WO2014132129A2

WO2014132129A2 - Autologous bone-marrow-derived mesenchymal stem cells for alcoholic cirrhosis

Info

Publication number: WO2014132129A2
Application number: PCT/IB2014/000727
Authority: WO
Inventors: Yoon Ok JANG
Original assignee: Pharmicell Co., Ltd.
Priority date: 2013-01-08
Filing date: 2014-01-08
Publication date: 2014-09-04
Also published as: KR20150100722A; WO2014132129A3

Abstract

A method of improving liver function in a subject with alcoholic cirrhosis, comprising administering to said subject a composition comprising autologous bone marrow derived mesenchymal stem cells (BM-MSCs) in a way to elucidate anitfibrotic effect of said BM-MSCs in said subject.

Description

BACKGROUND OF THE INVENTION

Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art to the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.

Liver cirrhosis, the end stage of progressive hepatic fibrosis, is characterized by distortion of the hepatic architecture and the formation of regenerative nodules, angiogenesis, and shunt formation, leading to loss of liver function and the development of hepatocellular carcinoma (1 -5). The main causes of cirrhosis are chronic alcohol abuse, and hepatitis B and C viruses. Alcoholic liver cirrhosis (ALC), one of the major medical complications of alcohol abuse, is a major cause of chronic liver disease worldwide (6-8). Cirrhosis may cause irreversible liver damage. If untreated, liver and kidney failure and gastrointestinal hemorrhage can occur, sometimes leading to death. In the United States, cirrhosis results in about 25,000 deaths annually. There remains a need for an effective method for treating liver cirrhosis and other forms of liver disease.

The most effective therapy for advanced cirrhosis is currently liver transplant. However, this procedure has several limitations, including lack of donors, surgical complications, immunological rejection, and high medical cost (9). Alternative approaches that circumvent the use of the whole organ, such transplantation of cells of various origins, have recently been accepted (10, 1 1 ). For example, stem-cell transplantation has been suggested as an effective alternate therapy for hepatic disease ( 12, 13).

Several previous studies using animal models of liver diseases have demonstrated that bone marrow (BM) cell transplantation may accelerate the liver regeneration process, reduce hepatic fibrosis, and improve liver function and the survival rate (14-17). The prospects for stem cell transplantation as a therapy for hepatic disease, as determined by initial translational pilot studies testing the direct hepatic administration of BM -derived stem cells, have been encouraging and have suggested enhanced liver regeneration prior to partial hepatectomy, showing improved liver function in advanced chronic liver disease (18-24). Among the stem cells, mesenchymal stem cells (MSCs) in particular have practical advantages in regenerative medicine due to their high capability for self-renewal and differentiation without ethical or tumorigenic problems.

REFERENCES 1. BOLONDI L, GRAMANTTERI L. From liver cirrhosis to HCC, Internal and emergency medicine 2011 ; 6 Suppl 1 : 93 -8.

2. IREDALE J P. Cirrhosis: new research provides a basis for rational and targeted treatments. BMJ 2003; 327(7407): 143-7.

3. KIM M Y, BAIK S K. YEA C J, et ai. Hepatic venous pressure gradient can predict the development of hepatocellular carcinoma and hyponatremia in decompensated alcoholic cirrhosis. European journal of gastroenterology & hepatology 2009; 21(11): 1241-6.

4. KIM M Y, SUK K T, BAIK S K, et al. Hepatic vein arrival time as assessed by contrast-enhanced ultrasonography is useful for the assessment of portal hypertension in compensated cirrhosis. Hepatology 2012; 56(3): 1053-62.

5. LEE S S, SHIN H S, KIM H J, et al. Analysis of prognostic factors and 5 -year survival rate in patients with hepatocellular carcinoma: a single- center experience. The Korean journal of hepatology 2012; 18( 1): 48-55.

6. GAO B, BATALLER R. Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology 2011 ; 141(5): 1572-85.

7. KIM M Y, CHO M Y, BAIK S K, et ai. Beneficial effects of candesartan, an angiotensin-blocking agent, on compensated alcoholic liver fibrosis - A randomized open- label controlled study. Liver international : official journal of the International Association for the Study of the Liver 2012.

8. KWON O S, JUNG Y K, KIM Y S, et al. Effect of alcohol on the development of hepatocellular carcinoma in patients with hepatitis B virus-related cirrhosis: a cross- sectional case-control study. The Korean journal of hepatology 2010; 16(3): 308-14. 9. SIN GAL A K, DUCHINI A. Liver transplantation in acute alcoholic hepatitis: Current status and future development. World journal of hepatology 2011 ; 3(8): 215-8.

10. STROM S C, BRUZZONE P, CAI H, et al. Hepatocyte transplantation: clinical experience and potential for future use. Cell transplantation 2006; 15 Suppl 1 : SI05-10.

11. STROM S, FISHER R. Hepatocyte transplantation: new possibilities for therapy. Gastroenterology 2003; 124(2): 568-71.

12. KAKINUMA S, NAKAUCHI H, WATANABE M. Hepatic stem/progenitor cells and stem-cell transplantation for the treatment of liver disease. Journal of gastroenterology

2009; 44(3): 167-72.

