WO2008140141A1

WO2008140141A1 - Pharmaceutical composition for preventing and treating liver fibrosis or hepatic cirrhosis comprising mesenchymal stem cell

Info

Publication number: WO2008140141A1
Application number: PCT/KR2007/002397
Authority: WO
Inventors: Kyung Sun Kang; Jung Chan Ra; Il Seob Shin; Ju Yeon Seo
Original assignee: Rnl Bio Co., Ltd.
Priority date: 2007-05-16
Filing date: 2007-05-16
Publication date: 2008-11-20

Abstract

Provided is a pharmaceutical composition for preventing and treating liver fibrosis and hepatic cirrhosis including a mesenchymal stem cell. The composition substitutes for a damaged liver cell, thereby recovering liver functions and reducing collagen fibrils deposited on the liver, and thus may be used for preventing and treating liver fibrosis or hepatic cirrhosis.

Description

PHARMACEUΉCAL COMPOSITION FOR PREVENTING AND TREATING LIVER FIBROSIS OR HEPATIC CIRRHOSIS COMPRISING MESENCHYMAL

STEM CELL

[Technical Held] The present invention relates to a pharmaceutical composition for preventing and treating liver fibrosis or hepatic cirrhosis comprising a mesenchymal stem cell. [Background Art]

A hepatic disease is one of the major causes of death in many countries (Corrao G et al., Int J Epidemiol. 1997 Feb;26(l):100-109). Generally, hepatic cirrhosis is a primary cause of death from liver diseases (Schlichting P et al., Scand J Gastroenterol. 1983 Oct;18(7): 881-888). In progression of hepatic cirrhosis, liver fibrosis is central and essential (Hu QW et al., Life Sci. 2006 M 4;79(6): 606-612). Liver fibrosis is a part of a wound healing process related to various types of liver damage, which is characterized by continuous deposition of collagen fibrils into an extra-cellular matrix (ECM) (Du WD et al., World J Gastroenterol. 1999 Oct; 5(5):397-403). Generally, liver fibrosis is reversible, which is different from hepatic cirrhosis, and has thin fibrils and no tuberculation. Thus, when the cause of the liver damage disappears, the liver can recover from liver fibrosis. However, chronically repetitive mechanisms of liver fibrosis may lead to irreversible hepatic cirrhosis in which crosslinking between connective tissues increases to accumulate thick fibrils, and a liver lobule loses its normal structure to cause tuberculation. Although a chronic liver disease may have various causes, it can eventually lead to liver fibrosis or hepatic cirrhosis irrespective of the cause. A liver disease may be asymptomatic in an initial state and thus be difficult to diagnose early. Furthermore, if the liver disease is discovered in a chronic state, it may be difficult to treat and have a high mortality rate. Also, effective medication has not yet been developed.

It has been noted that mesenchymal stem cells (MSCs) are derived from adult tissues including bone marrow (BM) (Cohnheim J. Ueber Entz W ndung und Eiterung, 1867; Ftedenstein AJ et al., J Embryol. Exp. Morphol, Dec 16 1996, 3:381-390; Pittenger MF et al., Science 1999 Apr 2, 284(5411):143-147), adipose Tissue (AD) (ZuJc PA et al., Tissue Eng. 2001 Apr; 7(2):211-228) and umbilical cord blood (UCB) (Eήces A et al., Br J Haematol. 2000 Apr; 109(l):235-242; Goodwin HS et al., Biol. Blood Marrow Transplant 2001;7(ll):581-588; Bieback K et al., Stem Cells 2004;22(4):625-634; Kogler G et al., J Exp. Med. 2004 M 19;200(2):l23-135; Lee OK et al., Blood 2004 Mar l;103(5):1669-1675). Human BM-derived multipotent adult progenitor cells (MAPCs) can be differentiated into hematocyte- like cells acquiring functional characteristics of hematocyte in vitro {.Schwartz RE et al., J Clin. Invest. 2002 May;109(10):1291-1302). Also, human MSCs directly xenotransplanted into a liver of a rat in vivo can be differentiated into human hematocytes without fusion (Sato Y. et al., Blood 2005 M 15; 106(2): 756-763). [Disclosure] [Technical Problem]

Studies which have been conducted for several years to develop medication for liver fibrosis and hepatic cirrhosis have found that a mesenchymal stem cell can substitute for a damaged liver cell, thereby recovering liver function and decreasing collagen fibrils deposited in the liver. Thus, the present invention is directed to a pharmaceutical composition for preventing and treating liver fibrosis including a mesenchymal stem cell.

