] (Method for preparing mesenchymal stem cells) The method for preparing mesenchymal stem cells with high TFPI expression is not particularly limited, but for example, That is, it can be prepared from tissues such as adipose tissue, umbilical cord, bone marrow, etc., by the method known to those skilled in the art. According to a known method, mesenchymal stem cells are isolated and cultured, and anti-TFP that specifically binds to TFPI is then administered. I antibody was used to separate TFPI-high expressing cells using a cell sorter or magnetic beads. In addition, by culturing in a specific medium, it is possible to obtain mesenchymal stem cells. By inducing TFPI expression, it is also possible to obtain mesenchymal stem cells with high TFPI expression. In the cell population obtained by this induction, more than 50% of the cell population highly expressed TFPI. It is preferable that 70% or more of the cells have high TFPI expression, and more preferable that 80% or more of the cells have high TFPI expression. More preferably, 90% or more of the subjects have high TFPI expression. It is particularly preferred that the cell population is a homogeneous cell population with substantially high TFPI expression. A method for preparing mesenchymal stem cells with high TFPI expression will be specifically described below.

[0029] Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. A method for preparing adipose tissue-derived mesenchymal stem cells is described below. It may be obtained, for example, by the manufacturing method described in U.S. Pat. No. 6,777,231, e.g. For example, it can be produced by a method including the following steps (i) to (iii): (i) obtaining a cell suspension by enzymatic digestion of adipose tissue; (ii) sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not exhibit binding to the solid surface.

[0030] The adipose tissue used in step (i) is preferably washed. using a physiologically compatible saline solution (e.g., phosphate-buffered saline (PBS)). This can be done by vigorously stirring and settling the adipose tissue. To remove debris (e.g. damaged tissue, blood, red blood cells, etc.) from tissue. Therefore, washing and settling are typically repeated until the supernatant is totally free of debris. The remaining cells are present as clumps of various sizes, so the cell In order to dissociate the cell aggregates while minimizing damage, the washed cell aggregates were subjected to a series of procedures that weakened the intercellular bonds or or by treating with enzymes that destroy collagen (e.g., collagenase, dispase, or trypsin, etc.). The amount of such enzyme and the duration of treatment vary depending on the conditions used. Alternatively or in combination with such enzyme treatment, cell masses may be treated with However, it can be broken down by other treatment methods such as mechanical agitation, ultrasonic energy, and thermal energy. However, in order to minimize damage to the cells, it is preferable to use only enzyme treatment. If used, it should be used after an appropriate period of time to minimize harmful effects on cells. It is preferable to inactivate the enzyme using a culture medium or the like.

[0031] The cell suspension obtained by step (i) may be a slurry or suspension of aggregated cells, and contains various contaminating cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. Then, the aggregated cells and the contaminant cells may be separated and removed. Since it can be removed by adhesion and cleaning in iii), the separation and removal are omitted. To separate and remove contaminating cells, centrifugation is used to forcefully separate the cells into a supernatant and a pellet. The resulting pellet containing the contaminating cells is suspended in a physiologically compatible solvent. The cells in suspension may contain red blood cells, but they may be selected by adhesion to solid surfaces as described below. Since red blood cells are excluded by the lysis step, the lysis step is not necessarily required. For example, the method of dissolving the cellulose in a hypertonic medium or a hypotonic medium by dissolving the cellulose in ammonium chloride is used. Methods well known in the art, such as incubation in culture medium, can be used. After lysis, the lysate is separated from the desired cells, for example by filtration, centrifugation, or density fractionation. Good too.

[0032] In step (ii), in order to increase the purity of mesenchymal stem cells in the suspension, The cells may be washed once or multiple times in succession, centrifuged, and resuspended in medium. Cells are separated based on cell surface marker profiles or based on cell size and granularity You may do so.

[0033] The medium used for resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells. However, such media may be supplemented with serum and / or albumin, thrombin, etc. Transferin, fatty acids, insulin, sodium selenite, cholesterol, collagen 1) 1. ... These media may be supplemented with one or more serum substitutes, if necessary. lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, Substances such as antioxidants, pyruvic acid, buffers, inorganic salts, etc. may also be added.

[0034] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle medium, and the like. e's Minimum Essential Medium (EMEM), αME M medium, Dulbecco's modified Eagle's Medium (D MEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's Examples of the medium include MCDB201 medium, MCDB201 medium, and a mixture of these media.

[0035] Examples of the serum include human serum, fetal bovine serum (FBS), bovine serum, and calf blood. Examples of serum include serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, and rat serum. When serum is used, it should be added at a concentration of 5 v / v% to 15 v / v% of the basal medium. % v / v, preferably 10% v / v.