13. KHARAZIHA P, HELLSTROM P M, NOORINAYER B, et al. Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem ceil injection: a phase I-II clinical trial. European journal of gastroenterology & hepatology 2009; 21(10): 1 199-205. 14. CHO K A, LIM G W, JOO S Y, ei al. Transplantation of bone marrow cells reduces CC14 -induced liver fibrosis in mice. Liver international : official journal of the

International Association for the Study of the Liver 201 1 ; 31 (7): 932-9.

15. SAKAIDA I, TERAI S, YAMAMOTO N, et al. Transplantation of bone marrow cells reduces CCM-induced liver fibrosis in mice. Hepatology 2004; 40(6): 1304-11.

16. HARD JO M, MIYAZAKI M, SAKAGUCHi M, et al Suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow. Cell transplantation 2009; 18(1 ): 89-99.

17. HIGASHIYAMA R, INAGAKI Y, HONG Y Y, et al. Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice.

Hepatology 2007; 45(1): 213-22.

18. MORMONE E, GEORGE J, NIETO N. Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches. Chemico-biological interactions 2011 ; 193(3): 225-31.

19. PAI M, ZACHAROULIS D, MILICEVIC M N, ei al. Autologous infusion of expanded mobilized adult bone marrow-derived CD34+ cells into patients with alcoholic liver cirrhosis. The American journal of gastroenterology 2008: 103(8): 1952-8.

20. ROCKEY D C. Current and future anti-fibrotic therapies for chronic liver disease. Clinics in liver disease 2008; 12(4): 939-62, xi.

21. SAKAIDA I, TERAI S, NISHINA H. OKJTA K. Development of cell therapy using autologous bone marrow cells for liver cirrhosis. Medical molecular morphology 2005;

38(4): 197-202.

22. TERAI S, ISHIKAWA T, OMORI K, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Ceils 2006;

24(10): 2292-98.

23. AM ESCH J S, 2ND, KNOEFEL W T, KLEIN M, et al. Portal application of autologous CD 133+ bone marrow ceils to the liver: a novel concept to support hepatic regeneration. Stem Cells 2005; 23(4): 463-70.

24. VAN POLL D, PAREKKADAN B, CHO C H, et al. Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in VIVO, Hepatology 2008: 47(5): 1634-43.

25. D'AMICO G, GARCIA-TSAO G, PAGLIARO L. Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 11 8 studies. Journal of hepatology 2006: 44(1): 217-31. 26. VELDT B J, LAINE F, GUILLYGOMARC'H A, et al. indication of liver

transplantation in severe alcoholic liver cirrhosis: quantitative evaluation and optimal timing. Journal of hepatology 2002; 36(1 ): 93-8.

27. HU Y, LIAO L, W ANG Q, et al. Isolation and identification of mesenchymal stem cells from human fetal pancreas. The J ournal of laboratory and clinical medicine 2003;

141(5): 342-9.

28. MARTIN J Y, DEAN D D, COCHRAN D L, SIMPSON J, BOY AN B D,

SCH W ARTZ Z. Proliferation, differentiation, and protein synthesis of human osteoblast- like cells (MG63) cultured on previously used titanium surfaces. Clinical oral implants research 1996; 7(1 ): 27-37.

29. KIM M Y, CHO M Y, BAT.K S K, et al. Histological subcfassification of cirrhosis using the Laennec fibrosis scoring system correlates with clinical stage and grade of portal hypertension. Journal of hepatology 201 1; 55(5): 1004-9.

30. WANLESS I R, SWEENEY G, DHILLON A P, et al. Lack of progressive hepatic fibrosis during long-term therapy with deferiprone in subjects with transfusion-dependent beta -thalassemia. Blood 2002; 100(5): 1566-9.

31 . CALVARIJSO V, BURROUGHS A K, STANDISH R, et al. Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient, Hepatology 2009; 49(4): 1236-44.

32. SUK K T, BAIK S K, YOON J H, et al. Revision and update on clinical practice guideline for liver cirrhosis. The Korean journal of hepatology 2012; 1 8(1 ): 1 -21.

33. PENG L, XIE D Y, LIN B L, et al. Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes. Hepatology 2011 ; 54(3): 820-8.

Figure Legends

Fig. L Flow chart of the inclusion of patients and the study design.

Fig. 2. Immunoplienotypes and differentiation potentials of the BM-MSCs. (A) The expressions of cell-surface antigens (CD 14, CD34, CD45, CD73, and CD 105) were evaluated by flow cytometry. (B) Representative histograms for the positive populations represent averages for 1 1 patients with, the indicated standard deviations. (C) BM-MSCs stained positively for endogenous alkaline phosphatase activity, indicating osteogenic differentiation within an osteogenic medium (OM; I), or stained negatively in control medium (CM; II). BM-MSCs stained positively for lipid droplets, indicating adipogenic differentiation within adipogenic medium (AM; III), or stained negatively in CM (IV) ( 200). Fig. 3. Histological and ImmuMohisiochensical Analysis.

Histological analysis was evaluated by (A, B) H&E and (C, D) MTC staining (x lOO). BM-MSC therapy induced an improvement of cirrhosis from (A, C) F4C pretherapy to (B. D) F4B posttherapy according to the Laennec fibrosis scoring system,

Picrosirius Red staining of a section from a liver biopsy specimen showed a change of collagen proportion stained as red from (E) pretherapy to (F) posttherapy (x4G). (G) The relative area of collagen stained by Picrosirius Red was analyzed with an image- analysis program. The percentage of collagen proportionate area decreased from

22.6i±8.39% (pretherapy) to 17.70±6.93% after BM-MSC therapy. Data are mean and SD values. */><0.001,

Fig. 4. Gene expression analysis associated with fibrosis.