The present invention is further directed to a pharmaceutical composition for preventing and treating hepatic cirrhosis including a mesenchymal stem cell. [Technical Solution] In one aspect, a pharmaceutical composition for preventing and treating liver fibrosis including a mesenchymal stem cell is provided.

In another aspect, a pharmaceutical composition for preventing and treating hepatic cirrhosis including a mesenchymal stem cell is provided.

Hereinafter, the present invention will be described in detail. The term "liver fibrosis" is a disease in which a thin fibril is abnormally proliferated in the liver, and may be caused by a variety of chronic toxic damage, including: chronic alcohol abuse; chronic exposure to drugs including acetaminophen, amiodarone, aspirin, azathioprine, isoniazid, methyldopa, methotrexate, nitrofurantoin, propylthiouracil and sulfonamide; chronic exposure to chemical agents including CCU, dimethyl nitrosamine, vinyl chloride, polychlorinated biphenyl, aflatoxin and insecticides; autoimmune diseases including viral infection, diabetes, primary sclerosing cholangitis, primary bile duct liver cirrhosis, autoimmune hepatitis, lupus hepatitis and inflammatory bowel disease; hemochromatosis; alpha-1-antotrypsin deficiency; chronic cholestasis; non-alcoholic steatohepatitis (NASH); chronic biliary atresia; Wilson's disease; and other states which have been known to cause cirrhosis.

The term "hepatic cirrhosis" is a disease caused by a hardened liver due to lesions of a parenchymal cell of the liver and increase of connective tissues. The term "mesenchymal stem cell (MSC)" refers to a type of adult stem

75 cell, which is a pluripotent cell having a potential to be differentiated into all kinds of cytotypes with self -restoring ability.

The present invention is the first to report that a damaged liver cell can be replaced by an MSC, thereby recovering liver function and reducing collagen fibrils deposited into the liver.

80 In order to demonstrate whether the MSC can prevent and treat liver fibrosis, the inventors used animal models having liver fibrosis which was induced by dimethylnitrosamine (DMN). The animal models had advanced liver fibrosis, low mortality and stable persistency, which thus had great potential as screen models for liver fibrosis medication {George J et al., Toxicology 2001 Jan. 2; 156(2-

85 3);129-138; Jezequel AM et al., J Hepatol. 1989 Jan;8(l):42-52).

In one exemplary embodiment of the present invention, DMN was administered to 6-week-old male SD rats for 8 weeks to induce liver fibrosis. Referring to Exemplary Embodiment <l-2> which will be described below, an accumulative survival rate of the DMN-induced group began to

90 decrease in the 5th week, and drastically fell from the 6th week, and eventually all rats were dead in the 8th week. Further, as can be seen in FIGS. 2A to 2D, the DMN-induced group exhibited common clinical symptoms commonly shown in rats having liver fibrosis, in which total protein and albumin levels are low and AST and ALT levels are high. Furthermore, it may be shown in D of FIG. 3 that

95 the DMN-induced group exhibits an approximately 50% decrease in liver weight per body weight (LW/BW), replacement of the damaged liver cell by a thick collagen fibril, and increase of collagen fibrils in number, as compared with the negative control. From the results described above, the inventors could confirm that liver

100 fibrosis may be effectively induced by DMN in rats. Accordingly, in Exemplary Embodiment 2, the inventors checked the effect of the MSCs on liver fibrosis in the DMN-induced liver fibrosis rats which were observed from the moment mortality began to decrease.

As can be seen in FIG. 4, the rats xenotransplanted with human MSCs

105 without administration of an immune inhibitor lived until the 4^th week after the cell injection. Also, as can be seen in FIG. 5, total protein and albumin levels indicating synthesis ability of a liver in the liver fibrosis rats increased due to the administration of MSCs, and ALT and AST levels indicating a degree of necrosis of liver cells decreased.

110 Further, FIGS. 7 and 8 show that the administration of MSCs may significantly lower collagen fibrils.

Accordingly, the above experimental results showed that the MSCs may substitute for damaged liver cells of a recipient, and decompose collagen fibrils around the damaged liver cell, thereby preventing and treating liver fibrosis and

115 hepatic cirrhosis.

Consequently, the present invention provides a pharmaceutical composition for preventing and treating liver fibrosis including MSCs.