[0036] The fatty acids include linoleic acid, oleic acid, linoleic acid, arachidonic acid, myris acid, Examples of such acids include acetic acid, palmitoyl acid, palmitic acid, and stearic acid. Lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, Examples of the amino acid include, but are not limited to, phosphatidylcholine. , L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine Contains L-cystine, L-glutamic acid, L-glutamine, L-glycine, etc. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and the like. Examples of polysaccharides include, but are not limited to, nectin and β2-microglobulin. Examples of glycosaminoglycans include hyaluronic acid, Examples of the growth factor include, but are not limited to, heparan sulfate. PDGF, basic fibroblast growth factor (bFGF), transformin Transforming Growth Factor Beta (TGF-β), Hepatocyte Growth Factor (HGF), Epidermal Growth Factor (EGF) , connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), etc. However, the present invention is not limited to these. The adipose-derived mesenchymal stem cells obtained in the present invention can be used for cell transplantation. From the viewpoint of the above, it is recommended to use a xeno-free medium that does not contain any xenogeneic components such as serum. Such a medium is preferably used. Logical Industries, Veritas, R&D Systems For mesenchymal stem cells (stromal cells), It is provided as a prepared medium.

[0037] Next, in step (iii), the cells in the cell suspension obtained in step (ii) are cultured without being differentiated. Cultivate the cells on a solid surface at the appropriate cell density and culture conditions using the appropriate cell culture medium described above. In the present invention, the term "solid surface" refers to a surface on which the adipose tissue-derived mesenchymal stem cells of the present invention are attached. In certain embodiments, such a material is any material that is capable of bonding or adhering. A plastic material that has been treated to promote the binding and adhesion of mammalian cells to the surface. The shape of the culture vessel having a surface is not particularly limited, but a petri dish or a flask is preferably used. After incubation, the cells are washed to remove unbound cells and cell debris. Wash the cells.

[0038] In the present invention, the cells that remain attached and bound to the solid surface are called adipose tissue-derived interferon. A cell population of mesenchymal stem cells can be selected.

[0039] The selected cells were confirmed to be adipose tissue-derived mesenchymal stem cells according to the present invention. In order to do so, surface antigens can be analyzed by conventional methods such as flow cytometry. In addition, the ability to differentiate into each cell lineage may be examined, and such differentiation may be This can be done in a conventional manner.

[0040] The mesenchymal stem cells of the present invention can be prepared as described above, but have the following characteristics: may be defined as a cell; (1) When cultured in standard medium, it exhibits adhesion to plastic. (2) Surface antigens CD44, CD73, and CD90 are positive, and CD31 and CD45 are negative and (3) They can be differentiated into bone cells, adipocytes, and chondrocytes under appropriate culture conditions.

[0041] From the mesenchymal stem cells obtained by the above step (iii), Selectively separate the cells by immunological techniques using a cell sorter, magnetic beads, etc. By doing so, it is possible to obtain mesenchymal stem cells that highly express TFPI protein. By culturing in a specific medium that can induce the expression of TFPI, mesenchymal stem cells To induce TFPI expression in mesenchymal stem cells and efficiently obtain mesenchymal stem cells with high TFPI expression. As an example, a specific method for selective separation by an immunological technique using a cell sorter is shown below. The method is explained below.

[0042] Cells obtained by treating the mesenchymal stem cells prepared above with trypsin-EDTA solution, etc. Centrifuge the suspension (room temperature, 400G, 5 minutes) and remove the supernatant. Add Buffer (1% BSA-PBS) and dilute to 1x10 6 cells / 500uL The cell suspension concentration was adjusted to a uniform level by pipetting, and then a new 1.5 mL Dispense 50uL into each microtube. Add the primary antibody (Mouse anti human TFPI, Sekisui diagnostics, A DG4903) was added at a concentration of 5-20 μg / mL, suspended, and then incubated in a dark, refrigerated container for 30 Incubate for 1 min to 1 hr. Wash 3 times with 1 mL of Staining Buffer. Add staining buffer to make 50uL and incubate with secondary antibody (Anti Mou se IgG alexar488, Thermofisher scientific A21202) was added at a concentration of 1-10 μg / mL and suspended in a refrigerator under light protection. Incubate for 30 minutes to 1 hour. Wash three times with 1 mL of Staining Buffer. After that, add PI Buffer (14.4 mL of staining buffer) pidium iodide solution (SIGMA, P4864)28. Add 300uL of the solution, suspend well, and pass through a tube equipped with a cell strainer. and fluorescence activated cell sorting (FA Separation can be achieved using the CS.

[0043] (Mesenchymal stem cell cryopreservation) The mesenchymal stem cells of the present invention can be appropriately cryopreserved and thawed as long as they have a disease treatment effect. In the present invention, cryopreservation is carried out by any method known to those skilled in the art. This can be done by suspending the mesenchymal stem cells in a preservation solution and cooling. The cells are then peeled off using a peeling agent such as cinnamate, transferred to a cryopreservation container, appropriately treated, and then frozen in a cryopreservation solution. This can be done by adding:

[0044] The cryopreservation solution contains DMSO (Dimethyl sulfoxide) as a cryoprotectant. ), but DMSO, in addition to being cytotoxic, has the potential to induce differentiation of mesenchymal stem cells. It is preferable to reduce the DMSO content because of its anti-inflammatory properties. Examples of suitable solvents include glycerol, propylene glycol, and polysaccharides. In this case, the concentration is 5% to 20%, preferably 5% to 10%, and more preferably 10%. In addition, the additives described in WO2007 / 058308 may be included. Examples of such cryopreservation solutions include those available from BioVerde, Inc. and Japan Genetics Co., Ltd. , ReproCell, Zenoac, Cosmo Bio, Kohjin Bio, Thermof A freezing medium provided by Fisher Scientific or the like may also be used.