After administration of BM-MSC therapy, the relative expressions (2--AACt values) of (A) TGF-β Ι , (B) collagen- 1 , and (C) a-SMA by real-time PGR decreased significantly from 3.81+1.39 to 2.65±1.21 (*p ===0.013), from 4.51±2.62 to 3.15+2.82 (*/>=0.021 ), and from 4.27+1 .89 to 2.34±1.60 (*p=0.0Q7), respectively. Data are mean and SD values.

BRIEF SUMMARY OF THE INVENTION

The invention provides stem cell treatment for liver disease in a subject in need thereof. In one aspect, the invention provides therapies comprising the introduction into a patient of mesenchymal stem cells (MSCs) to treat patients with alcoholic cirrhosis. In another aspect, the invention relates to antifibrotic effects of autologous bone marrow- derived mesenchymal stem cells (BM-MSCs) on human alcoholic cirrosis. In another aspect, the in vention relates to a procedure of applying hematologic examination or histological examination as a means to predict the effect of stem cells in liver cirrhosis. Yet in another aspect, the invention relates to a high purity stem cells (98-99%) and the method of making thereof to produce therapeutic effects of stem cells which is much more superior and. has better performance than other stem cells.

DETAILED DESCRIPTION

Before the present plasma modified medical devices and methods are disclosed and described, it is to be understood, that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used, for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. The references discussed herein are provided, solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this in vention belongs.

In describing and claiming the present inveiUion, the following terminology will be used in accordance with the definitions set out below.

As used herein, "comprising," "including." "containing." "characterized by," and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method, steps. "Comprising" is to be interpreted as including the more restrictive terms "consisting of and "consisting essentially of."

As used herein, "consisting of and grammatical equivalents thereof exclude any element, step, or ingredient not specified in the claim.

As used herein, "consisting essentially of and grammatical equivalents thereof limit the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic or characteristics of the claimed invention.

The invention relates to antifibrotic effects of bone marrow mesenchymal stem eells(BM-MSCs) therapy on humans with alcoholic liver cirrhosis(ALC). In specific embodiments of the methods described herein, the human mesenchymal stem cells are autologous, allogeneic, or HLA compatible with the subject. The number of mesenchymal stem cells administered to an individual afflicted alcoholic liver cirrhosis will vary according to the severity of the cirrhosis, the duration of the cirrhosis condition, the size of the tissue that is liver cirrhosis, and the method of delivery. In one embodiment of the methods described herein, the therapeutically effective amount of enriched human mesenchymal stem cells is a safe and effective amount. In another specific embodiment, the amount of bone marrow mesenchymal stem ceils(BM-MSCs) is at least 1x10 ⁴ cells. In another embodiment, the amount of bone marrow mesenchymal stem cells(BM-MSCs) administered to the subject in the methods described herein is between about 1x10 ⁴ and about 5x10 ⁸ cells. In certain embodiments, the number of cells may be between 1x10 ⁴ and 5xl 0⁷, 1x10 ⁴ and 5x10 ⁶, 1x 10 ⁵ and 5x10 ⁷, 1x10 ⁶ and 5x10 ^"'.times, 1x10 ⁷ and 5x10 ^s.

When a combination of cell types is administered, both cells types of cells maybe autologous, allogeneic, or HLA compatible with the subject, whereas in other

embodiments one cell type may be autologous and the other allogenic or HLA compatible with the subject.

The amount of cells administered to the subject will depend on the mode of administration and the site of administration. For example, a therapeutically effective cell dose via the Edmonton protocol may be lower than that for intra-arterial injection.

In embodiments of the methods described herein, administering to the subject is effected via an infusion of cells into the subject. The intusion may comprise a systemic infusion of cells into the subject, or it may comprise an infusion of cells in the proximity of the fibrotic tissue, so as to facilitate the migration of ceils to the fibrotic tissue, or it may comprise both. The infusion may also be performed on the blood vessels that supply blood to the fibrotic tissue, such as the portal vein or a hepatic artery, or to blood vessels which remove blood, from the fibrotic tissue, such as a hepatic vein. In specific embodiments of the methods described herein, the intusion of ceils into the subject comprises an infusion into bone marrow, an intra-arterial infusion, an

intramuscular infusion, an intravenous infusion or an intradermal infusion. In one embodiment of the methods described herein, the cells are administered to the subject by infusion into at least one artery.

The present invention provides a method of improving liver function in a subject with alcoholic cirrhosis, comprising administering to said subject a composition comprising autologous bone marrow derived mesenchymal stem cells (BM-MSCs) in a way to elucidate antifibrotic effect of said BM-MSCs in said subject.

The subject has baseline biopsy-proven alcoholic cirrhosis.

The subject has been alcohol free for at least 6 months before the administering

BM-MSCs.

The BM-MSCs being isolated, from said subject's bone marrow and being amplified, for I month.

The way of administering BM-MSC is injecting 5x107 BM-MSCs to the subject twice at weeks 4 and. 8, through hepatic artery. The subject being taken biopsy and laboratory tests at 12 weeks to evaluate histological and quantitative improvement of hepatic fibrosis.

In some embodiments of the methods described herein, administration of the cells to the subject is performed using an intra-arterial catheter, such as but not limited to a balloon catheter, or by using a stent. Any method currently available for delivering cells to a subject may be used to administer cells to a subject in the methods described herein.

