Preventive or therapeutic agent for organ fibrosis

Abstract

The present invention aims to provide a cell preparation for the prevention and / or treatment of organ fibrosis such as liver fibrosis that is safe and easy to prepare, and provides a cell preparation for the prevention and / or treatment of organ fibrosis such as liver fibrosis, which contains SSEA-3-positive pluripotent stem cells (Muse cells) derived from biological mesenchymal tissue or cultured mesenchymal cells.

Description

[Technical field]

[0001] The present invention relates to a cell preparation for regenerative medicine, more specifically to a cell preparation containing pluripotent stem cells that is effective for repairing and regenerating organs suffering from fibrosis. [Background technology]

[0002] Organ fibrosis is a disease that occurs when organs are damaged or necrotic due to infection, inflammation, accumulation of substances in the body such as fat, and degeneration of tissues and cells caused by a variety of factors (including unknown causes) such as reactions in the body such as microorganisms, chemicals, and immune reactions, dietary habits, the environment, and genetic background, and when the organs do not regenerate sufficiently, the connective tissue increases and becomes fibrotic. Organ fibrosis is known to occur in many organs and tissues, including the liver, lungs, heart, kidneys, and nervous system, as well as muscles, bones, and skin.

[0003] Cirrhosis, a type of fibrosis that occurs in the liver, is a pathological condition that is ultimately reached as a result of chronic liver disease caused by various factors, and results in a significant decline in liver function due to a decrease in functional liver cells and an increase in fibrous tissue. The number of patients with cirrhosis is estimated to be approximately 300,000 in Japan and approximately 20 million worldwide, but there is no established effective medical treatment for serious liver failure caused by cirrhosis. At present, the main treatment is to slow the progression of chronic liver disease to cirrhosis using various symptomatic treatments.

[0004] Pulmonary fibrosis includes fibrosis caused by lung damage associated with pneumonia such as interstitial pneumonia, as well as idiopathic pulmonary fibrosis of unknown cause, which is a disease with poor prognosis that causes cough, chest pain, dyspnea, etc. Myocardial fibrosis is a condition in which the tissues of the myocardium and heart valves become fibrotic due to underlying diseases such as coronary circulatory failure, cardiomyopathy caused by infection or immune reaction, and valvular disease, and if it progresses, it can lead to heart failure. In the kidney, when fibrosis occurs in kidney tissue with the progression of chronic kidney disease, kidney function rapidly deteriorates and reaches a state where recovery is impossible. As such, it is known that fibrosis occurs in various organs and tissues with the progression of the primary disease, but in any disease, the progression of fibrosis leads to an irreversible deterioration of the function of the organ or tissue, and is a pathology with an extremely poor prognosis that is difficult to treat with current medical science.

[0005] The only effective treatment for liver cirrhosis is liver transplantation, but this has many problems, such as a shortage of organ donors, high medical costs, and in the case of living donor liver transplants, the risk to the donor. In addition, despite the fact that the number of people waiting for liver transplants is increasing year by year, there is a shortage of organ donors, and so the increase in the number of people who die while waiting for a transplant is also a major issue.

[0006] In recent years, stem cell transplantation has been attracting attention as a possible alternative treatment to transplantation of pathological organs. For example, it has been reported that mesenchymal stem cells (MSCs) suppress liver fibrosis and inflammation in chronic liver disease (e.g., Non-Patent Document 1). However, MSCs have a low frequency of engraftment in damaged liver tissue and formation of new liver cells, and most of the effects of suppressing fibrosis and reducing inflammation were due to the anti-inflammatory action, fibrosis inhibitory factors, and protective factor production based on MSCs (e.g., Non-Patent Document 2). In addition, research is also progressing on inducing hepatic progenitor cells from embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) and transplanting them into the liver, but there are still important issues to be overcome, such as the presence of mixed undifferentiated cells and cancer due to genomic instability. Therefore, it is hoped that a fundamental solution will be provided by an efficient stem cell therapy that can replenish functional liver cells.

[0007] Research by Idezawa, one of the present inventors, has revealed that pluripotent stem cells (Multilineage-differentiating Stress Enduring cells; Muse cells) that are present in the mesenchymal cell fraction and express SSEA-3 (Stage-Specific Embryonic Antigen-3) as a surface antigen, which can be obtained without gene transfer or induction using cytokines, etc., are responsible for the pluripotency of the mesenchymal cell fraction and may be applicable to disease treatment aimed at tissue regeneration (Patent Document 1; Non-Patent Documents 3 to 5). However, there have been no examples that have demonstrated that the expected therapeutic effects can be obtained by using Muse cells for the prevention and / or treatment of fibrosis. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5185443 specification [Non-patent literature]

[0009] [Non-Patent Document 1] Transplantation 2004; 78: 83-88 [Non-Patent Document 2] The Journal of surgical research 2014; 186: 408-416 [Non-Patent Document 3] Proc. Natl.Acad.Sci.USA,2010; 107: 8639-8643 [Non-Patent Document 4] Proc. Natl.Acad.Sci.USA,2011; 108: 9875-9880 [Non-Patent Document 5] Nat. Protc., 2013; 8: 1391-1415 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a cell preparation for the prevention and / or treatment of organ fibrosis. [Means for solving the problem]

[0011] The inventors have discovered that in a liver fibrosis model in immunodeficient mice that do not reject human cells, intravascular administration of human Muse cells causes the Muse cells to accumulate and engraft in the damaged liver, reconstructing and repairing the damaged liver, thereby improving or restoring liver function. They have therefore discovered that Muse cells can be suitably used for the treatment and prevention of organ fibrosis, including liver fibrosis, and have thus completed the present invention.

[0012] That is, the present invention is as follows. [1] A cell preparation for preventing and / or treating organ fibrosis, comprising SSEA-3-positive pluripotent stem cells derived from mesenchymal tissue of a living body or cultured mesenchymal cells. [2] The cell preparation described in [1], wherein the organ fibrosis is fibrosis occurring in the digestive system, respiratory system, circulatory system, urogenital system, musculoskeletal system, cranial nervous system, endocrine organs, or skin. [3] The cell preparation described in [2], wherein the organ fibrosis is fibrosis occurring in the digestive tract. [4] The cell preparation described in [3], wherein the organ fibrosis is liver fibrosis. [5] The cell preparation described in [4], wherein the pluripotent stem cells have the ability to differentiate into hepatoblasts or cells expressing hepatocyte markers. [6] A cell preparation described in [4] or [5], which, when administered to a subject, improves serum total bilirubin and / or serum albumin levels compared to a non-administered control. [7] The cell preparation described in [2], wherein the organ fibrosis is fibrosis occurring in the skin. [8] The cell preparation described in [2], wherein the organ fibrosis is pulmonary fibrosis. [9] The cell preparation according to any one of the above [1] to [8], wherein the pluripotent stem cells are pluripotent stem cells having all of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability.