The present invention also provides a pharmaceutical composition for preventing and treating hepatic cirrhosis including MSCs.

120 The MSC, the active ingredient of the pharmaceutical composition of the present invention, is a cell having an unexpressed histocompatibility antigen (HLA- DR; class II), the most critical cause of tissue or organ rejection. Thus, the MSC, which may be autologous or allogeneic, may not cause or may minimize the chance of immune reaction such as rejection, which can be a problem in 125 conventional transplant operations. Preferably, the MSC may be isolated from cord blood or adipose tissue of mammals including human.

The term "cord blood" used herein refers to blood collected from the umbilical vein which connects a placenta to a fetus of a mammal.

The term "adipose tissue" used herein refers to tissue including plural 130 cytotypes such as an adipocyte, a microvascular cell and the like. Also, the adipose tissue includes connective tissue storing adipose.

Cord blood may be easily collected from the umbilical vein of a donor after delivery. More particularly, in spontaneous vaginal delivery (SVD), cord blood may be collected from the umbilical vein which is completely out of a birth 135 canal, but the placenta still remains in a womb after delivery. Also, in Caesarean delivery, cord blood may be collected from the umbilical vein while the placenta is out of the womb after delivery.

In the present invention, cord blood is collected from the umbilical vein connecting the placenta to a fetus after delivery by aseptic treatment. Here, the 140 cord blood may be collected before or after placental abruption in or out of the womb after the delivery. In a Caesarean delivery, the cord blood may be collected outside the womb after the placental abruption. After securing the umbilical vein, the cord blood is extracted from a cord blood collection bag containing warfarin using a probe 145 The adipose tissue may be obtained by a predetermined method well known to those of ordinary skill in the art. For example, the adipose tissue may be obtained by suction-assisted liposuction, ultrasonic-assisted liposuction, adipose tissue removal or combinations thereof. In suction-assisted liposuction, the adipose tissue is collected by inserting a cannula in or around an adipose tissue storage

150 existing in a patient and sucking out lipids into suction equipment. Adipose tissue removal includes the steps of incidentally collecting tissue containing adipose tissue (e.g., skin), that is, target tissue for an operation (e.g., skin in lipectomy or cosmetic surgery) together with the adipose tissue.

The MSCs may be isolated from the cord blood or the adipose tissue

155 which are obtained according to the method well known to those of ordinary skill in the art. The MSCs may be isolated by any conventional method well known to those of ordinary skill in the art.

For example, the methods may include density gradient fractionation, immunoselection and differential adhesion separation.

160 Isolating and culturing MSCs from cord blood may be performed by any conventional method including one disclosed in Korean Patent application No. 10- 2002-0008639 (Pittinger MF, Mackay AM et al., Science 1999, 284:143-7; Lazarus HM, Haynesworth SE et al., Bone Marrow Transplant 1995, 16: 557-64).

In one aspect, the isolation of MSCs from cord blood may be performed

165 by a method to be described below. The MSC may be isolated by the steps of isolating cord blood cells into low-density mononuclear fractions, culturing the mononuclear cells to result in a confluency of 80% to 90% in appropriate conditions and subculturing the cells after being treated with trypsin.

The isolation and culture of the MSCs from the adipose tissue may be

170 performed by any method well known to those of ordinary skill in the art.

In another aspect, the MSCs may be isolated by the steps of treating collagenase to the adipose tissue at a sufficient concentration, culturing the adipose tissue in appropriate conditions (temperature and time), isolating floating fat cells by centrifugation or another method well known to those of ordinary skill in the art, and tissue-culturing precipitating stromal fractions.

The isolated MSCs may be cultured in a cell culture medium well known to those of ordinary skill in the art, which may include DMEM medium, McCoys 5 A medium (Gibco), Eagle's basal medium, CMRL media, Glasgow minimal essential medium, Ham's F- 12 medium, Iscove's modified Dulbecco's medium, Liebovitz's L- 15 medium, and RPMI 1640 medium, but the present invention is not limited thereto. Also, in the present invention, at least one auxiliary element may be added when required, which may include: serum of calf, horse and human; antibiotics such as streptomycin sulfate and penicillin G for preventing contamination of microorganisms; and antifungal agents such as amphotericin B, gentamicin and nystatin.