In some embodiments of the methods described herein, at least one treatment is further administered to the subject. In one embodiment, the treatment comprises vegetable protein-rich diet, abstinence from alcohol, bed rest, vitamin B, vitamin E, or vitamm C or any combination thereof. In preferred, embodiments, the treatment promotes decreased need for liver transplant or resection or increased survival.

In some preferred embodiments of the methods described herein, the mesenchymal stem cells, are enriched prior to administration. By enrichment it is meant that the concentration of the cells relative to that of other cells is increased. Enrichment may be accomplished by removing other types of cells from the composition containing these cells, by culturing the cells under conditions which improve their proliferation over those of other cells, or by any method known in the art for enriching one cell type over another. In some embodiments, the cells used in the methods described herein are enriched at least about two-fold, about five-fold, about twenty-fold, about fifty-fold, about one hundredfold, about five hundred-fold, about one thousand-fold, about five thousand-fold, about ten thousand-fold, or by about fifty thousand fold.

In some embodiments of the methods described herein, the cells which are to be administered to the subject are incubated in a buffer, such as a saline buffer. In one preferred embodiment, the buffer comprises human blood serum isolated from the same subject who is the recipient of the therapy. Human serum may be isolated using standard procedures. A solution comprising human blood serum may also be used to thaw a sample of cells that has been cryopreserved. In some embodiments, the solution comprising human serum comprises between 1-20% human serum, or more preferably 5-15%,

One aspect of the invention provides methods for treating liver disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of human mesenchymal stem cells. Mesenchymal stem cells are the formative pluripotent blast cells found in the bone marrow and peripheral blood that are capable of differentiating into any of the specific types of connective tissues (i.e., the tissues of the adipose, areolar, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various environmental influences. Mesenchymal stem cells are also commonly referred to as "marrow stromal cells" or just "stromal cells". Mesenchymal progenitor cells, are derived from mesenchymal stem cells and have a more limited differentiating potential, but are able to differentiate into at least two tissues (see for example, FIG. 1 of Minguell et al. 2001 Exp Biol Med (Maywood); 226(6):507-20). As used herein, the term "mesenchymal stem cells" comprises mesenchymal stem cells, mesenchymal progenitor cells and marrow stromal cells.

Although hMSCs are rare, comprising about 0.01-0,0001% of the total nucleated cells of bone marrow, Applicants have perfected a cell culture methodology for their isolation from bone marrow, purification to homogeneity from other bone marrow cells and mitotic expansion in culture without loss of their stem cell potential (Haynesworth S E et al. Bone 13, 81 -88 (1992)).

M esenchymal stem cells for use in the methods according to the in vention can be isolated from peripheral blood or bone marrow. A method for preparing hMSC has been described in U.S. Pat. No. 5,486,359. In a preferred embodiment of the methods described herein, when the mesenchymal stem cells are isolated from bone marrow or peripheral blood of the subject who will be the recipient of the treatment. Several techniques are known for the rapid isolation of mesenchymal stem cells including, but are not limited to, leucopheresis, density gradient fractionation, immunoselection, differential adhesion separation, and the like. For example, immunoselection can include isolation of a population of hMSCs using monoclonal antibodies raised against surface antigens expressed by bone marrow-derived hMSCs.

The hMSC for use in the methods according to the invention can be maintained in culture media which can be chemically defined serum free media or can be a "complete medium", such as Dulbecco's Modified Eagles Medium supplemented with 10% serum (DMEM). Suitable chemically defined serum free media are described in U.S. Pat. No. 5,908,782 and WG96/39487, and complete media are described in U.S. Pat. No.

5,486,359. Chemically defined medium comprises a minimum essential medium such as Iscove's Modified Dulbecco's Medium (IMDM), supplemented with human serum albumin, human Ex Cyte lipoprotein, transferrin, insulin, vitamins, essential and. nonessential amino acides, sodium pyruvate, giutamine and a mitogen. These media stimulate mesenchymal stem cell growth without differentiation. Culture for about 2 weeks results in 10 to 14 doublings of the population of adherent cells. After plating the cells, removal of non-adherent cells by changes of medium e very 3 to 4 days results in a highly purified culture of adherent cells that have retained their stem cell characteristics, and can be identified and quantified, by their expression of cell surface antigens identified, by monoclonal antibodies.

In the methods described herein, the therapeutically effective amount of amount hMSCs, can range from the maximum number of cells that is safely received by the subject to the minimum number of cells necessary for either induction of antifibrotic effects. Generally, the therapeutically effective amount of each endothelial generating cells and hMSCs is at least lx lO⁴ per kg of body weight of the subject and, most generally, need not be more than 5x 10⁶ of each type of cell per kg. Although it is preferable that the hMSCs are autologous or HLA-compatible with the subject, the hMSCs can be isolated from other individuals or species or from geneticaily-engmeered inbred donor strains, or from in vitro cell cultures.