[10] A cell preparation for inhibiting tissue fibrosis and / or dissolving fibrotic tissue, comprising SSEA-3-positive pluripotent stem cells derived from a living body's mesenchymal tissue or cultured mesenchymal cells. Effect of the Invention

[0013] In the present invention, Muse cells are administered to a subject suffering from fibrosis via a blood vessel or the like, or directly into the target organ or its surrounding area. The Muse cells that accumulate in the fibrotic organ suppress the progression of fibrosis or dissolve the fibrous tissue that has formed, and through a regenerative mechanism in which they differentiate themselves into cells that constitute the organ, the fibrous tissue in fibrosis is eliminated and the function of the organ is improved.

[0014] Muse cells can efficiently migrate to and engraft in damaged organs such as the liver, and spontaneously differentiate into hepatic cells such as hepatocytes at the engrafted site, making it unnecessary to induce differentiation into the target cells prior to transplantation. They are also non-tumorigenic and have excellent safety. Furthermore, since Muse cells are not subject to immune rejection, treatment with allogeneic preparations produced from donors is also possible. Therefore, Muse cells with the excellent properties shown above can provide an easily feasible means for the treatment of patients with organ fibrosis. [Brief description of the drawings]

[0015] [Figure 1]Characterization of Muse cells. (A) SSEA-3(+)-Muse cells (gate P3) and SSEA-3(-)-non-Muse cells (gate P6) sorted from human BM-MSCs. Top: no staining, middle: secondary antibody only, bottom: SSEA-3 antibody. (B) Q-PCR results for OCT4, SOX2, and Nanog in M-clusters, Muse cells, and non-Muse cells. *: P<0.05, **: P<0.01, ***: P<0.001. (C) Micrographs of cells grown from a single M-cluster on gelatin-coated dishes. (D) Micrographs of immunostaining of cells grown from a single M-cluster on gelatin-coated dishes. Staining was performed with markers DLK, α-fetoprotein, cytokeratin 19, and cytokeratin 18. Scale bar: 50 μm. [Diagram 2] In vitro results of Muse cell migration to serum and liver from animals before (intact), 1, 24, and 48 hours after carbon tetrachloride (CCl4) administration are shown. Compared to the migration observed with non-Muse cells, Muse cells migrated more efficiently to (A) serum and (B) liver tissue. ***p<0.001. [Diagram 3] The results show the in vivo dynamics of Muse and non-Muse cells in a 24-hr-CCl4-treated liver injury model. (A) Quantitative results of human genome-specific Alu sequences present in the liver 2 weeks after intravenous injection of human Muse or non-Muse cells into CCl4-treated and non-injured groups. (B) Micrographs showing the results of human golgi (H-Golgi) immunostaining (reflecting the localization of human Muse cells engrafted in mouse liver to label the Golgi apparatus of human cells) in the liver 30 days after cell injection. (C) The number of H-Golgi (+) cells relative to the total number of cells per mm2 in liver sections is shown. ***: P<0.001. (D,E) Micrographs showing the results of double staining for H-Golgi / human mitochondria and hepatocyte marker HepPar-1. Scale bar: 50 μm. [Figure 4]Functional and histological evaluations of the liver fibrosis model are shown. (A) Procedures for generating liver fibrosis model mice by CCl4 treatment and administration of Muse cells are shown. (B,C) Serum total bilirubin (B) and serum albumin (C) concentrations in the Muse, vehicle, and non-Muse groups 8 weeks after the start of CCl4 treatment are shown. (D,E) Microscopic images showing the evaluation of liver fibrosis area by Sirius red staining (D) and Masson's trichrome staining (E). The graph shows the results of quantifying the area. **: P<0.01, ***: P<0.001. Scale bar: 50 μm. [Diagram 5] The results of differentiation of human Muse cells into liver tissue in a liver fibrosis model induced by CCl4 treatment are shown. (A) The number of H-Golgi(+) cells present in the liver at 8 weeks after the start of CCl4 treatment is shown. In the Muse group, a considerable number of H-Golgi(+) cells were observed around blood vessels in the liver. In contrast, few of these cells were observed in the non-Muse group. (B) The number of H-Golgi(+) cells relative to the total number of cells per mm2 in liver sections is shown. ***: P<0.001. (C) Immunostaining results (microscope photograph) showing the expression of HepPar-1 (hepatocyte marker) in H-Golgi(+) cells. (D) Immunostaining results (microscope photograph) showing the expression of human albumin in H-Golgi(+) cells. (E) Immunostaining results (microscope photograph) showing the expression of human antitrypsin (hepatocyte marker) in human mitochondria(+) cells (indicating that they are human cells). Scale bar: 50 μm. [Figure 6]The results of FISH and functional analysis of the Muse group 8 weeks after the start of CCl4 treatment are shown. (A) The upper row shows the results of FISH, and the lower row shows the results of immunostaining for H-Golgi / HepPar-1 in adjacent sections (microscope photographs). In FISH, mouse chromosomes are colored green, and human chromosomes are colored red. In the results, the cell marked *1 is considered to be a mouse cell, *2 and *3 are human cells that have not fused with mouse cells, and *4 is a human-mouse fusion cell. Since the sections were prepared with a thickness of 8 to 10 μm, the position of the nucleolus and the shape of the cytoplasm do not strictly match between adjacent sections. However, the cells marked *1, *2, and *3 in FISH can be projected onto the corresponding cells in immunostaining. The cell marked *4 was not projected in immunostaining. The cell marked *1 is negative for H-Golgi. The cells in *2 and *3 were double positive for H-Golgi(+) and HepPar-1, suggesting that H-Golgi(+)-human Muse cells differentiated into hepatocyte marker HepPar-1(+) cells without fusing with mouse hepatocytes. Scale bar: 25 μm. (B) RT-PCR results for human specific-albumin, -CYP1A2, -Glc-6-Pase, and human and mouse beta-actin are shown (photo). Human liver was used as a positive control, and the liver of the solvent group was used as a negative control. [Figure 7] FIG. 1 shows the amount of collagen in the skin in the control group, the bleomycin (BLM) for 28 days + Muse cell administration group, and the BLM for 28 days + vehicle administration group. [Figure 8] FIG. 13 is a graph showing the thickness of the dermis layer in the control group, the BLM28 day + Muse cell administration group, and the BLM28 day + vehicle administration group. [Figure 9] A diagram showing the results of hematoxylin-eosin staining of skin sections from the control group, the BLM28 day + Muse cell administration group, and the BLM28 day + vehicle administration group (microscope photographs, 100x magnification). The double arrows indicate the thickness of the skin. [Figure 10] FIG. 1 shows fibrosis scores in the left lung lobe and all lung lobes in the control group and the Muse cell administration group. [Figure 11]FIG. 1 shows the results of hematoxylin-eosin staining of lung sections from the control group and the Muse cell-injected group (microscope photographs). [Figure 12] Graph showing the time course of frequency of rapid breathing in the control group and the Muse cell administration group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention relates to a cell preparation for preventing and / or treating organ fibrosis, comprising SSEA-3-positive pluripotent stem cells (Muse cells). The present invention is described in detail below.

