WO2016001839A1

WO2016001839A1 - Management of liver disease using pooled mesenchymal stromal cells

Info

Publication number: WO2016001839A1
Application number: PCT/IB2015/054909
Authority: WO
Inventors: Gurbind SINGH; Mathiyazhagan RENGASAMY; Sudha BALASUBRAMANIAN; Anoop CHULLIKANA HOUSE; Raviraja Neelavar Seetharam; Pawan Kumar Gupta; Anish Sen Majumdar
Original assignee: Stempeutics Research Pvt. Ltd.
Priority date: 2014-06-30
Filing date: 2015-06-30
Publication date: 2016-01-07

Abstract

The present disclosure relates to composition comprising pooled Mesenchymal Stromal cells (MSCs) for management of liver diseases. In particular, the disclosure relates to bone marrow derived and pooled MSC compositions with specific efficacy dosage ranges and modes/route of administration for effective management of said liver diseases.

Description

MANAGEMENT OF LIVER DISEASE USING POOLED MESENCHYMAL

STROMAL CELLS"

TECHNICAL FIELD

The present disclosure relates to the field of stem cell therapy. In particular, the disclosure relates to compositions comprising pooled mesenchymal Stromal cells (MSCs)for management of liver disease.

BACKGROUND OF THE DISCLOSURE

Liver has several critical functions and any disease or injury to it can lead to loss of those functions, thus causing significant damage to the body. The term Liver disease, also referred to as hepatic disease, is a broad term that covers all conditions that causes failure of liver functions. Generally, symptoms of liver diseases include weakness, fatigue, weight loss, nausea, vomiting and yellow discoloration of the skin (jaundice). The liver can be damaged in a variety of ways. For instance, cirrhosis occurs when normal liver cells are replaced by scar tissue as a result of chronic liver disease, hepatitis occurs when liver cells become inflamed, obstruction in bile flow can lead to cholestasis. Further, liver tissue can also be damaged by chemicals, minerals or alcohol. Alcohol abuse is the most common cause for liver damage. Alcohol is toxic to liver cells and can cause liver inflammation, referred to as alcoholic hepatitis.

Liver diseases are a major health problem worldwide. About 1.4 million liver disease deaths occur each year, and over 55% of them are due to liver cirrhosis. Prominent liver diseases include alcoholic hepatitis, liver fibrosis, liver cirrhosis, idiopathic liver cirrhosis, chronic hepatitis B or C, non-alcoholic steatohepatitis, primary biliary cirrhosis and autoimmune hepatitis. Hepatic cirrhosis results from chronic damage to the liver and is characterized by excessive accumulation of extracellular matrix (ECM) and scar formation that surround the nodules of hepatocytes. Accumulation of ECM results from both its increased synthesis and decreased degradation. Hepatic stellate cells (HSC) are the main ECM-producing cells in injured liver. HSCs transdifferentiate into myofibroblast-like cells, acquiring contractile, proinflammatory and ECM secreting properties. In advanced stages of cirrhosis, the liver contains approximately 6 times more ECM than normal liver. Cirrhosis leads to hepatocellular dysfunction and increased intrahepatic resistance to blood flow, which result in hepatic insufficiency and portal hypertension, respectively. Majority of patients with hepatic cirrhosis die from life-threatening complications at an early age. Likewise, the number of people with other liver diseases is also ever- increasing.

Under physiological conditions, liver in mammals does not need any external source of cells to repair injury as the resting hepatocytes have the ability to re-enter the cell cycle rapidly and efficiently after an injury has occurred. However, in cases of persistent liver injury, as in the case of chronic liver diseases in humans, the sustained proliferative stress exhausts their ability to replicate. Further, acute liver failure may or may not be reversible, based on if there is a treatable cause and if the liver is able to recover and resume its normal functions.

Liver transplantation is an effective alternative but unfortunately there are shortage of available donors, risk of immunorejection, high cost and several other post-operative complications. Transplantation of adult hepatocytes is another alternative which has been used in clinical studies, predominantly in hereditary metabolic disorders or as a bridging therapy for patients with liver failure awaiting liver transplantation. Again, a major limitation for broader use of hepatocyte transplantation is the lack of availability of primary human hepatocytes and decrease in its metabolic effect with time. Therefore, there is a critical need to find effective alternative therapy for this life threatening disease. The existing treatment for liver disease is only symptomatic and involves treating only symptoms of the disease. It does not play a role in regeneration of the damaged liver.

The present invention aims to overcome the drawbacks observed in the currently available treatments for liver diseases, by providing efficient Mesenchymal Stromal cell therapy for regeneration of the tissue.

STATEMENT OF DISCLOSURE

Accordingly, the present disclosure relates to a composition for managing liver disease comprising pooled mesenchymal stromal cells in an amount ranging from about 1 million cells to about 1000 million cells, optionally along with pharmaceutically acceptable excipient; a method of managing liver disease in a subject having or suspected of having the liver disease, said method comprising act of administering a composition comprising pooled mesenchymal stromal cells at a dose ranging from 0.5 million cells per kg of body weight of the subject to 8 million cells per kg of body weight of the subject, optionally along with pharmaceutically acceptable excipient to the subject; a kit for managing liver disease in a subject in need thereof comprising the composition as described above, optionally along with an instruction manual; a method of assembling a kit comprising act of combining a composition as above, optionally along with an instruction manual; and a composition comprising pooled mesenchymal stromal cells in an amount ranging from about 1 million cells to about 1000 million cells, optionally along with pharmaceutically acceptable excipient for use as a medicament for managing liver disease.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 depicts morphological appearance of bone marrow derived mesenchymal stromal cells showing spindle fibroblastic shape.