The therapeutically effective amount of the MSCs can be suspended in a

pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to basal culture medium plus 1 % serum albumin, saline, buffered saline, dextrose, water, and combinations thereof. The formulation should suit the mode of administration.

hi a preferred embodiment, the hMSC preparation or composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous, intra-arterial or intracardiac administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed, together in unit dosage form, for example, as a cryopreserved concentrate in a hermetically sealed container such as an ampoule indicating the quantity of active agent. When the composition is to be administered by infusion, it can be dispensed, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered, by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

A variety of means for administering cells to subjects will, in view of this specification, be apparent to those of skill in the art. Such methods include injection of the cells into a target site in a subject. Cells may be inserted into a delivery device which facilitates introduction by injection, implantation, or contact with the subjects. Such delivery devices may include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred, embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. In a preferred embodiment, cells are formulated for administration into a blood vessel via a catheter (where the terra "catheter" is intended, to include any of the various tube- like systems for delivery of substances to a blood vessel). The cells may be prepared for delivery in a variety of different forms. For example, the cells may be suspended, in a solution or gel. Cells may be mixed with a pharmaceutically acceptable carrier or diluent in which the ceils of the invention remain viable.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid, and will often be isotonic. Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

Modes of administration of the hMSCs include but are not limited to intravenous or intra-arterial injection and injection directly into the tissue at the intended site of activity. The preparation can be administered by any convenient route, for example by infusion or bolus injection and. can be administered together with other biologically active agents. Administration is preferably systemic.

The practice of the present invention will employ, where appropriate and unless otherwise indicated, conventional techniques of ceil biology, cell culture, molecular biology, transgenic biology, microbiology, virology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 3rd Ed,, ed, by Sambrook and Russell (Cold Spring Harbor Laboratory Press: 2001 ); the treatise, Methods hi

Enzymology (Academic Press, Inc., N.Y.); Using Antibodies, Second Edition by Harlow and Lane, Cold Spring Harbor Press, New York, 1999; Current Protocols in Cell Biology, ed. by Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York, 1999.

EXAMPLES

The invention now being generally described, it will be more readily understood, by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention, as one skilled in the art would, recognize from the teachings hereinabove and the following examples, that other stem cell sources and selection methods, other culture media and culture methods, other dosage and treatment schedules, and other animals and/or humans, all without limitation, can be employed, without departing from the scope of the invention as claimed.. Other aspects of the invention will be apparent to those skilled, in the art to which the invention pertains.

Example 1 Antifibrotic effects of bone marrow mesenchymal stem ceilsiBM- MSCs) therapy on humans with alcoholic liver cirrhosis(ALC).

Patients between 37 and 60 years of age with biopsy-proven ALC and discontinued alcohol intake for at least 6 months prior to participating in this study (to exclude the influence of inflammation by recent alcohol consumption) were considered eligible for the study (25, 26), We presumed that 6 months of abstinence from alcohol might further reduce the active inilammation and allow stabilization of the liver histology in most of the participants, and play an essential role in eliminating the bias that may occur with severe inflammation resulting from recent consumption of alcohol.

hi addition, patients younger than 20 or older than 65 years, with hepatitis B or C virus-related cirrhosis, severe liver failure (serum bilirubin >85 umol/L), hepatic encephalopathy, hepatorenal syndrome, recurrent gastrointestinal bleeding, spontaneous bacterial peritonitis, presence of liver tumor or history of other cancer, and pregnant or lactating were also excluded from this study (Fig. 1).

Radiological imaging studies including ultrasonography and computed- tomography scan, and laboratory studies were conducted on each patient to determine the Child-Pugh score and the model for end-stage liver disease (MELD) score. Other causes of liver disease were excluded, by performing serologic tests for hepatitis B and C infection, and autoantibodies.

According to these criteria, 12 consecutive patients were initially considered for the study. One patient who had continued, to consume alcohol (continuous alcohol intake of more than 60 g/week) was withdrawn from this study. Thus, 1 1 patients were ultimately enrolled. The general characteristics of the patients are listed in Table 1.

transferase; MELD, Model for End-stage Liver Disease BM Aspiration, Isolation of MSCs, and Cell Culture

All manufacturing and product testing procedures for the generation of clinical- grade autologous MSCs were performed under good manufacturing practice conditions (FCB-Pharmicell Co., Ltd., Sungnam, Korea). Approximately 10-20 mL of BM was aspirated from the posterior iliac crest of the patients under local anesthesia. BM mononuclear ceils were isolated by density-gradient centrifugation (Histopaque-1077,

Sigma-Aldrich, St. Louis, MO, USA). Mononuclear cells (2-3x 105 cells/cm2) were plated in a 75-cm2 flask (Falcon, Franklin Lakes, NJ, USA) with low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibeo, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin^streptomycin (Gibco), and cultured at 37°C in a 5% C02 atmosphere. After 5 days, nonadherent cells were removed by replacing the medium. When the cultures approached 80% confluence, the cells were harvested by treatment with a trypsm/EDTA solution (Gibco) and replated at a density of 4-

5 x 103 celis/cm2 in 175-cm2 flasks. Cells for injection were serially subcuiiured up to passages four or five. During culture, some passage-2 cells were harvested and cryopreserved in 10% dimethyl sulfoxide (Sigma- Aldricb) and 90% fetal bovine serum (FBS) for the second injection.

Immunophenotypes and Differentiation Assays of BM-MSCs

The immunophenotypes of the BM-MSCs (CD14, CD34, CD45, CD73, and CD105) were analyzed on the day of injection, and their differentiation potentials were determined (osteogenesis and adipogenesis; Fig. 2). For immunopheriotype analysis, BM- MSCs were stained with antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (FE): CD14-FITC, CD34-F1TC, CD45-FITC, CD73-PE, and CD105-PE (BD Biosciences, San Jose, CA, USA). Briefly, 5 x105 cells were resuspended in 0.2 mL phosphate-buffered saline (PBS) and incubated with FITC- or PE-conjugated antibodies for 20 minutes at room temperature. FITC- or PE-conjugated mouse IgGs were used as the control isotype at the same concentration as the specific primary antibodies. The fluorescence intensity of the cells was evaluated by flow cytometry (Epics XL; Beckman Coulter, Miami, FL, USA).