The isolated MSCs may be stored by a method well known to those of ordinary skill in the art before use. Generally, the MSCs may be cold-stored after cyroprotection treatment. The cyroprotection treatment may be performed using a cyroprotective agent such as dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidine, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose or choline chloride.

Also, the pharmaceutical composition of the present invention including the MSC as an active ingredient may further include a pharmaceutically available carrier and diluent. The pharmaceutically available carrier and diluent may be biologically and physiologically friendly to MSCs and a recipient. The pharmaceutically available diluents may include saline water, soluble buffer, solvent and/or dispersing agent, but the present invention is not limited thereto. The pharmaceutical composition of the present invention may be suitable

200 for transdermal, muscular, abdominal, vascular, hypodermic, nasal, spinal or oral administration, but the present invention is not limited thereto. Also, the pharmaceutical composition of the present invention may be applied using a catheter. For example, in a peripheral vein method, cells may be injected through a catheter in a single lump or several smaller amounts. The injection of

205 the cells using a catheter may include delivery into a vein through, for example, a standard peripheral vein catheter, a central vein catheter or a pulmonary catheter.

The effective administration amount of the pharmaceutical composition of 210 the present invention may be determined by one skilled in the art according to the specific purpose described above in consideration of a variety of factors such as administration route, the number of treatments, and age, weight, health condition, gender, seriousness of the illness, diet and an evacuation rate of a patient.

215 Hereinafter, particular methods of the present invention will be described in detail with reference to exemplary embodiments, but the scope of the present invention is not limited to the embodiments described herein. [Advantageous Effects]

A composition including a mesenchymal stem cell according to the present 220 invention can substitute for a damaged liver cell to recover liver function and reduce collagen fibrils deposited to the liver, and thus can be used for prevention and treatment of liver fibrosis or hepatic cirrhosis. [Description of Drawings J FIG. 1 illustrates an experimental method designed to induce liver fibrosis 225 in rats using dimethylnitrosamine (DMN).

FIG. 2 illustrates a measurement result of levels of total protein (A), albumin (B), alanine transaminase (ALT) (C) and aspartic acid transaminase (AST) (D) which are measured in second and fifth weeks after administration of DMN to rats. 230 NC: Negative control

DMN-induced: DMN-induced liver fibrosis group

* denotes a significant difference from a value of the NC (P<0.05).

FIG. 3 is a graph (D) illustrating per-area % (C) of collagen fibrils and liver weight per body weight in a DMN-induced group after the livers of 5 week 235 old DMN-induced rats are stained by Masson's trichrome staining (A: negative control, B: DMN-induced group).

NC: Negative control

DMN-induced: DMN-induced liver fibrosis group

* denotes a significant difference from a value of the NC (P<0.05).

240 FIG. 4 illustrates an experimental method designed to measure an effect of administration of mesenchymal stem cells (MSC) to DMN-induced liver fibrosis rats.

FIG. 5 is a graph illustrating an accumulated survival rate according to administration of MSCs into DMN-induced liver fibrosis rats.

245 FIG. 6 illustrates a measurement result of levels of total protein (A), albumin (B), ALT (C) and AST (D) according to injection of MSCs into DMN- induced liver fibrosis rats. FIG. 7 illustrates a measurement result of liver weight per body weight according to administration of MSCs into DMN-induced liver fibrosis rats. 250 FIG. 8 shows a photograph of a liver stained by Masson's trichrome staining in one week after administration of MSCs into a DMN-induced liver fibrosis rat.

A: Liver of normal rat

B: Group into which only cell carriers are administered, Arrow: Collagen 255 fibril deposition and bridge necrosis

C: Cord blood-derived MSC administered group, Arrow: Decreased collagen fibril deposition and bridge necrosis

D: Adipose tissue-derived MSC administered group, Arrow: Decreased collagen fibril deposition and bridge necrosis

260 FIG. 9 shows a photograph of a liver stained by Masson's trichrome staining in 4 weeks after administration of MSCs into a DMN-induced liver fibrosis rat.