[0045] When the above-mentioned suspended cells are frozen and stored, they are stored at a temperature between -80°C and -100°C (e.g., -80℃) and can be stored in any freezer that can achieve that temperature. Although not limited to this, a programmable freezer should be used to avoid sudden temperature changes. The cooling rate may be appropriately controlled depending on the components of the cryopreservation solution. This can be done according to the manufacturer's instructions for the cryopreservation medium.

[0046] The storage period is as follows: after thawing, cells cryopreserved under the above conditions retain the same properties as before freezing. As long as the period is within the limit, there is no particular upper limit. For example, one week or more, two weeks or more, three weeks or more , 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more Storage at lower temperatures can suppress cell damage. Therefore, they are transferred to the gas phase above liquid nitrogen (below -150°C to below -180°C) and stored. When storing in the vapor phase above liquid nitrogen, storage containers well known to those skilled in the art may be used. Although not particularly limited, for example, when storing for 2 weeks or more, the gas phase above liquid nitrogen can be used. It is preferable to store it at .

[0047] The thawed mesenchymal stem cells may be appropriately cultured until the next cryopreservation. The culture is carried out using a medium capable of culturing the above-mentioned mesenchymal stem cells, and is not particularly limited thereto. The culture is carried out at a culture temperature of about 30 to 40°C, preferably about 37°C, in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. Confluency appropriate for the vessel (e.g., 50% to 80% for culture vessels) should be maintained. After reaching the cell-free stage, the cells are detached by a detaching agent such as trypsin. The cells may be detached and seeded at an appropriate cell density in a separate culture vessel for continued culture. A typical cell density for seeding is 100 cells / cm. 2 ~100,000 cells / cm 2 , 500 cells / cm 2 ~50,000 cells / cm 2 , 1,000~10,0 00 cells / cm 2 , 2,000~10,000 cells / cm 2 Examples include: In this study, the cell density was 2,000–10,000 cells / cm. 2 The appropriate confluency It is preferable to adjust the time until the culture reaches the plateau within 3 to 7 days. During cultivation, the medium may be replaced as needed.

[0048] Cryopreserved cells can be thawed by methods well known to those skilled in the art. For example, thawing at 37°C An example of the method involves leaving the mixture at rest or shaking it in a thermostatic chamber or a hot water bath.

[0049] The mesenchymal stem cells of the present invention may be in any state, for example, cells in culture. The cells may be cells that have been detached and collected, or may be cells that have been frozen in a cryopreservation solution. By using cells from the same lot obtained by expansion and freezing, the cells were aliquoted and stored stably. It is preferable in that the same action and effect can be obtained by freezing and that it is easy to handle. The mesenchymal stem cells in the cryopreservation state are thawed immediately before use and then suspended in the cryopreservation solution and infused or cultured. Alternatively, the freezing solution may be removed by centrifugation or other methods before mixing. The cells may be suspended in a solution such as a liquid infusion or a culture medium. The term refers to a solution used in the treatment of a patient, and is not particularly limited, for example, physiological saline. , Japanese Pharmacopoeia Physiological Saline Solution, 5% Glucose Solution, Japanese Pharmacopoeia Glucose Injection, Ringer's Solution, Japanese Pharmacopoeia Ringer's Solution , Lactated Ringer's solution, Acetated Ringer's solution, No. 1 solution (starting solution), No. 2 solution (dehydration replenishment solution), No. 3 solution (maintenance fluid), No. 4 fluid (post-operative recovery fluid), etc.

[0050] [Liver disease treatment] The therapeutic agent for liver disease of the present invention contains the above-mentioned mesenchymal stem cells highly expressing TFPI of the present invention. The therapeutic agent for liver disease of the present invention can effectively suppress liver fibrosis. Regarding mesenchymal stem cells contained in the current liver disease treatment agent, the explanation in the mesenchymal stem cells section above can be applied. Cut.

[0051] The therapeutic agent for liver disease of the present invention may contain the above-mentioned mesenchymal stem cells within a range that does not impair the effects of the present invention. In addition, depending on the purpose and form, pharma- ceutically acceptable carriers and additives may be included in the formulation in accordance with the usual methods. Such carriers and additives may include, for example, isotonicity agents, thickeners, sugars, sugars, etc. Alcohols, preservatives, germicides or antibacterial agents, pH regulators, stabilizers, chelating agents Agents, oil bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, Foaming agents, fluidizing agents, dispersing agents, emulsifying agents, buffering agents, solubilizing agents, antioxidants, sweetening agents, acidulants, Examples of the coloring agent, flavoring agent, fragrance, or refreshing agent, but are not limited to these. Examples of the components include the following carriers and additives.