The osteogenic differentiation was determined by first plating the cells at

2.x 104 cells/cm2 in six-well plates and then leaving them in the following osteogenic medium for 2-3 weeks: low-glucose DMEM medium supplemented with 10% FBS,

10 mM β-glycerophosphate, 10-7 M dexamethasone, and 0.2 mM ascorbic acid (Sigma- Aldrich) (27). The osteogenic differentiation was quantified from the release of p- nitrophenoi from p-nitrophenyl phosphate by the enzyme alkaline phosphatase (28).

For adipogenic differentiation, BM-MSCs were plated, at 2x104 cells/cm2 in six- well plates, cultured for 1 week, and then differentiation was induced with an adipogenic medium (10% FBS, 1 μΜ dexamethasone, 0.5 mM 3-isobutyl-l-methylxanthine, 10 .ug/mL insulin, and. 100 μ.Μ mdomethacin in high-glucose DMEM) for an additional 3 weeks. The differentiated cells were fixed in 4% paraformaldehyde for 10 minutes and stained with fresh Oil Red-0 solution ( Sigma- Aldrich) to show lipid droplets (Fig. 2C). Autologous BM-MSC Injections

On the day of the injection, MSCs were harvested, using trypsiii/EDTA, washed, twice with PBS and once with saline solution, and then resuspended at a final concentration of 5x 106 ce1ls/mL in 10 ml, of than 80%, absence of microbial contamination (bacteria, fungus, virus, or mycoplasma) when tested 3-4 days before administration, expression of CD73 and CD 105 by more than 90% of the cells, and absence of CD 14, CD34, and CD45 in less than 3% of the cells, as assessed by flow cytometry. Each time the 5x 107 cells in 10 mL of saline were injected at weeks 4 and 8 via the right hepatic artery by a coaxial angiographic catheter (Boston Scientific, Natick, MA, USA). Injections of BM-MSCs were performed using coaxial angiographic catheters (Boston Scientific) placed in the hepatic arteiy. Continuous monitoring was performed via leukapheresis as well as the injection procedure, and. any hemodynamic changes were recorded.

Follow-up

After enrollment, patients were foilowed-up according to the study design. The patients did not receive any other medications during the study. Alcohol side effects were monitored, every week by phone call to the patients and. their family, and during their monthly visits to the hospital. Clinical and biochemical tests for the assessment of the side effects present were also performed at each hospital visit. Any evidence of drinking alcohol would result in the subject being withdrawn from the study. A follow-up liver biopsy was performed 12 weeks after the second therapeutic BM-MSC injection (Fig. 1 ).

Primary and Secondary Outcomes

The primary outcome was the improvement of histological findings, which required improvement of at feast 1 point on the Laennec fibrosis scoring system. The secondary outcome included, changes in the Child-Pugh score, direct markers related to hepatic fibrosis, and the development of side effects.

Fibrosis Assessment

Histomorphological and Immunohistochemical Analysis

Paired liver biopsies were performed at baseline and 12 weeks after the second, therapeutic BM-MSC injection. Five-micrometer- thick sections of paraffin -embedded liver biopsy samples were prepared and stained with hematoxylin and eosin (H&E), Masson's trichrome (MTC), a-smooth muscle actin (a-SMA), and Picrosirius Red. Fibrosis was evaluated using both the Laennee fibrosis scoring system (Table 2, Fig. 3) and the METAVIR fibrosis scoring system by a liver pathologist (M. Y.C.) who was blinded to the clinical data of the patients (29, 30). Liver biopsy specimens that were >15 mm long and >1.2 mm wide were required for the study. In fragmented biopsies, the total length was estimated, by adding the maximum dimensions of each individual fragment. In using the Laennee system, the thickness of the predominant type of septae in each specimen was chosen and the smallest nodule was selected for scoring. The Laennee fibrosis scoring system was used because this system divides cirrhosis into three subclasses, allowing for a more detailed estimation of fibrosis after intervention (29). In addition, to estimate any treatment-induced change in Hver fibrosis, the fibrosis area was quantified as a percentage of the total area that was positive for MTC stain in the digital photomicrographs using a computerized image-analysis system (analySIS 3.0, Soft Imaging System, Minister, Germany). To quantify the fibrosis area, microscopic areas were selected randomly at an original magnification of x 100. For immunohistochemical analysis, tissue sections were incubated with primary antibody against a-SMA (diluted

1 :800, Neomarkers, Fremont, CA, USA) for 90 minutes at room temperature after washing with buffer. Tissue sections were incubated with the chrornogen 3~amino-9-ethylcarbazo1e (BioGenex, San Ramon, CA, USA) for 5-7 minutes. Prior to mounting, the sections were counterstained. with hematoxylin and then dehydrated. An UltraVision LP Large Volume Detection System (Lab Vision, Runcorn, UK) was used as the detection system.

Morphological analysis of immunopositive cells was also performed with a computerized image-analysis system.