A: Liver of normal rat

B: Group into which only cell carriers are administered

265 C: Cord blood-derived MSC administered group, Black arrow: Decreased collagen fibril deposition and bridge necrosis

D: Adipose tissue-derived MSC administered group, White arrow: Decreased collagen fibril deposition and bridge necrosis

FIG. 10 illustrates image analysis results of livers stained by Masson's 270 trichrome staining in 1 week and 4 weeks after administration of MSCs into DMN- induced liver fibrosis rats. * denotes a significant difference from a value of the NC (P<0.05). FIG. 11 illustrates hybridization results in tissues in one week after administration of cord blood-derived MSCs to DMN-induced liver fibrosis rats. 275 A: H&E slide of liver tissue of cord blood-derived MSCs administered group in one week after the cell injection

B: Positive reaction (arrow) in the liver tissue of the cord blood-derived- MSCs injected group in one week after the cell injection [Mode for Invention]

280 Hereinafter, exemplary embodiments of the present invention will be described in detail. However, the present invention is not limited to the exemplary embodiments disclosed below, but can be implemented in various types. Therefore, the present exemplary embodiments are provided for complete disclosure of the present invention and to fully inform the scope of the present 285 invention to those of ordinary skill in the art. <Exemplary Embodiment 1> Induction of liver fibrosis using DMN <1-1> Animals and Experiment Design

Sprague-Dawley (SD) rats (5-weeks old) were purchased (SLC INC.,

290 Shizuoka-ken, Japan) and adapted to circumstances for 5 days. All rats were bred at a temperature of 22 + 3¹¹C and a humidity of 50 ±20%, and in a 12-hour lighting cycle. Water and feed were supplied to the rats ad libitum. Animals and experimental procedures were approved by the Korean Association for Laboratory

Animal Science (KALAS; Approval No. SNU-051227-2, Seoul, Korea).

295 After the 5-day adaptation, 69 rats were separated into 2 groups. Group

I (n=15) was a negative control (NC), and Group II was a group having dimethylnitrosamine (DMN)-induced liver fibrosis. The DMN diluted with 1% saline was applied to Group II at a concentration of l(l/g by abdominal injection for the first three days in each of 8 weeks (Hu QW et al., Life Sci. 2006 M 4;79(6):606- 300 612). Referring to FlG. 1, the DMN induction was terminated in the 8^th week when all rats were dead.

<l-2> General Test and Accumulated Survival Rate To check whether liver fibrosis was induced in the rats by the DMN in Embodiment <1-1>, clinical symptoms of liver fibrosis, the primary characteristics, 305 including ascites, jaundice, epistaxis, bilirubin urea and weight loss were checked every day, and survival rate was calculated. Meanwhile, autopsies were performed on animals within 24 hours after death. The animals that received an autopsy were not included in the measurement of accumulated survival rate.

As a result of the test, the accumulated survival rate of the DMN- 310 induced liver fibrosis group began to decrease in the 5th week and abruptly decreased in the 6th week, and all of the remaining rats died in the 8th week.

<l-3> Measurement of Total Protein, Albumin, aspartic acid transaminase (AST) and alanine transminase (ALT) Levels

The liver fibrosis SD rats exhibited general clinical symptoms of low total

315 protein and albumin levels and high AST and ALT levels. Here, in the 2nd and

5th weeks after the DMN injection described in Embodiment <1-1>, blood was collected from ophthalmic venous plexus of three rats in Groups I and II, and their total protein, albumin, AST and ALT levels were measured.

The procedures described above were repeated three times separately, 320 and the results obtained therefrom are shown as average ± standard deviations. Statistical comparison was performed using a Turkey test after an ANOVA test. It is considered herein that P<0.05 is significant. As can be seen in A and B of FIG. 2, the total protein and albumin levels indicating synthesis ability of a liver were significantly lowered in the DMN-

325 induced group until the 5th week. In contrast, as can be seen in C and D of FIG.

2, the AST and ALT levels indicating a hepatocellular necrosis level were increased in the DMN-induced group.

<l-4> Measurement of Organ Weight

Autopsies were performed on the laboratory animals used in Embodiment 330 <1-1> within 24 hours after death, and then their weights of liver, spleen and kidney were measured.

In the results, the weights of spleens and kidneys in the DMN-induced group did not show significant differences from those in the negative control (the result is not illustrated). Meanwhile, liver weight per body weight (LWIEW) in the

335 DMN-induced group was decreased by approximately 50% of the negative control in the 5th week after the DMN induction.

<l-5> Histopathology and Image Analysis

Liver tissue of the rats used in Embodiment <1-1> was immobilized with 10% neutral formalin, embedded and fragmented to stain it using H&E staining 340 and Masson's trichrome staining. To measure the area of collagen fibrils with respect to other tissues, the collagen fibrils were analyzed using Image-Pro Plus software (Media, Cybernetics, LP). Per-area % indicates a percentage of the area selected as a collagen fibril with respect to the total area.