[0017] 1.Applicable diseases The present invention uses a cell preparation containing SSEA-3-positive pluripotent stem cells (Muse cells) to prevent and / or treat organ fibrosis. Here, the term "organ fibrosis" refers to any disease in which fibrosis occurs in organs and tissues due to various causes (including unknown causes) such as reactions in the body, such as microorganisms, chemicals, and immune reactions, dietary habits, environment, and genetic background, and the functions of these organs and tissues are impaired. Examples of such fibrosis include fibrosis occurring in the digestive system, such as the liver (hepatic fibrosis), pancreas (pancreatic fibrosis), and large intestine, as well as fibrosis occurring in the respiratory system, such as the lungs (pulmonary fibrosis), fibrosis occurring in the circulatory system, such as the heart (myocardial fibrosis), bone marrow (marrow fibrosis), and spleen, fibrosis occurring in the urogenital system, such as the kidney (renal fibrosis), fibrosis occurring in the musculoskeletal system, such as muscles, and various fibroses occurring in the brain and nervous system, endocrine organs, skin, etc.

[0018] 2. Cell Preparation (1) Pluripotent stem cells (Muse cells) The pluripotent stem cells used in the cell preparation of the present invention are cells that one of the present inventors, Idezawa, discovered in the human body and named "Muse (Multilineage-differentiating Stress Enduring) cells". Muse cells can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)), dermal connective tissue of the skin, etc., and are also known to exist widely in tissues and organs. In addition, these cells have the properties of both pluripotent stem cells and mesenchymal stem cells, and are identified as, for example, cells positive for the cell surface marker "SSEA-3 (Stage-specific embryonic antigen-3)", preferably double positive cells positive for SSEA-3 and CD-105. Therefore, Muse cells or cell populations containing Muse cells can be isolated from biological tissues, for example, using the expression of SSEA-3 alone or SSEA-3 and CD-105 as an indicator. Details of the isolation method, identification method, and characteristics of Muse cells are disclosed in International Publication No. WO2011 / 007900. In addition, by utilizing the high resistance of Muse cells to various external stresses, Muse cells can be selectively enriched by proteolytic enzyme treatment, culturing under various external stress conditions such as low oxygen conditions, low phosphate conditions, low serum concentration, low nutrition conditions, exposure to heat shock, in the presence of harmful substances, in the presence of active oxygen, under mechanical stimulation, and under pressure treatment. In this specification, pluripotent stem cells (Muse cells) or cell populations containing Muse cells prepared from mesenchymal tissue of a living body or cultured mesenchymal tissue using SSEA-3 as an indicator as a cell preparation for treating fibrosis may be simply referred to as "SSEA-3 positive cells". In addition, in this specification, "non-Muse cells" may refer to cells contained in mesenchymal tissue of a living body or cultured mesenchymal tissue, other than "SSEA-3 positive cells".

[0019] Muse cells or cell populations containing Muse cells can be prepared from biological tissues (e.g., mesenchymal tissues) using cell surface markers SSEA-3 or SSEA-3 and CD-105 as indicators. Here, the term "biological organism" refers to a mammalian organism. In the present invention, the biological organism does not include fertilized eggs or embryos at a developmental stage earlier than the blastula stage, but includes embryos at developmental stages after the blastula stage, including fetuses and blastulas. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparation of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are directly isolated from biological tissues with markers. Furthermore, the term "mesenchymal tissue" refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, umbilical cord blood, and amniotic membrane, as well as tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, fat tissue, blood, dental pulp, umbilical cord, umbilical cord blood, and amniotic membrane. For example, it is preferable to collect mesenchymal tissue from a living organism, prepare Muse cells from this tissue, and use the same. Furthermore, Muse cells may be prepared from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells using the above-mentioned preparation means.

[0020] Furthermore, the cell population containing Muse cells used in the cell preparation of the present invention can also be prepared by a method comprising applying an external stress stimulus to mesenchymal tissue of a living body or cultured mesenchymal cells, selectively proliferating cells resistant to the external stress, and recovering cells with an increased abundance ratio. The external stress may be any one or a combination of protease treatment, culture at a low oxygen concentration, culture under low phosphate conditions, culture at a low serum concentration, culture under low nutrient conditions, culture under exposure to heat shock, culture at low temperature, freezing treatment, culture in the presence of a harmful substance, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical impact. The treatment time with the protease is preferably 0.5 to 36 hours in total in order to impart external stress to the cells. The protease concentration may be any concentration used when detaching cells adhered to a culture vessel, disintegrating a cell clump into single cells, or recovering single cells from tissue. The protease is preferably a serine protease, an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a glutamic acid protease or an N-terminal threonine protease, and more preferably, the protease is trypsin, collagenase or dispase.

[0021] In the cell preparation of the present invention, the Muse cells used may be autologous or allogeneic to the recipient of the cell transplantation.