Picrosirius Red staining was performed to quantify the total amount of collagen. Five-micrometer-thick sections of paraffin-embed d ed liver biopsy specimens were deparaffinized and rehydrated with distilled water and stained with a Picrosirius Red staining kit (Poiysciences, Warrington, PA, USA) according to the manufacturer's instructions.

In addition, the amount of collagen (the main component of fibrous tissue) was estimated from the collagen proportionate area, expressed as the percentage of the total area that was positive for Picrosirius Red stain on microscopy (Olympus BX51 , Tokyo, Japan) using a computerized image-analysis system (TMT i solution, Vancouver, Canada). While measuring the collagen proportionate area, we eliminated image artifacts and structural collagen in large portal tracts and blood vessel walls (31).

Gene Expressions of Transforming Growth Factor βΐ , Type 1 Collagen, and a-SMA

Total RNA was isolated, from paired. liver biopsy samples using TRIzoI reagent

(Invitrogen. Carlsbad, CA. USA) according to the manufacturer's protocol. RNA purity and concentration were determined using a spectrophotometer (Ultrospec 2100 pro UV/Visible, Amersham Bioscience, Freiburg, Germany). cDNAs were then synthesized using the GeneAmp RNA PGR Kit (Applied. Biosystems, Foster City, CA, USA) with random hexamers. For polymerase chain reaction (PGR) amplification, sequence-specific oligonucleotide primers for the genes of interest were designed based on human-specific sequences in the GenBank database (Supplementary table 1). Quantitative real-time PGR was carried out using SYBR GreenER qPCR SuperMix for ABI PRISM (Invitrogen) and perfonned in an ABI PRISM 7900HT Fast Real-Time PGR System (Applied Biosystems) according to the manufacturer's instructions. Data were analyzed using SDS2.2.2 software (Applied. Biosystems). The cycle threshold. (Ct) values of the target genes were normalized, to the Ct values of the endogenous control (glyceraldehyde-3 -phosphate dehydrogenase). Relative changes were calculated using the equation 2-AACt. Statistical Analysis

The outcomes were analyzed by per-protocol (PP) analysis. Comparisons were made using Fisher's exact test, Mann- Whitney U test, and Wilcoxon signed-rank test. Results are expressed as mean±SD values. The level of statistical significance was set at p<0.05. A medical statistician (E.H.C.) supported the study design and analysis of the data.

Results

The characteristics of the patients in this study are listed, in Table 1. All patients had paired biopsy-proven cirrhosis, and the cohort had a median age of 50 years (range 37-60 years). There was a male-to-female ratio of 10: 1. The immunophenotypes for CD14, CD34, CD45, CD73, and CD105 cells were determined and osteogenic or adipogenic differentiation was induced on the day of autologous BM-MSC injection (Fig. 2). In both the first and second injected cell populations, CD73 or CD 105 (which are positive markers of BM-MSCs) was expressed in more than 98% of the cells, and CD 14, CD34, or CD45 (which are known to be negative markers of BM-MSCs) was expressed in less than 1% of the cells (Fig. 2B). BM-MSCs from all patients could be differentiated into osteocytes and adipocytes (Fig, 2C).

Primary Outcome

Histological and Immunohistoehemicai Analysis

The Laennec fibrosis scoring system revealed detailed individual changes within the cirrhotic tissue (F4A-F4C; Table 2). Histological analysis was evaluated by H&E and MTC staining (Fig. 3), According to the Laennec fibrosis system, histological improvements were observed in six patients (54.5%) following BM-MSC therapy (Table 3).

Liver Disease

These results were further confirmed by immunohistochemical staining revealing a-SMA expression and Picrosirius Red staining (Fig. 3). The relative expression of the collagen proportionate area stained by Picrosirius Red was analyzed using an image- analysis program. The percentage of collagen proportionate area decreased significantly from 22.61±8.39% to 17.70+6.93% following BM-MSC therapy (p<0.001) (Fig. 3G} (Table 4).

Secondary Outcome

Changes in Laboratory Data After BM-MSC Therapy

The Child-Pugh score were used to evaluate overall liver function; scores improved in 10 out of the 1 1 patients (90.9%), from 7.1±Q.9 (pretherapy) to 5.4 . 0.7 (posttherapy; p<0.001 ; Tables 1 and 3). Changes in the value of the other relevant laboratory parameters [albumin, alanine aminotransferase (ALT), aspartate ammotransferase (AST), bilimbin, and creatinine] according to BM-MSC therapy are listed in Table 1. MELD scores decreased from 9.2 . 2.8 to 8.3÷2.4 following BM-MSC therapy (p<0,005; Tables 1 and 3).

Gene Expression Associated With Fibrosis After stem-cell administration, the relative expressions (2-AACt values) of transforming growth factor βΐ (TGF-β Ι), type 1 collagen (collagen- 1), and a-SMA in realtime PGR significantly decreased from 3.81±1.39 to 2.65±1,21 (p=0.013), from 4,51±2.62 to 3.15±2.82 (p=0.021), and from 4.27±1.89 to 2,34+1.60 (p=0.007), respectively (Fig. 4).

Safety

All 1 1 patients tolerated, the therapy. They were monitored regularly for any sign of possible side effects of the stem-cell therapy such as fever, hypersensiti vity reaction, and acute rejection fever. There was no evidence of stem-cell-therapy-related tumor developing during the follow-up period..