The H&E staining result shows dilatations and thicknesses of portal triads, 345 arterioles and bile ducts. As shown in the results of the test, a damaged liver cell of the DMN-induced group was replaced by a thick collagen fibril (not illustrated). The Masson's trichrome staining shows that in the 5- week-old DMN- induced group collagen fibrils increased by a large amount over the negative

350 control (A and B of FIG. 3). It also shows that per-area % of the collagen fibrils in the DMN-induced group reached approximately 9% in the 5th week (C of FIG.3).

Effects of Administration of MSCs to Liver Fibrosis-hiduced Rats

355 <2-l> Animals and Experiment Design

In order to demonstrate an effect of mesenchymal stem cells (MSCs) in liver fibrosis rats, by the same method described in Embodiment <1-1>, 36 rats were treated with DMN to induce liver fibrosis. Referring to Embodiment <2-2> which will be described below, survival rate of the DMN-induced group began to

360 decrease in the 5th week, and drastically decreased in the 6th week, and thus the DMN induction was terminated in the 8th week. The rats with liver fibrosis induced by DMN were separated into three groups. Into Group III (n=12), only cell-vehicles (VC group) were administrated, and into Groups IV (n=12) and V (n=12), cord blood-derived MSCs (UCB-MSCs group) and adipose tissue-derived

365 MSCs (AD-MSCs group) were administrated, respectively. The MSCs were injected into a tail vein at a concentration of IxIO⁶ cell/100(l PBS per rat in the first week. Further, in the VC group, the same volume of vehicles (100(1 PBS) was injected into the same route.

The UCB-MSCs were treated by a method which has been disclosed by

370 the present inventors (Kim SW et al., Stem Cells 2006 Jun;24(6):1620-1626). A UCB sample was donated from a woman undergoing full-termed delivery at Boramae Hospital, Seoul. This experiment was approved by Boramae Hospital and the Seoul National University Institutional Review Board (IRB) (SNUIRB No. 0603/001-002, Seoul, Korea). Cord blood was defrosted at 37^C and separated into

375 low-density mononuclear fractions using Ficoll-Paque Plus (GE Healthcare AB, Uppsala, Sweden, www. amersham.com). After that, the mononuclear cells were washed and suspended with Dulbecco's modified Eagle's medium low glucose (Gibco, Grand Island, NY, www.invitrogen.com) containing 20% fetal bovine serum (Gibco), and then inoculated to a T-25 flask at a concentration ranging from

380 4xlO⁶ to 5xlO^δ cells/cm². This remained at 37"C in a humidified atmosphere containing 5% CO₂. Three days later, the suspended cells were moved to a new flask. Every seven days, the medium was changed. The cells reaching a confluency of 80 to 90% were trypsinized with 0.005% trypsin/ethylene diamine tetra-acetic acid (Gibco) and subcultured, and then replated at 5xlO⁴

385 cells/cm². The cells were cultured and proliferated for 4 to 6 weeks after the initial culture, and subcultured once or twice before use for a subsequent clinical experiment.

The human AD-MSCs were isolated from subcutaneous adipose tissue. The adipose tissue was obtained from tissue removed after selective

390 cosmetic liposuction. The post-liposuction disposal tissue was periodically agitated for an hour at 37 ± to be decomposed using lmg collagenase type I per ml of Dulbecco's modified Eagle's medium (DMEM; Gibco). Floating adipose cells were isolated from precipitating stromal fractions by centrifugation. The stromal cells were cultured in a tissue culture flask containing DMEM (Gibco) having 10% fetal

395 bovine serum (Gibco). Twelve hours later, the culture medium was substituted by defined Keratinocyte-SFM (Gibco) containing 5% fetal bovine serum. Every other day, the medium was exchanged with new one, and the cells were cultured until reaching a predetermined confluency. One week later, a sufficient number of cells were generated for trypsinization, and then stored in liquid nitrogen or

400 subcultured before use for a subsequent clinical experiment. <2-2> General Test and Accumulated Survival Rate In each group in Embodiment <2-l>, as primary characteristics of liver fibrosis, clinical symptoms including ascites, jaundice, epistaxis, bilirubin, urea, and weight loss were checked every day, and a survival rate was calculated.