[0022] As described above, Muse cells or cell populations containing Muse cells can be prepared from biological tissues using, for example, SSEA-3 positivity or double positivity for SSEA-3 and CD-105 as an indicator, but human adult skin is known to contain various types of stem cells and progenitor cells. However, Muse cells are not the same as these cells. Such stem cells and progenitor cells include skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs), melanoblasts (MBs), perivascular cells (PCs), endothelial progenitor cells (EPs), and adipose-derived stem cells (ADSCs). Muse cells can be prepared using the "non-expression" of markers specific to these cells as an indicator. More specifically, Muse cells can be separated using non-expression of at least one, for example, two, three, four, five, six, seven, eight, nine, ten or eleven of eleven markers selected from the group consisting of CD34 (EP and ADSC marker), CD117 (c-kit) (MB marker), CD146 (PC and ADSC marker), CD271 (NGFR) (NCSC marker), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and Dct (MB marker) as an indicator. For example, but not limited to, the non-expression of CD117 and CD146 can be used as an indicator, and the non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be used as an indicator, and the non-expression of the above 11 markers can be used as an indicator.

[0023] Furthermore, the Muse cells having the above-mentioned characteristics used in the cell preparation of the present invention are as follows: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) Self-renewing ability The Muse cells used in the cell preparation of the present invention may have at least one property selected from the group consisting of: Preferably, the Muse cells used in the cell preparation of the present invention have all of the above properties. Here, in the above (i), "low or no telomerase activity" means that, for example, when telomerase activity is detected using a TRAPEZE XL telomerase detection kit (Millipore), the telomerase activity is low or undetectable. "Low" telomerase activity means, for example, that the telomerase activity is the same as that of human fibroblasts, which are somatic cells, or that the telomerase activity is 1 / 5 or less, preferably 1 / 10 or less, compared to HeLa cells. In the above (ii), Muse cells have the ability to differentiate into three germ layers (endodermal, mesodermal, and ectodermal) in vitro and in vivo, and can, for example, be differentiated into hepatocytes (including hepatoblasts or cells expressing hepatocyte markers), nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, adipocytes, etc., by induction culture in vitro. In addition, when transplanted into the testis in vivo, they may also show the ability to differentiate into three germ layers. Furthermore, when transplanted into a living body by intravenous injection, they have the ability to migrate and engraft in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells according to the tissue. Regarding (iii) above, Muse cells grow at a growth rate of about 1.3 days in suspension culture, but in suspension culture, they grow from a single cell, create embryoid-like cell clusters, and growth stops in about 14 days. However, when these embryoid-like cell clusters are brought to adhesion culture, cell growth begins again, and the cells that grow from the cell clusters spread. Furthermore, when transplanted into the testis, they have the property of not becoming cancerous for at least half a year. Regarding (iv) above, Muse cells have the ability to self-renew (self-replicate). Here, "self-renewal" refers to the fact that differentiation into three germ layer cells can be confirmed from cells contained in an embryoid-like cell mass obtained by culturing a single Muse cell in suspension culture, and at the same time, by bringing a single cell from the embryoid-like cell mass back into suspension culture, a next-generation embryoid-like cell mass can be formed, from which differentiation into the three germ layers and the formation of an embryoid-like cell mass in suspension culture can again be confirmed.The self-renewal may be repeated one or more times.

[0024] (2) Preparation and use of cell preparations The cell preparation of the present invention can be obtained by suspending the Muse cells or a cell population containing Muse cells obtained in (1) above in physiological saline or an appropriate buffer solution (e.g., phosphate-buffered saline), although it is not limited thereto. In this case, when the number of Muse cells isolated from autologous or allogeneic tissues is small, the cells may be cultured before cell transplantation and allowed to grow until a predetermined number of cells is obtained. As already reported (International Publication No. WO2011 / 007900), Muse cells do not become tumorigenic, so that even if cells collected from biological tissues are contained in an undifferentiated state, there is a low possibility of canceration and it is safe. In addition, the culture of the collected Muse cells can be performed in a normal growth medium (e.g., α-minimal essential medium (α-MEM) containing 10% calf serum, etc.), although it is not particularly limited thereto. For more details, with reference to the above International Publication No. WO2011 / 007900, a medium, additives (e.g., antibiotics, serum), etc. can be appropriately selected for the culture and growth of Muse cells, and a solution containing Muse cells at a predetermined concentration can be prepared. When the cell preparation of the present invention is administered to a human subject, bone marrow fluid is collected from the human ilium, and for example, bone marrow mesenchymal stem cells are cultured as adherent cells from the bone marrow fluid to increase the cell amount to obtain an effective therapeutic amount of Muse cells, and then Muse cells are isolated using the SSEA-3 antigen marker as an indicator to prepare autologous or allogeneic Muse cells as a cell preparation. Alternatively, for example, bone marrow mesenchymal stem cells obtained from the bone marrow fluid are cultured under external stress conditions to grow and concentrate the Muse cells to obtain an effective therapeutic amount, and then autologous or allogeneic Muse cells can be prepared as a cell preparation.

[0025] Furthermore, when Muse cells are used in a cell preparation, the cell preparation may contain dimethyl sulfoxide (DMSO) or serum albumin to protect the cells, and antibiotics to prevent bacterial contamination and proliferation. Furthermore, the cell preparation may contain other components acceptable for formulation (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) and cells or components other than Muse cells contained in mesenchymal stem cells. Those skilled in the art can add these factors and drugs to the cell preparation at appropriate concentrations. In this way, Muse cells can also be used as a pharmaceutical composition containing various additives.

[0026] The number of Muse cells contained in the cell preparation prepared above can be appropriately adjusted in consideration of the subject's sex, age, weight, condition of the affected area, condition of the cells used, etc., so that the desired effect in the treatment of fibrosis (e.g., in the case of liver fibrosis, recovery of serum total bilirubin and albumin, and reduction of fibrosis, etc.) can be obtained. The subject individual includes, but is not limited to, humans. The cell preparation of the present invention may be administered multiple times (e.g., 2 to 10 times) at appropriate intervals (e.g., twice a day, once a day, twice a week, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months) until the desired therapeutic effect is obtained. Therefore, although it depends on the condition of the subject, the therapeutically effective amount is, for example, 1 × 10 per individual per administration. 3 cells ~1×10 8 The preferred dose is 1 to 10 times the amount of cells. The total dose per individual is not limited, but is preferably 1×10 3 cells ~1×10 8 cells, 1 x 10 4 cells ~5×10 7 cells, 2 x 10 4 cells ~2×10 7 cells, 5 x 10 4 cells ~5×10 6 cells, 1 x 10 5 cells ~1×10 9 Cells, etc.