[0052] Examples of the carrier include aqueous carriers such as water and aqueous ethanol; Examples of suitable chlorides include sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. Examples of polyhydric alcohols include glycerin, propylene glycol, and polyethylene. glycols, etc.; thickeners include, for example, carboxyvinyl polymers, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, alginic acid, Polyvinyl alcohol (completely or partially saponified), polyvinylpyrrolidone, macrogol Examples of sugars include cyclodextrin and glucose; examples of sugar alcohols include Examples of such saccharides include xylitol, sorbitol, and mannitol (which may be d-, l- or The preservative, bactericide or antibacterial agent may be, for example, dibutyl Hydroxytoluene, butyl hydroxyanisole, alkyldiaminoethyl glycine hydrochloride Benzalkonium chloride, benzethonium chloride, glycerol, sodium benzoate, ethanol Chlorhexidine disodium EDTA, chlorobutanol, sorbic acid, potassium sorbate, thoro Metamol, sodium dehydroacetate, methyl parahydroxybenzoate, parahydroxybenzoic acid Ethyl, propyl parahydroxybenzoate, butyl parahydroxybenzoate, oxyquinoline sulfate , phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhydrochloride, Rehexanide (polyhexamethylene biguanide), etc.), Gloquil (Rhodia product) Examples of pH regulators include hydrochloric acid, boric acid, aminoethylsulfonic acid, ip Silylaminocaproic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, hydroxyl Calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, Triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, malate Magnesium, phosphoric acid, polyphosphoric acid, propionic acid, oxalic acid, gluconic acid, fumaric acid, milk Acid, tartaric acid, malic acid, succinic acid, gluconolactone, ammonium acetate, etc.; stabilizers Examples of the butyl hydroxytoluene include dibutyl hydroxytoluene, trometamol, and sodium formaldehyde. Aldehyde sulfoxylate (Rongalit), tocopherol, sodium pyrosulfite, Monoethanolamine, Aluminum Monostearate, Glyceryl Monostearate, Sodium hydrogen sulfite, sodium sulfite, etc.; oily bases, e.g., olive oil , vegetable oils such as corn oil, soybean oil, sesame oil, cottonseed oil, and medium-chain fatty acid triglycerides. Examples of aqueous bases include Macrogol 400, etc. Examples of gel bases include Ca Examples of surfactants include polysorbate 8. 0, hydrogenated castor oil, glycerin fatty acid ester, sorbitan sesquioleate, etc.; Suspension Examples of the additives include beeswax, various surfactants, gum arabic, and gum arabic. powder, xanthan gum, soy lecithin, etc.; binders, e.g., hydroxyethyl cellulose, cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, calcium Sodium carboxymethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. excipients include, for example, sucrose, lactose, starch, corn starch, and crystalline cellulose , light anhydrous silicic acid, etc.; lubricants include, for example, sucrose fatty acid esters, stearic acid magnesium, talc, etc.; disintegrants include, for example, low-substituted hydroxypropylcellulose. Examples of the foaming agent include crospovidone, croscarmellose sodium, etc.; , sodium bicarbonate, etc.; as a fluidizing agent, for example, sodium aluminometasilicate, and light anhydrous silicic acid.

[0053] The therapeutic agent for liver disease of the present invention can be in various forms, such as a solid agent, a semi-solid agent, or a liquid agent, depending on the purpose. For example, it can be provided in a variety of dosage forms, such as solid formulations (tablets, powders, powders, granules, Capsules, etc.), semi-solid preparations [ointments (hard ointments, soft ointments, etc.), creams, etc.], liquid preparations [ Lotions, extracts, suspensions, emulsions, syrups, injections (infusions, implant injections) , sustained release injections, and ready-to-use injections), dialysis agents, aerosols, soft capsules, The present invention can be used in the form of a drink, a patch, a cataplasm, etc. The composition of the present invention can also be used in the form of a solution or emulsion in an oily or aqueous vehicle. The liver disease therapeutic agent of the present invention can be applied to the affected area by spraying. The present invention can also be used in the form of a gel or sheet that is applied to the affected area after application. The agent can also be applied to the affected area after forming the above-mentioned mesenchymal stem cells into a sheet or three-dimensional structure. Cut.

[0054] The therapeutic agent for liver disease of the present invention is preferably a physiological saline solution, a Japanese Pharmacopoeia physiological saline solution, a 5% glucose solution, a Japanese Pharmacopoeia glucose solution, Glucose injection, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetated Ringer's solution, bicarbonate Ringer's solution Gel liquid, No. 1 liquid (initiation liquid), No. 2 liquid (dehydration rehydration liquid), No. 3 liquid (maintenance liquid), No. 4 liquid (postoperative recovery liquid) The cells are suspended or diluted in infusion fluids such as erythrocyte reconstitution fluid or cell culture media such as DMEM. Preferably, physiological saline, 5% glucose solution, or No. 1 solution (starting solution) is used. Alternatively, it can be suspended or diluted with 5% glucose solution or No. 1 solution (starting solution) and used.