405 As can be seen in FIG. 5, survival rates of the UCB-MSC group and the

AD-MSC group were lower than that of the VC group to which only cell vehicles are administrated The survival rates of the VC group, the UCB-MSC group and the AD-MSC group in the 4th week after the cell injection were 66.7%, 33.3% and 22.2%, respectively.

410 The rats to which human MSCs were transplanted lived for 4 weeks after the cell injection. It has been reported that a xenotransplanted recipient can have a variety of complications and may not live for more than one week. Thus, the reason for low survival rates of the UCB-MSC group and the AD-MSC group is graft versus host rejection due to the transplantation of the MSCs.

415 <2-3> Measurement of Total Protein, Albumin, AST and ALT Levels

In order to demonstrate whether liver function is recovered by administration of MSCs, blood samples were collected from an abdominal vein, and a clinical and biochemical experiment was performed by the same method as described in Embodiment <l-3>. The blood was collected from the abdominal

420 vein in the first to fourth weeks after the cell injection.

In the results, total protein and albumin levels in all groups began to increase after the cell injection. As can be seen in A and B of FIG. 6, the total protein and albumin levels of the UCB-MSC group in the fourth week after the cell injection were higher than those of the AD-MSC group and the VC 425 group. As can be seen in C and D of FIG. 6, the AST and ALT levels begin to decrease after the cell injection. The UCB-MSC group exhibited the most excellent recovery characteristic in the fourth week after the cell injection.

<2-4> Measurement of Organ Weight

In the first and fourth weeks after the cell injection described in 430 Embodiment <2-l>, liver weight per body weight (LW/BW) of each group was measured.

As a result of the test, LW/BW increased only in the AD-MSC group in the first week after the cell injection. LW/BW decreased in the AD-MSC group in the fourth week after the cell injection, and highly increased in the VC group

435 and the UCB-MSC group (refer to FIG. 7). The MSC-administrated group and the

VC group did not show any significant difference.

<2-5> Histopathology and Image Analysis

The samples obtained from the respective groups in Embodiment <2-l> were immobilized with 10% neutral formalin to be used for histopathological

440 analysis. The histopathology and image analysis were performed by the same method as described in Embodiment <l-5>. In order to measure the area of collagen fibrils with respect to another tissue, three slide-based images from the respective groups were analyzed using Image-Pro Plus software. Per-area % indicates a percentage of the area selected as collagen fibrils with respect to the

445 total area.

The H&E staining result shows that the collagen fibril of the MSCs- administered group was thinner than that of the VC group. The Masson's trichrome staining shows that the collagen fibrils in the UCB-MSCs group and the AD-MSCs group significantly decreased in number in the 450 first week after the cell injection (C and D of FIG. 8), however the collagen fibrils in the VC group slightly decreased in number (B of FIG. 8). In the UCB- MSC group and the AD-MSC group, the significant decrease of collagen fibrils was maintained until the fourth week after the cell injection (C and D of FIG. 9).

FIG. 10 shows that the per-area % of the collagen fibril in the UCB-MSC 455 group and the AD-MSC group is decreased 6% and 2% in the first week after the cell injection, respectively.

It can be seen from the results described above that MSCs can substitute for damaged liver cells of a recipient, and decompose collage fibers existing around the damaged liver cell. 460 <2-6> In situ Hybridization

In order to demonstrate homing of the administrated MSCs toward the damaged liver tissue, a series of tissue fragments were stained by H&E staining, and hybridized with a human-specific AIu gene in the tissue.

A DNA probe suitable for the human-specific AIu gene was formed by a

465 method which will be described below. Genome DNAs of human UCB-MSCs were extracted using a DNAeasy tissue kit (Qiagen, Hilden, Germany). A PCR primer was located at the most compatible area of the human AIu sequence, and produced a 224 bp PCR product {Kim J et al., Cell 1998 Aug 7;94(3):353-362; Cho

JJ et a/., Nat. Med. 2002 Sep;8(9):1033-1036; Walker JA et al., Anal. Biochem. 2003

470 Apr 1;315(1):122-128). In order to amplify the AIu sequence using PCR, primers to be listed below were used.

AIu sense primer (sequence No. 1) - Id ¬

s' ACG CCT GTA ATC CCA GCA CTT-3' AIu anti-sense primer (sequence No. 2) 475 5'-TCG CCC AGG CTG GAG TGCA-3'

The PCR was performed under the following conditions: 25 cycles of 10 min at 95⁰C , 30 sec at 95V , 45 sec at 58r and 45 sec at 72⁰C , and 10 min at 72¹C .