[0027] The Muse cells used in the cell preparation of the present invention have the property of migrating to and engrafting in an injured organ. Therefore, the administration site of the cell preparation and the type of blood vessel (vein or artery) to which the cell preparation is administered are not limited.

[0028] The cell preparation of the present invention can improve or restore the function of an injured organ in a fibrosis patient to normal (or to normal levels). As used herein, "improving" organ function means alleviating various symptoms associated with fibrosis and inhibiting its progression, and preferably alleviating symptoms to a level that does not interfere with daily life. Furthermore, "restoring organ function to normal" means that all symptoms caused by fibrosis return to the state before organ injury.

[0029] For example, in the case of hepatic fibrosis, liver function after administration of the cell preparation of the present invention can be evaluated by measuring serum total bilirubin levels, albumin levels, or expression of liver marker genes.

[0030] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0031] Materials and Methods Preparation of human Muse cells Human Muse cells were prepared according to the method described in International Publication No. WO2011 / 007900. More specifically, the Muse cells used in Example 1 were obtained by culturing mesenchymal cells having adhesive properties from human bone marrow fluid, proliferating them, and separating Muse cells or a cell population containing Muse cells as SSEA-3 positive cells by FACS. The Muse cells used in Examples 2 to 4 were obtained by expanding and enriching mesenchymal stem cells by culturing them under stress conditions. In addition, non-Muse cells were a group of SSEA-3 negative cells among the above mesenchymal cells, and were used as a control. Thereafter, the cells were adjusted to a predetermined concentration using phosphate buffered saline or culture medium, and used in the following experiments.

[0032] <Example 1> Transplantation of human Muse cells into a mouse model of liver fibrosis CB17 / Icr-Prkdc <scid> / CrlCrlj (SCID) mice were used in all the studies. All animal experiments were conducted in accordance with the guidelines for the humane care and use of laboratory animals at Tohoku University (Sendai, Japan). The experimental procedures for establishing the liver fibrosis model were also referred to International Journal of Molecular Science 2012; 13: 3598 - 3617 and Journal of Biochemistry 2003; 134: 551 - 558. Specifically, a liver fibrosis model in SCID mice was established by intraperitoneal injection of CCl4 (0.5 ml / kg). Human Muse cells or non - Muse cells (5×10 4 cells) were injected into the tail vein of the liver fibrosis model mice.

[0033] Statistical analysis The significant differences between two groups were evaluated using Student's t - test. The statistical significant differences among three or more groups were evaluated using one - way analysis of variance (one - way ANOVA) with Bonferroni's multiple comparison test. A P - value of < 0.05 (indicated by * in the figure) was determined to be significant, and < 0.01 ( ** ) or < 0.001 ( *** ) was determined to be highly significant.

[0034] Results <Characterization of Muse cells> SSEA-3 positive human Muse cells (approximately 2% of human BM-MSCs) and SSEA-3 negative non-Muse cells (control group) were isolated by FACS (Figure 1A). When these were cultured in single-cell suspension, only Muse cells generated single-cell-derived clusters (M clusters) similar to ES cell-derived embryoid bodies formed during suspension culture, whereas non-Muse cells did not form such clusters at all (data not shown). Gene expression of pluripotency markers in adherent cultured Muse cells, adherent cultured non-Muse cells, and M clusters was examined, and it was shown that adherent cultured Muse cells showed higher gene expression of pluripotency markers OCT4, SOX2, and Nanog compared to adherent cultured non-Muse cells. Furthermore, the expression of SOX2 and Nanog in adherent cultured non-Muse cells was below the detection threshold. In particular, the expression levels of OCT4, SOX2, and Nanog in the M cluster were approximately 9-fold, 54-fold, and 35-fold higher, respectively, than those in adherent cultured Muse cells, showing statistically significant differences (Fig. 1B). The ability of Muse cells to differentiate from a single cell into triploblastic lineage cells was confirmed as described in Proc Natl Acad Sci USA 2010; 107: 8639-8643. Muse cells have already been reported to differentiate into alpha-fetoprotein / albumin-positive cells upon induction with hepatocyte growth factor (HGF) and fibroblast growth factor 4 (FGF-4) (Proc Natl Acad Sci USA 2011; 108: 9875-9880). We therefore examined whether cells grown from M-clusters formed in single-cell suspension culture on gelatin-coated culture dishes (Fig. 1C) spontaneously differentiated into cells expressing hepatoblast and hepatocyte markers. These cells included cells positive for DLK (1.5±0.6%), alpha-fetoprotein (3.0±0.8%), cytokeratin 19 (1.7±0.4%), and cytokeratin 18 (2.0±0.9%) (Fig. 1D). Because non-Muse cells do not form clusters in single-cell suspension culture, they were directly seeded on gelatin-coated culture dishes immediately after isolation and cultured for the same length of time as M-clusters.However, non-Muse cells did not show any expression of hepatoblast / hepatic cell lineage markers (data not shown). Therefore, it is considered that Muse cells have a high differentiation potential into hepatoblast / hepatic cell lineage cells, while non-Muse cells do not have such ability.

[0035] <Muse cells efficiently migrate to and engraft in the injured liver> Next, the ability of human Muse cells to migrate to the serum and liver tissues of a liver injury model was tested in vitro. For this purpose, a single intraperitoneal administration of carbon tetrachloride (CCl4) was performed in immunodeficient mice (SCID mice) that do not reject human cells, and serum and injured liver tissues were collected at 1, 24, and 48 hours later. As a control, serum and liver tissues from non-carbon tetrachloride (CCl4)-administered SCID mice were collected. For the evaluation of migratory ability, the Boyden chamber method was used. The collected serum or tissue was placed on the lower side of the insert, and human Muse cells or non-Muse cells were added to the upper side, and the number of human Muse cells or non-Muse cells that passed through the insert was counted. As a result, the migration of Muse and non-Muse cells was very slight in the serum of non-injured mice, and there was no statistical difference between the two (Figure 2A). In the serum 1 hour after CCl4 injection (1hr-CCl4), the migration of both Muse cells and non-Muse cells increased slightly, but there was no statistical difference between the non-injured case and 1hr-CCl4 for both Muse cells and non-Muse cells. However, in the serum of 24hrs-CCl4, the number of migrated Muse cells increased significantly, and there was a statistically significant difference compared to non-injured and 1hr-CCl4 (both p < 0.001). Also, the increase in the number of migrated cells at the 24-hour time point was approximately 12 times greater than that of Muse cells in 1hr-CCl4. On the other hand, such dramatic changes were not observed in non-Muse cells. At the 24-hour time point, the migration rate of Muse cells was 3 to 4 times higher than that of non-Muse cells, and a statistically significant difference was observed between the two (p < 0.001). In the serum of 48hrs-CCl4, the level was not as high as that seen in 24hrs-CCl4, but a statistical difference was still observed between Muse cells and non-Muse cells (P < 0.01) (Figure 2A).