[0055] When the hepatic disease therapeutic agent of the present invention is a liquid preparation, the pH of the hepatic disease therapeutic agent is determined based on medical and pharmacological viewpoints. There are no particular limitations as long as it is within a pharma- ceutical or physiologically acceptable range, but For example, 2.5 to 9.0, preferably 3.0 to 8.5, more preferably 3.5 to 8.0 The range in which

[0056] When the hepatic disease therapeutic agent of the present invention is a liquid preparation, the osmotic pressure of the hepatic disease therapeutic agent should be acceptable to the living body. As long as it is within the acceptable range, there is no particular limitation. An example of the osmotic pressure ratio of the composition of the present invention is as follows: Preferably, it is 0.7 to 5.0, more preferably, it is 0.8 to 3.0, and further preferably, it is 0.9 to The range where the osmotic pressure is 1.4 is one example. Osmotic pressure can be adjusted using inorganic salts, polyhydric alcohols, and sugar alcohols. The osmolarity ratio can be determined by any method known in the art, using, for example, sugars, etc. Based on the revised Japanese Pharmacopoeia, the permeability of 286 mOsm (0.9 w / v% sodium chloride solution) The osmotic pressure is the ratio of the osmotic pressure of the sample to the pressure of the sample itself. The osmotic pressure is measured by the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. ) is used as a reference for the measurement. Aqueous solution) is prepared by heating sodium chloride (Japanese Pharmacopoeia standard reagent) at 500-650℃ for 40-50 minutes. After drying for 1 hour, cool it in a desiccator (silica gel) and weigh 0.900 g accurately. Dissolve in purified water to make exactly 100 mL, or use a commercially available standard solution for measuring osmolality (0 .9 w / v% sodium chloride aqueous solution) is used.

[0057] The administration route of the therapeutic agent for liver disease of the present invention to a subject is oral administration, subcutaneous administration, intramuscular administration, or intravenous administration. Intravenous, intraarterial, intrathecal, intraperitoneal, sublingual, rectal, vaginal, intraocular For administration via nasal route, inhalation, transdermal administration, implantation, spraying onto the liver surface, and application as sheets, etc. From the viewpoint of the effectiveness of the therapeutic agent for liver disease of the present invention, preferred are Implants, hepatic artery administration, intravenous administration, spraying on the liver surface, or applying sheets, etc. It is a direct administration, and from the viewpoint of reducing the burden on the subject, it is more preferably an intravenous administration.

[0058] The dosage of the therapeutic agent for liver disease of the present invention is determined according to the condition of the patient (body weight, age, disease, etc.). Although it may vary depending on the patient's condition, physical condition, etc., and the dosage form of the hepatic disease therapeutic agent of the present invention, From the viewpoint of exerting the therapeutic effect of the therapeutic agent, a large amount tends to be preferable. From the viewpoint of suppressing the onset of side effects, a small amount tends to be preferable. When administered, the cell number is 1x10 3 ~1x10 12 Pieces per time, preferably 1x1 0 4 ~1x10 11 Pieces / time, preferably 1x10 5 ~1x10 10 pcs / time, even better Preferably 5x10 6 ~1x10 9 The dosage per patient's weight is is 1x10~5x10 10 Pieces / kg, preferably 1x10 2 ~5x10 9 Pieces / kg, More preferably 1x10 3 ~5x10 8 / kg, more preferably 1x10 4 ~5x10 7 This dosage may be administered multiple times as a single dose, or multiple doses of this dosage may be administered multiple times as a single dose. It may be administered in divided doses.

[0059] The therapeutic agent for liver disease of the present invention may be administered together with one or more other drugs. The drug may be any drug that can be used as a liver treatment agent, for example: Hepatitis B drugs (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon Interferon preparations (interferon α, interferon α-2b, interferon β, pegylated interferon α-2a, pegylated interferon α-2b, etc.), hepatitis C treatment drugs ( Bavirin, telapirevir, simeprevir, vaniprevir, daclatasvir, asunaprevir corticosteroids (prednisolone, methylprednisolone chloride, etc.), sodium citrate, etc.), anticoagulants (dried concentrated human antithrombin III, Gabexa thrombomodulin α, etc.), antidotes (edetate calcium disodium Hydrate, Glutathione, Dimethicone, Sodium Thiosulfate Hydrate, Sugamadesucina thorium, etc.), human serum albumin, liver extract, ursodeoxycholic acid, glycyl Lithic acid, azathioprine, bezafibrate, amino acids (glycine, L-cysteine , L-isoleucine, L-leucine, L-valine, L-threonine, L-serine, L-amino Lanine, L-Methionine, L-Phenylalanine, L-Tryptophan, L-Lysine, L -histidine, L-arginine and their salts, etc.), vitamins (tocopherol, flavin Adenine dinucleotide, thiamine disulfide phosphate, pyridoxine, cyanocobalamin amine and their salts, etc.), antibiotics (sulbactam sodium, cefoperazone sodium Examples of such antibacterial agents include vancomycin, meropenem hydrate, and vancomycin hydrochloride.