The PCR product was electrophoresed in a 2% Agaros gel, and stained 480 with ethidium bromide (lOng/ml). A 224bp DNA band was extracted using a QIAquick gel extraction kit (Qiagen). The PCR product was marked with DIG using a DIG probe synthesis kit (Roche Diagnostics, Basel, Switzerland). The PCR was performed on the 50ng extracted DNA using the same method as the above- described PCR protocol. The marked probe was purified by ethanol precipitation 485 according to the PCR protocol for the DIG probe synthesis.

The hybridization in tissue was performed by a method which will be described below. Right before performing the hybridization in tissue, tissue fragments were deparaffinated in xylene and then rehydrated with PBS. Subsequently, the rehydrated slide was cultured together with PBS 490 containing 0.3% tryptone X-100, and further cultured together with TE buffer containing 2mg/ml proteinase K for 30 min at 37¹C , and then was washed three times for 5 min. The slide was acetylated two times for 5 min using TEA buffer containing 0.25% (v/v) acetic acid dehydrate (Sigma-Aldrich, St. Louis) in order to reduce non-specific background staining. After pre-hybridization for 3 hours at 495 85⁰C using a hybridization buffer in which formamide (Sigma-Aldrich), 0.1% sodium lauryl sarcosine (Sigma-Aldrich), 0.02% SDS (Sigma-Aldrich) and 2% blocking agent (Roche) are dissolved in 50% 5xSSC, the slide was further cultured for 10 min at 9AV together with a new hybridization buffer containing denaturalized DIG- marked DNA probe (10-200ng/ml). Then, the slide remained in ice for 10 min

500 and overnight-cultured at A2V . The pre-hybridization and hybridization steps were performed in a moist chamber containing 50% formamide. After the hybridization, the slide was simply washed with 2xSSC at room temperature, and washed three times with 0. IxSSC for 15 min at 42V . Visualization of the DIG- marked DNA probe was performed according to the DIG hexane detection kit

505 (Roche) protocol. The slide was blocked for 30 min with blocking buffer [1% blocking agent (Roche) dissolved in maleic acid buffer (0.1M maleic acid, 0.15M NaCl, pH 7.5)], and cultured for an hour together with an antibody solution coupled with alkaline phosphate [both (alkali and antibody) are dissolved in a blocking buffer containing 0.1% tryptone X-IOO at a ratio of 1:2000]. Then, the

510 slide was washed four times for 15 min with a maleic acid buffer, and stabilized for 5 min in a Tris buffer (pH 9.5). Color development was performed using a substrate solution [(nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (Roche; pH 9.5) which were dissolved in a Tris buffer). The reaction was terminated with termination buffer (1OmM Tris-HCl, pH 8.0, ImM EDTA) after

515 enzyme reaction for 2 to 6 hours. The slide was washed three times for 15 min, and counterstained with 0.1% nuclear fast red (Sigma- Aldrich). The slide was washed three times with PBS.

As can be seen in the result shown in FIG. 11, in the first week after the cell injection, a positive signal (black arrow) was detected from the UCB-MSC

520 group. The signal was not detected or was weakly detected from the AD-MSC group in the first week, and the UCB-MSC group and AD-MSC group in the fourth week after the cell injection. [Industrial Applicability]

A composition including a mesenchymal stem cell according to the present 525 invention can substitute for a damaged liver cell to recover liver function and reduce collagen fibrils deposited to the liver, and thus can be used for prevention and treatment of liver fibrosis or hepatic cirrhosis.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those of ordinary

530 skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

[CLAIMS] [Claim 1]

535 A pharmaceutical composition for preventing and treating liver fibrosis comprising a mesenchymal stem cell. [Claim 2]

The composition according to claim 1, wherein the mesenchymal stem cell is derived from cord blood or adipose tissue. 540 [Claim 3]

The composition according to claim 2, wherein the cord blood or the adipose tissue is isolated from a human. [Claim 4]

A pharmaceutical composition for preventing and treating hepatic cirrhosis 545 comprising a mesenchymal stem cell. [Claim 5]

The composition according to claim 4, wherein the mesenchymal stem cell is derived from cord blood or adipose tissue. [Claim 6]

550 The composition according to claim 5, wherein the cord blood or the adipose tissue is isolated from a human.

555