[0036] As with serum, the number of Muse cells migrated to the liver tissue of 24hrs-CCl4 was the highest, and showed statistically significant differences when compared with non-Muse cells (p<0.001) or with Muse cells from non-injured, 1hr-CCl4, or 48hrs-CCl4 liver tissues (p<0.001 for each). The number of Muse cells that migrated to the liver of 24hrs-CCl4 was about 7-fold higher than that of 1hr-CCl4 Muse cells and about 4-fold higher than that of 24hrs-CCl4 non-Muse cells. No significant migration was detected in non-Muse cells (Figure 2B). These results indicate that, unlike non-Muse cells, Muse cells have strong migration activity toward serum and the liver of the CCl4-liver injury model.

[0037] We analyzed the in vivo dynamics of human Muse and non-Muse cells injected via the tail vein. Using SCID mice, we prepared non-injured mice and mice 24 hours after a single intraperitoneal administration of carbon tetrachloride (CCl4) (24hrs-CCl4-liver injury mice), and injected human Muse or non-Muse cells into the tail vein. We performed Q-PCR of human-specific Alu sequences in each organ of non-injured and 24hrs-CCl4-liver injury mice 2 weeks after Muse cell administration to examine the distribution of human Muse and non-Muse cells. In non-injured mice, low signals of Alu sequences were detected in the lungs in both Muse cell-injected mice and non-Muse cell-injected mice, and the signals were below the detection limit in other organs (Figure 3A). In the 24hrs-CCl4-liver injury model with Muse cell injection, the signals were maximum in the liver, lower in the lungs, and below the detection limit in other organs. In contrast, in mice injected with non-Muse cells, signals were below the detection threshold in both the liver and other organs except the lungs (Figure 3A).

[0038] Next, the engraftment and differentiation of human Muse cells on the 30th day after cell administration in a liver injury model (24-hr CCl4-induced liver injury mice) were verified using an anti-human Golgi complex (H-Golgi) antibody or an anti-human mitochondria antibody. Since the histological images of the injured liver were extremely heterogeneous, 10 different regions were arbitrarily selected from both the injured area and the seemingly normal area, and measurements were taken for all of them. The number of H-Golgi(+) cells detected in the Muse group (1.89 ± 0.65% of all cells in a 1-mm 2 liver section) was approximately 48 times that in the non-Muse group (0.04 ± 0.08%), showing a highly significant statistical difference (p < 0.001) (Figures 3B, C). According to histological analysis, H-Golgi(+) cells were mainly distributed around blood vessels in the liver in the Muse group, suggesting that intravenously injected Muse cells engrafted from blood vessels into the liver (Figure 3B). The engraftment of human Muse cells in the injured liver was also confirmed by the detection of human-specific mitochondria (Figure 3D). Double staining for H-Golgi / human mitochondria and hepatocyte markers showed that 49.8 ± 1.9% of human mitochondria(+) cells were positive for human-specific albumin, and 80.4 ± 3.2% of H-Golgi(+) cells were positive for the human progenitor / mature hepatocyte marker HepPar-1 (Figures 3D, E).

[0039] All these results indicate that Muse cells have a very high ability to migrate and accumulate in the injured liver both in vitro and in vivo, and to differentiate into human-specific albumin- and HepPar-1-positive cells in vivo, while non-Muse cells did not show such an ability.

[0040] <Muse cells improve function and reduce fibrosis in a liver fibrosis model> The experimental procedures for establishing the liver fibrosis model, as well as the number and timing of cells to be injected, are shown in Figure 4A. Specifically, a liver fibrosis model in SCID mice was established by performing intraperitoneal injections of CCl4 (0.5 ml / kg) twice a week until 8 weeks. 5×10 4 Human Muse cells (Muse group, n = 8) or non-Muse cells (non-Muse group, n = 8), or an equal volume of phosphate-buffered saline (PBS) (vehicle group; n = 8) were injected into the tail vein of liver fibrosis model mice at 2, 4, and 6 weeks, and data were collected at 8 weeks.

[0041] No tumor formation was observed up to 8 weeks in the Muse, non-Muse, and vehicle groups (data not shown). At week 8, serum total bilirubin was significantly lower in the Muse group (0.26 ± 0.05 mg / dl) than in the vehicle group (0.74 ± 0.05 mg / dl, p < 0.001) and non-Muse group (0.48 ± 0.12 mg / dl, p < 0.001), and the Muse group was approximately 2.8 times lower than the vehicle group. Although not as low as the Muse group, the non-Muse group's total bilirubin was 1.5 times lower than the vehicle group, a statistically significant difference (p < 0.001). This suggests a moderate recovery (Figure 4B). The serum albumin concentration in the Muse group was the highest among the three groups (2.99 ± 0.11 g / dl), and there was a highly statistically significant difference compared to the vehicle group (2.65 ± 0.08 g / dl, p < 0.001) and the non-Muse group (2.81 ± 0.06 g / dl, p < 0.01). Although not as high as the Muse group, the non-Muse group showed a moderate recovery of serum albumin compared to the vehicle group (p < 0.01) (Figure 4C).