[0060] The mesenchymal stem cells of the present invention can be used for various liver diseases and liver disorders. These include autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, and non- alcoholic fatty liver disease (nonalcoholic fatty liver disease) sease (NAFLD), nonalcoholic fatty liver disease (nonalcoholic s teatohepatitis (NASH), nonalcoholic fatty liver disease holic fatty liver (NAFL)), liver fibrosis, liver cirrhosis, liver cancer, fat Liver, drug-induced allergic liver disease, hemochromatosis, hemosiderosis, Wilson's disease, Primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic Examples of liver diseases include active hepatitis and chronic persistent hepatitis. Since it has an anti-fibrosis effect, it is effective for diseases accompanied by fibrosis of liver tissue, such as liver fibrosis and liver cirrhosis. It can be suitably used as such.

[0061] <Treatment methods for liver disease> According to yet another aspect of the present invention, the present invention provides a tissue factor pathway inhibitor (TFPI) The present invention also includes a method for treating liver disease, which is characterized by using mesenchymal stem cells that highly express the above-mentioned gene. That is, according to the present invention, a mesenchymal stem cell line in which tissue factor pathway inhibitor (TFPI) is highly expressed is Treating liver disease, especially liver disease accompanied by fibrosis, by administering stem cells to patients with liver disease. The mesenchymal stem cells used in the treatment method of the present invention can be improved. The explanations in the above sections [Mesenchymal stem cells] and [Treatment agent for liver disease] are applicable. EXAMPLES

[0062] The present invention will be described in detail below with reference to examples and test examples. The examples are not intended to be limiting.

[0063] 1. Study on the effect of rTFPI in suppressing fibrosis in human hepatic stellate cell lines [Culture of human hepatic stellate cell line] Cryopreserved human hepatic stellate cell line Cells (HHSteC), ScienCell Research Labora A cell suspension (manufactured by Tories, product number: 5300) was immersed in a 37°C incubator and thawed. Add Stellate Cells to the thawed HHSteC suspension until the total volume is 2 mL. Medium(ScienCell Research Laboratories) Add 15 μL of the HHSteC suspension to an equal amount of Trypa Mix with 0.4% Blue Solution and count the number of live and dead cells. The amount of cell suspension required for seeding was dispensed into a new 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd.). The HHSteC suspension was diluted with Stellate C to a total volume of 30 mL. Add 10 mL of POLY-L-Lysine medium to each well and place on three sheets of POLY-L-Lysine coa ted 100mm dish (hereinafter, "100mm dish", IWAKI Co., Ltd., product The cells were seeded on a 100 mm dish (No. 4020-040). Place in an incubator (37℃, 5% CO2) and after 1 day, replace with a new Stellate Cell. The medium was replaced with ll Medium and culture was continued. Four days after seeding of HHSteCs The medium was removed from the 100 mm dish and the inside of the dish was washed with 10 mL of PBS. After removing BS, StemPro Accutase Cell Dissociation n Reagent (Thermo Fisher Scientific, A11 Add 2 mL of 105-01, Lot. 1750154 to each 100 mm dish and incubate with CO 2 Incubate in the incubator for 5 minutes. Centrifuge 50 mL of the HHSteC suspension. After transferring to a tube, add Stellate Cell Medium 1 to each 100 mm dish. Add 100 mL of centrifuge tube and collect the remaining HHSteC in the same 50 mL centrifuge tube. The mixture was centrifuged for 5 minutes (Eppendorf, 5702). The supernatant was removed and the HHSteC was Resuspend in 10 mL of tellate Cell Medium, and aliquot 15 μL. Mix with a small amount of Trypan Blue Solution (0.4%) to measure the number of live cells and dead cells. The number of cells was counted. After calculating the amount of cell suspension required for seeding, Stellate Cell Medium up to 7.6×10 4H to obtain a concentration of 1.2g / mL A suspension of HSteC was prepared using poly-L-lysine coated microfibers. Plate 12 well (hereinafter referred to as "12 well plate", IWAKI Co., Ltd., product number: 4815-040) in 1 mL (7.6 x 10 4 cell) per HHSte C was seeded and placed in a CO2 incubator (37°C, 5% CO2) to start culturing. .

[0064] [Suppression of fibrosis-related gene expression by rTFPI] One day after the start of culture, the 12-well plate seeded with HHSteCs was incubated in a CO2 incubator. Remove from the incubator, remove the medium from the wells corresponding to group number 1, and add Stellat e 1 mL of Cell Medium was added (Control group). Corresponding to group number 2 Remove the medium from the wells to be incubated and add Recombinant TFPI (R&D System s Company, 2974-PI-010) 1ng / mL and Stellate Cell Me The culture medium in the wells corresponding to group 3 was added with 1 ng / mL TFPI. The medium was removed, and the cells were incubated with 1,000ng / mL recombinant TFPI and Stell. Cell Medium (TFPI 1,000ng / mL added group) was added. After culturing for one day in a CO2 incubator, the total RNA of HHSteC was A was collected and the mRNA expression levels of ACTA2 and COL1A1, which are fibrosis-related factors, were quantified. The results were measured by PCR. The primers used were those listed in the table below. The results are shown in Figure 1 (ACTA 2) and 2(COL1A1).