Discussion

At the overt cirrhotic stage, irreversible liver disease is a mandatory process that necessitates liver transplantation (32). However, it has several limitations, such as shortage of organ donors, high medical cost, a widening donor-recipient gap, and life-long dependence on immunological rejection. Hence, stem-cell transplantation has been suggested as an effective alternate therapy for hepatic disease (10, 12, 13). Stem-cell therapies have shown promising benefits for hepatic fibrosis in experimental and clinical studies (13, 14, 19, 22). BM comprises two main populations of stem cells, hematopoietic stem cells and MSCs, of which the latter have been considered as alternative cell sources for liver or hepatocyte transplantation (24). In liver damage, MSCs are able to differentiate into hepatocytes, stimulate the regeneration of endogenous parenchymal ceils, migrate to damaged sites, and enhance fibrous matrix degradation (antifibrotic effects). Furthermore, several clinical studies have demonstrated favorable effects such as BM-MSCs improving the liver function in patients with hepatitis B or C virus-related cirrhosis (22, 33),

However, no previous study has examined the effect of autologous BM-MCs on hepatic fibrosis in patients with alcoholic cirrhosis.

After autologous BM-MSCs injection, histological improvements were observed in 6 of the 1 1 patients (54.5%). The Child-Pugh score improved in ten patients (90.9%), and the expressions of TGF-βΙ, a-SMA. and collagen- 1 were significantly decreased (p<0.05). Importantly, no significant complications or side effects were observed during this study. These results indicate that BM-MSC therapy has potential as an antifibrotic treatment in cirrhosis and a bridging therapy for liver transplantation in advanced cirrhosis with hepatic insufficiency. Current antiviral therapies can improve the hepatic fibrosis in patients with B or C virus-related cirrhosis, but the only treatment for alcoholic cirrhosis is alcohol abstinence. Therefore, this study results suggest a novel strategy for the treatment of alcoholic cirrhosis.

There is a clear histological variability of severity within cirrhosis classified as F4 by the METAVIR system. Cirrhosis is currently considered to be potentially reversible if the cause of the injury is removed. The lack of subclassification within cirrhosis can be problematic when assessing the antifibrotic effect of agents such as antiviral drugs. For instance, even though antifibrotic therapy leads to improvement of hepatic fibrosis from F4C to F4A in the Laennec system, the lack of change under the conventional METAVIR system will lead to the false conclusion that treatment is ineffective. Hence, further histological subclassification of cirrhosis is required (4, 29). In the present study, we applied the new Laennec fibrosis scoring system to provide a more detailed classification of F4 cirrhosis.

The quality of interventional studies of alcoholic liver disease patients is crucially- dependent on monitoring the alcohol intake (6, 7). In the present study we did our best to monitor the subjects' alcohol re in Sake and. alcohol abstinence, using serologic liver function tests including serum AST/ALT ratio and y-glutamyl transpeptidase, and mean corpus volume in complete blood counts. Furthermore, alcohol intake was monitored every week by phone calls and in monthly face-to-face interviews with patients and their family members. This was effective in preventing alcohol reintake in most of the participants due to the formation of a good rapport.

In addition, we incorporated other methods to assess hepatic fibrosis in this study- to improve the validity of our morpheme trie analysis and measurement of TGF-β Ι , collagen- 1, and a-SMA by quantitative real-time PGR of the biopsied liver tissues (31 ). The final limitation of our study was that we did. not track the injected MSCs or their localization in patients' bodies. Although tracing injected stem cells in the body is a complicated procedure and the interpretation of tracing studies has recently created considerable controversy, it is very important to understand the way stem cells act to improve liver function and liver volume.

This is the first study to determine the effect of BM-MSCs on hepatic fibrosis in patients with ALC. The obtained results support the approval of this class of agents as a therapy for hepatic fibrosis. In conclusion, BM-MSC therapy leads to the histological improvement of hepatic fibrosis, and. so may provide a new strategy for antifibrosis therapy of ALC.

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. The entire disclosures of all references, applications, patents, and publications cited above, and of the corresponding applications, are hereby incorporated by reference. It is to be understood that the above-described embodiments are only illustrative of application of the principles of the present invention. Numerous modifications and alternative embodiments can be derived without departing from the spirit and scope of the present invention and the appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been shown in the drawings and fully described above with particularity and. detail in connection with what is presently deemed to be the most practical and preferred embodiment(s) of the invention, it will be apparent to those of ordinary skill in the art that numerous modifications can be made without departing from the principles and concepts of the invention as set forth in the claims.

Claims

1. A method of improving liver function in a subject with alcoholic cirrhosis, comprising administering to said subject a composition comprising autologous bone marrow derived mesenchymal stem ceils (BM-MSCs) in a way to elucidate antifibrotic effect of said BM-MSCs in said subject.

2. The method of claim 1 , wherein the subject has basehiie biopsy-proven alcoholic cirrhosis.

3. The method of claim 2, wherein the subject has been alcohol free for at least 6 months before the administering BM-MSCs.

4. The method of claim 3, wherein the BM-MSCs being isolated from said subject's bone marrow and being amplified for 1 month.

5. The method of claim 4, wherein the way of administering BM-MSC is injecting 5x 107 BM-MSCs to the subject twice at weeks 4 and 8, through hepatic artery.

6. The method of claim 1, wherein the subject being taken biopsy and

laboratory tests at 12 weeks to evaluate histological and quantitative improvement of hepatic fibrosis.