[0042] The degree of fibrosis mainly composed of type I / III collagen was evaluated by Sirius red staining and Masson's trichrome staining. At 8 weeks, an expansion of fibrotic areas with typical internodular septa was observed in the solvent group. On the other hand, the fibrotic area was minimal in the Muse group. According to Sirius red staining, the Muse group (0.75 ± 0.15% of the total area per section) showed the smallest fibrotic area compared to the solvent group (2.91 ± 0.35%) and the non-Muse group (1.86 ± 0.13%), and there were statistically significant differences (p < 0.001) compared to both. This corresponds to a 75% improvement in fibrosis compared to the solvent group (Figure 4D). A statistical difference was observed between the non-Muse group and the solvent group (p < 0.001), and in the non-Muse group, a 36% equivalent improvement in fibrosis was seen compared to the solvent group. This suggests a moderate effect in the non-Muse group (Figure 4D). Similar results were also observed by Masson's trichrome staining. The Muse group showed the smallest fibrotic area (0.73 ± 0.15%) among the three groups, and there were highly statistically significant differences compared to the solvent group (1.90 ± 0.12%, p < 0.001) and the non-Muse group (1.11 ± 0.15%, p < 0.01). This corresponds to a 62% improvement in fibrosis compared to the solvent group (Figure 4E). Although not as much as the Muse group, the non-Muse group also showed a statistical difference (p < 0.001) compared to the solvent group, corresponding to a 42% improvement compared to the solvent group (Figure 4E).

[0043] These results indicate that the human Muse group is more effective than the non-Muse group in the improvement of liver function measured by total serum bilirubin and albumin, and also in the reduction of fibrosis in the mouse liver fibrosis model up to 8 weeks.

[0044] <Muse cells provide new hepatocytes by spontaneous in vivo differentiation in a liver fibrosis model> At the 8-week time point, a large number of human Muse cells were detected in the perivascular region, while only a very small number of non-Muse cells were detected (Figure 5A). 1 mm 2 The number of H-Golgi(+) cells in the total number of cells in the sections was high in the Muse group (5.78±2.39%), but extremely low in the non-Muse group (0.27±0.12%), with a statistically significant difference (p<0.001). The proportion in the Muse group was approximately 21 times higher (Figure 5A,B).

[0045] Immunostaining was further performed in the Muse group. Muse cells positive for H-Golgi and human mitochondria were detected in the liver, and the expression of HepPar-1 (71.1 ± 15.2% of H-Golgi(+) cells) (Figure 5C), human albumin (54.3 ± 8.2% of H-Golgi(+) cells) (Figure 5D), and human antitrypsin (47.9 ± 4.6% of human mitochondria(+) cells) (Figure 5E) was observed. Thus, it is suggested that the engrafted human Muse cells subsequently spontaneously differentiate into hepatocyte marker-positive cells.

[0046] Previous studies have revealed that bone marrow-derived hepatocytes can be formed by cell fusion in injured livers. To investigate whether the aforementioned differentiation in this study was the result of cell fusion, we performed fluorescence in situ hybridization (FISH) analysis to examine the presence of cell fusion between host hepatocytes (mouse origin, colored green) and injected Muse cells (human origin, colored red) (Figure 6A). We performed double staining for H-Golgi and HepPar-1 on sections adjacent to each FISH sample to infer whether FISH signals could be obtained from differentiated human Muse cells. The results suggested that only 2.6 ± 0.2% of H-Golgi(+) / HepPar-1(+)- human Muse cells, which were almost identical to the cells in FISH, were generated by cell fusion. Conversely, this suggests that about 97% of human Muse cells incorporated into mouse liver tissue differentiated into hepatocyte marker-positive cells without cell fusion.

[0047] The expression of human-specific markers of mature functional hepatocytes, such as human-specific albumin, human cytochrome P450 1A2 (CYP1A2), an enzyme involved in drug metabolism, and human glucose-6-phosphatase (Glc-6-Pase), an enzyme involved in free glucose formation, was examined by RT-PCR and found to be highly expressed in the livers of the Muse group. On the other hand, these markers were not expressed in the non-Muse and vehicle groups. Notably, the signal of human beta-actin was below the detection limit in the non-Muse transplanted liver (Figure 6B), which is consistent with the histological data of the non-Muse group in Figures 5A and B.

[0048] One of the mechanisms underlying the improvement of fibrosis is thought to be that proteases such as metalloproteases dissolve collagen and other fibers.

[0049] These results suggest that Muse cells can differentiate into hepatoblast and hepatocyte lineages in vitro. Muse cells showed high migration ability toward serum and liver of CCl4-treated mice in vitro, and specifically accumulated in the injured liver but not in other organs in vivo. After engraftment in the liver, Muse cells spontaneously differentiated in vivo into HepPar-1 (71.1±15.2%), human albumin (54.3±8.2%), and antitrypsin (47.9±4.6%)-positive cells without fusion with host hepatocytes, and expressed mature and functional human markers such as CYP1A2 and Glc-6-Pase at 8 weeks. Furthermore, significant recovery of serum total bilirubin and albumin, and reduction of fibrosis were observed. These results suggest that Muse cells may be effective in preventing and treating liver fibrosis, and further suggest that they may also be effective in preventing and treating other types of fibrosis.

[0050] <Example 2> Evaluation of Muse cells in a skin fibrosis model A mouse model of BLM-induced skin fibrosis was created according to the method described in Sci Rep. 2015 Aug 20;5:12466. doi: 10.1038 / srep12466. Female C57BL / 6J mice (Japan SLC, Inc.) were used. In addition, saline was administered instead of BLM to the control group. On day 14 after BLM administration, 1 × 10 6 Muse cells were administered at 100x the dose of 100x the dose per 1000 mice (cells / body weight (kg)). On the other hand, the vehicle-administered group received vehicle administration via the tail vein on day 14. On day 28 after BLM administration, skin tissue was isolated and subjected to collagen quantification, hematoxylin-eosin (HE) staining, and dermal thickness analysis.

[0051] Skin collagen analysis was carried out as follows. The skin pieces were homogenized with 0.5 M acetic acid / pepsin solution and stirred overnight at 4° C. The collagen concentration of the resulting extract was measured using a Sircol collagen assay kit (Biocolor, Cat No.: S1000).

[0052] For the analysis of HE staining and dermal layer thickness, first, skin sections (4 μm) were prepared, HE stained, and photographs were taken of 5 fields per section at 100x magnification using an optical microscope. The distance from just below the epidermis to the subcutaneous fat was taken as the thickness of the dermis, and was measured using ImageJ.