[0065] [Table 1]

[0066] As shown in Fig. 1 and 2, the mRNA expression of ACTA2 and COL1A1 in HHSteCs A expression was suppressed by rTFPI treatment.

[0067] 2. Comparison of TFPI expression levels in various cell lines Human adipose-derived mesenchymal stem cells (L-ADSC, manufactured by Lonza), human dermal fibroblasts ( hDFa, Human Dermal Fibroblasts, adult, Ther mo Fisher Scientific), human aortic smooth muscle cells (hASMS , Human Aortic Smooth Muscle Cells, Thermo Fisher Scientific) and HeLa cells (Hela, Inc.) were 5,000 cells / cm in a well plate (Corning, #3335) 2 Sowing in The medium suitable for each cell type was serum-free medium for mesenchymal stem cells (Rohto), C ascade Biologics Medium106+LSGS(Thermo F isher Scientific, #M-106-500), 10%FBS DMEM Culture medium (Sigma, #D5796), Smooth muscle cell growth After culturing for 3 days in th medium (promocell, #C-22062), Total RNA was collected and TFPI mRNA expression was confirmed by quantitative PCR. The primers used were those listed in the table below (custom primers for real-time PCR ( (Manufactured by Lofin Genomics, Inc.) The results are shown in Figure 3.

[0068] [Table 2]

[0069] As shown in Figure 3, mesenchymal stem cells cultured in Rohto's medium outperformed other cells (human human dermal fibroblasts (hDFa), human aortic smooth muscle cells (hASMS), HeLa cells TFPI mRNA was found to be highly expressed in comparison with Roht The expression of TFPI mRNA in mesenchymal stem cells cultured in Company O's medium was significantly higher than that in hDFa. 1.34-fold for hASMS, 1.65-fold for hASMS, and 2.14-fold for HeLa cells. The expression was high, 2-fold.

[0070] 3. Enhancement of TFPI expression in adipose-derived mesenchymal stem cells [Preparation of adipose-derived mesenchymal stem cells] After obtaining informed consent from human donors, subcutaneous adipose tissue obtained by liposuction was washed with saline. To achieve the disruption of the extracellular matrix and the isolation of the cells, collagenase (Roche d iagnostics) (solvent is physiological saline) was added, and the mixture was shaken at 37°C for 90 minutes. The suspension was then centrifuged at 800 g for 5 minutes to precipitate the stromal vascular cells. A serum-free medium for mesenchymal stem cells (Rohto) was added to the cell precipitate, and the cells were cultured. The cell suspension was centrifuged at 400 g for 5 minutes, and the supernatant was removed. Serum-free medium for mesenchymal stem cells (Ro The cells were resuspended in 5% CO2 (HTO) and seeded in flasks. The cells were incubated at 37°C for several days in 5% CO2 After a few days, the cultures were washed with PBS to remove blood cells and lipids contained in the culture medium. Remaining tissues were removed, and mesenchymal stem cells attached to the plastic container were obtained.

[0071] [Analysis of cell surface markers (flow cytometry)] Evaluation of various surface markers on adipose tissue-derived mesenchymal stem cells by flow cytometry The adipose tissue-derived mesenchymal stem cells were resuspended in FACS staining buffer. The antibodies used in the FACS analysis were FITC (fluorescent isocyanine) or PE (phycoerythrin). Phosphorus-labeled mouse anti-human antibodies CD45, CD73, CD90, and the corresponding mouse Ig G1 isotype control antibody. Cells were stained for 30 min at room temperature and then washed. , BDFADSCantoII (BD Biosciences, San Jose, C A) was used for analysis. Data was analyzed using BD FACSDiva Software (BD As a result, adipose tissue-derived mesenchymal stem cells (hereafter referred to as The ADSCs (below, "ADSC") were negative for CD45 and positive for CD73 and CD90.

[0072] [Cryopreservation of adipose tissue-derived mesenchymal stem cells] The obtained ADSCs were detached using trypsin, transferred to a centrifuge tube, and centrifuged at 400 × g for 5 min. After removing the supernatant, the cells were frozen in a cell freezing medium (STEM-CEL An appropriate amount of LBANKER (Zenoak) was added to suspend the cells. After dispensing into tubes, store in a freezer at -80°C, then transfer to the vapor phase above liquid nitrogen. The storage continued.

[0073] [Enhanced TFPI expression in adipose-derived mesenchymal stem cells] ADSCs were treated with serum-free medium for mesenchymal stem cells (Rohto) from P2 to P4, respectively. roAD (Procal; manufactured by Rohto), serum-free medium (manufactured by Lonza), 10% F The P4 cells were cultured in BS serum medium and frozen stocks were prepared. 5,000 cells / cm2 and cultured in each of the four media for 3 days. After culturing, total RNA, protein, and culture supernatant were collected. Expression and protein expression were analyzed by quantitative PCR (Figure 4) and Western blotting (Figure 5), respectively. 5) was used to detect TFPI concentrations in the culture supernatant. Using the ikine ELISA Kit (R&D systems, #DTFP10) The measurements were made (Figure 6).