[0053] Plasma hyaluronic acid analysis Plasma hyaluronan concentrations were measured using plasma obtained from mice by ELISA (Quantikine Hyaluronan ELISA Kit, R&D systems, Inc.).

[0054] result 1. Skin collagen content The analysis results of skin collagen content are shown in Figure 7. A significant increase in skin collagen content was observed in the BLM28 days + vehicle administration group compared to the control group not administered BLM. In addition, a significant decrease in skin collagen content was observed in the BLM28 days + Muse cell administration group compared to the BLM28 days + vehicle administration group.

[0055] 2. HE staining and dermal thickness The HE staining images are shown in Figure 9, and the thickness of the dermis layer is shown in Figure 8. A significant increase in the thickness of the dermis layer was observed in the BLM28 day + vehicle administration group compared to the control group. In addition, a significant decrease in the thickness of the dermis layer was observed in the BLM28 day + Muse cell administration group compared to the BLM28 day + vehicle administration group.

[0056] 3. Plasma hyaluronic acid concentration No significant difference was observed in plasma hyaluronan concentration between the control group and the BLM28 days + vehicle administration group.On the other hand, the BLM28 days + Muse cell administration group showed a tendency for an increase in plasma hyaluronan concentration compared to the BLM28 days + vehicle administration group.

[0057] <Example 3> Evaluation of Muse cells in a pulmonary fibrosis model BLM solution (14 μg / 25 μL / lung) was administered intratracheally to create a BLM pulmonary fibrosis model mouse (Medicine and Pharmacology, 62(4):661-668, 2009). The mice used were Crl:CDl (ICR), male (Charles River Biosciences Japan). 21 days after BLM administration, 1 × 10 6 Muse cells or vehicle were administered at 100x cells / kg body weight.

[0058] Formalin-fixed lungs were removed and stained with Masson's Trichrome stain. Fibrosis was scored for each lung lobe using Masson's Trichrome stain. The lung fibrosis score was classified according to the following lung fibrosis evaluation criteria. Then, photographs of the images close to the average lung fibrosis score for each group were taken (Figure 11). The fibrosis score was evaluated using lung tissues 21 and 35 days after BLM administration.

[0059] Assessment criteria for pulmonary fibrosis (numbers are scores) O: Normal 1: Mild fibrous thickening of the alveoli or bronchial walls. 2: Moderate fibrous thickening of the alveoli or bronchial walls, but no obvious structural changes in the lungs 3: Obvious structural changes in the lungs and the formation of small fibrotic foci are observed. 4: Severe structural changes in the lungs and the formation of large fibrotic lesions are observed. 5: The entire area is replaced by fibrosis.

[0060] To examine the effect of Muse cells on respiratory distress that occurred after BLM administration, the number of episodes of respiratory distress that occurred was counted from 21 to 35 days after BLM administration in the control group and the Muse cell-administered group.

[0061] result 1. Pulmonary fibrosis score The results of the pulmonary fibrosis scores are shown in Figure 10. As a result, fibrosis was formed 21 days after BLM administration, and the degree of fibrosis did not change even after 35 days. On the other hand, 35 days after BLM administration, the fibrosis score was lower in the Muse cell-administered group compared to the vehicle-administered group, and when the fibrosis scores were compared for each lung lobe, a tendency toward suppression was observed in the left lung (p=0.086).

[0062] 2. Number of respiratory distress occurrences As shown in FIG. 12, in the Muse cell-administered group, the frequency of episodes of rapid breathing was significantly reduced compared to the vehicle-administered group from 29 to 35 days after BLM administration.

[0063] <Example 4> Evaluation of Muse cells in a liver fibrosis model Mice were administered 10 mL / kg of 10% carbon tetrachloride intraperitoneally twice a week for 12 weeks to induce liver fibrosis. Female BALB / c mice (Charles River Biosciences Japan, Ltd.) were used. 1 × 10 on the 57th day after the first administration of carbon tetrachloride 6 Cells / body weight (kg) (low dose group) and 1 × 10 7 Muse cells were administered via the tail vein at 100 cells / body weight (kg) (high dose group).

[0064] On the 84th day after the initial administration of carbon tetrachloride, the liver was removed and stained with HE and Masson's trichrome staining. The degree of fibrosis was observed under a light microscope. The observed changes were evaluated using a grading scale (0, none; 1, minimal; 2, mild; 3, moderate; 4, severe).

[0065] Blood was collected 84 days after the first administration of carbon tetrachloride, and plasma ALT (GPT), AST (GOT), ALB (albumin), CHE (cholinesterase), and TBIL (total bilirubin) were measured using DriChem (Fuji Medical Systems Co., Ltd.).

[0066] result 1. Fibrosis level The degree of fibrosis is shown in Table 1. The degree of fibrosis in the vehicle control group was level 2 in 10 out of 10 cases. The degree of fibrosis in the low-dose Muse cell group was level 1 in 9 of 10 cases and level 2 in 1 case. The degree of fibrosis in the high-dose Muse cell group was level 1 in 7 of 10 cases and level 2 in 3 cases. Both Muse cell-administered groups showed significant improvement in fibrosis compared with the vehicle control group.

[0067] [Table 1]

[0068] 2. Measurement of plasma ALT (GPT), AST (GOT), ALB, CHE and TBIL In the vehicle control group, changes indicative of hepatitis were observed in ALT, AST / ALT ratio, and TBIL, but in the Muse cell administration group, both low and high doses showed significant improvements in ALT, AST / ALT ratio, and TBIL. In addition, a slight tendency for improvement was observed in ALB and CHE compared to the vehicle control group. [Industrial Applicability]

[0069] When administered to a patient suffering from fibrosis, the cell preparation of the present invention can reconstruct and repair tissue at the injured site and restore function, and can be applied to the prevention and treatment of fibrosis.< / scid>

Claims

[Claim 1] A cell preparation for treating pulmonary fibrosis, comprising as an active ingredient a cell fraction enriched in SSEA-3-positive and CD-105-positive pluripotent stem cells, the cell fraction being isolated from a mesenchymal tissue or cultured mesenchymal cells of a living body using an antigen marker of SSEA-3 as an indicator, or being enriched by culturing the cells under external stress conditions, The cell preparation contains 1×10 Muse cells per subject per administration. 3 Cell ~1×10 8 A cell for intravenous administration, The pluripotent stem cells are a cell preparation having all of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability.

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