[0074] Compared with the 10% FBS serum medium, all serum-free media significantly increased TFPI mRNA expression. The expression level of TFPI (Figure 4) and protein expression (Figure 5) were high in the 14-HT10 cells. As shown in the figure, the secretion of IL-1 in the culture supernatant was greater in all serum-free media than in the 10% FBS serum medium. Specifically, the amount of TFPI detected in the mesenchymal stem cells of Rohto was significantly higher than that in the control group. The expression of TFPI mRNA in mesenchymal stem cells cultured in serum-free medium was 1.16 times higher than when cultured in Al medium, and 1.16 times higher than when cultured in Lonza medium. The expression was 2.64 times higher than when cultured in 10% FBS serum medium, and 6.92 times higher than when cultured in 10% FBS serum medium. In addition, the amount of TFPI secreted into the culture supernatant was measured using Rohto's mesenchymal stem cell The amount of TFPI secreted into the culture supernatant of serum-free medium (pg / mL) was 2.54 times higher than the culture supernatant of Lonza medium, 5.43 times higher than the culture supernatant of Lonza medium, and 1 This was 5.83 times higher than that of the culture supernatant in 0% FBS serum medium.

[0075] 4. Enhancement of anti-fibrotic activity of adipose-derived mesenchymal stem cells As in 3 above, ADSCs were cultured in serum-free medium for mesenchymal stem cells (Ro The medium was cultured in serum-free medium (Lonza), 10% FBS serum medium (Lonza), and 10% FBS serum medium. and prepared frozen stocks.

[0076] Cryopreserved ADSCs were immersed in a 37℃ incubator to thaw the cell suspension, and then 15 mL The cells were separated into centrifuge tubes and placed in serum-free medium for mesenchymal stem cells (Rohto) and serum-free medium (L Onza), and then dilute to 10 mL with 10% FBS serum medium at room temperature and 400 × g. After centrifugation, the supernatant was removed and each medium was Add 6 mL of Trypan Blue Solution to the cell suspension, and then add 15 μL of the cell suspension to the cell suspension. The cells were mixed with 0.4% cation and the number of live and dead cells was counted. Add each medium to a new 15 mL centrifuge tube until the viable cell concentration is 1.52 x 10 4 Cells The transwell insert was placed on each well of a 12-well plate at a concentration of 1 / mL. A 100-well plate (corning, #3460) was placed in the lower compartment, and 1 mL of each medium was placed in the lower compartment. In addition, the transwell insert contained 1.52 × 10 viable cells. 4 Prepared in cells / mL 0.5 mL of each of the prepared ADSCs was added and the cells were seeded. The cells were then placed in a CO2 incubator (7°C, 5% CO2) and culture was started. One day after the start of culture, the cells were placed in a CO2 incubator. Remove the plate with the cells from the incubator (37°C, 5% CO2) and place it in the lower compartment. Discard the medium in the compartment and wash with 1 mL of PBS. Remove the PBS and place Ste 1 mL of Late Cell Medium was added to each transwell insert. Discard the medium, wash twice with 0.5 mL of PBS, remove the PBS, and then Stellate C. 0.5 mL of Cell Medium was added.

[0077] As in 1 above, HHSteCs were seeded on a 12-well plate and cultured for 1 day. After removing the wells from the incubator and removing the medium, the cells were l 1mL of Medium was added. On the wells corresponding to Group No. 1 (Control group) Place an empty transwell insert in Stellate Cell Medium 0.5 mL of the medium for mesenchymal stem cells was added to the wells corresponding to groups 2 to 4. Serum medium (Rohto, Rohto group), serum-free medium (Lonza, Lonza group) ), and ADSCs cultured in 10% FBS serum medium (10% FBS group) were seeded on the transgenic mice. The plate with the transwell insert was placed in a CO2 incubator. Place the plate in an incubator (37℃, 5% CO2) and start co-culture of HHSteCs and ADSCs. One day after the start of co-culture, total RNA of HHSteC was collected and lineaged. The mRNA expression levels of ACTA2 and COL1A1, which are fibrosis-related factors, were measured by quantitative PCR. The results are shown in Figures 7 (ACTA2) and 8 (COL1A1).

[0078] The expression of ACTA2 and COL1A1 mRNA in HHSteCs was significantly different from that in ADSCs. The co-culture significantly suppressed the inflammatory response. The suppressive effect was greater than that of ADSCs cultured in serum medium. In addition, ADSCs cultured in serum-free medium for mesenchymal stem cells (Rohto) showed significantly higher was confirmed. [Industrial Applicability]

[0079] INDUSTRIAL APPLICABILITY The present invention provides novel mesenchymal stem cells and therapeutic agents for liver diseases containing the same.

Claims

[Claim 1] The invention described in the specification.

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