Figure 2(a) depicts flow cytometry analysis of molecular marker expression for BM-MSCs showing high level of expression of positive markers.

Figure 2(b) depicts flow cytometry analysis of molecular marker expression for BM-MSCs showing very low expression of CD14, CD19, CD34, CD45, whereas HLADR expression is 3.3%.

Figure 2(c) depicts flow cytometry analysis of molecular marker expression for WJ-MSCs showing high level of expression of positive markers.

Figure 2(d) depicts Flow cytometry analysis of molecular marker expression for WJ-MSCs showing very low expression of CD14, CD19, CD34, CD45, and HLADR.

Figure 3 depicts the results of in-vitro quality check VEGF assay. The levels of VEGF (a potency marker for consistent quality check) in the composition of the present pooled MSC cell composition/IMP are evaluated. Figure 4 depicts the growth hormone secretion profile of individual MSCs versus pooled MSCs measured by ELISA. The individual donor cells are represented as Donor 1(D1), Donor 2 (D2), Donor 3 (D3), whereas the pooled cells are represented as 'pool' in the figure.

Figure 5 depicts schematic diagram showing liver fibrosis induction, grouping of animals, cell dosages and route of delivery.

Figure 6 depicts histopathology of normal and cirrhotic liver before MSCs transplantation: Masson's Trichome staining showing extensive fibrous extracellular matrix proteins in cirrhotic group.

Figure 7 depicts gross pathology of liver before and after MSCs transplantation showing better lobular architecture at week 8 and 12 in treated groups as compared to plasmalyte A group.

Figure 8 depicts histopathology of liver before and after MSCs treatment: Masson's trichome staining showing reduction in extracellular matrix protein in week 8 in treatment group.

Figure 9 depicts histopathology of liver before and after MSCs treatment: Sirus red staining showing reduction in collagen fibres at week 8 in MSCs treatment group.

Figure 10 depicts hydroxyproline levels in liver tissues of sham control, vehicle, BM-MSCs and WJ-MSCs treated groups on day 30 and 70. Statistically significant differences between following groups is observed: At day 30 (A) : vehicle vs BM-MSCs, *p<0.05; vehicle vs WJ- MSCs, ns; vehicle vs sham control, **p<0.01. Day 70 (B) : vehicle vs BM-MSCs, *p<0.05; vehicle vs WJ-MSCs, ns; vehicle vs sham control, ***p<0.001. ns, not significant.

Figure 11 depicts histopathological analysis of liver sections from sham control (day 30) by using H&E (Hematoxylin + eosin), Sirius red and Masson's trichrome staining revealed normal architecture with hepatocytes arranged in the form of cords radiating away from the central vein. The portal triad contained an artery, vein and the bile.

Figure 12 depicts histopathological analysis of liver sections from disease control animals (day 30) revealed fibrous expansion of most portal areas. Some of the fibrous bands extend from one portal area to the adjacent one or to the central vein with bridging in between forming pseudo-lobules.

Figure 13 depicts histopathological analysis of liver sections from BM-MSCs treated animal (day 30) revealed lesser number of fibrous bands extending from the portal area to the central veins; the bends are also much thinner than the pathological control. The number of pseudo lobules formed are also lesser.

Figure 14 depicts histopathological analysis of liver sections from sham control animals (day 70) revealed normal architecture with hepatocytes arranged in the form of cords radiating away from the central vein. The portal triad contained an artery, vein and the bile duct. Normal distribution of sinusoidal spaces is also observed.

Figure 15 depicts histopathological analysis of liver sections from disease control animals (day 70), the fibrous bands found in this group revealed numerous pseudo lobule formations which are lesser than the day 30 disease control animals.

Figure 16 depicts histopathological analysis of liver sections from BM-MSCs treated animals (day 70), the fibrous bands found in this group revealed much lesser pseudo lobule formation and thinner fibrous bands.

Figure 17 depicts microphotographs (10 x 20, 200 X magnification) of liver section taken from rats. A - Normal control group; B - CC1₄ induced disease group (about 0.1 ml/kg); C - MSCs treated group; D - Silymarin treated group; E - Silymarin+MSCs treated group.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates a composition for managing liver disease comprising pooled mesenchymal stromal cells in an amount ranging from about 1 million cells to about 1000 million cells, optionally along with pharmaceutically acceptable excipient.

The present disclosure also relates to a method of managing liver disease in a subject having or suspected of having the liver disease, said method comprising act of administering a composition comprising pooled mesenchymal stromal cells at a dose ranging from 0.5 million cells per kg of body weight of the subject to 8 million cells per kg of body weight of the subject, optionally along with pharmaceutically acceptable excipient to the subject.

In an embodiment of the present disclosure, the mesenchymal stromal cells are derived from source selected from group comprising bone marrow, adipose tissue and dental pulp, or any combination thereof, preferably bone marrow.

In another embodiment of the present disclosure, the mesenchymal stromal cells are obtained by ex-vivo cell culturing; and wherein at least 80% of the mesenchymal stromal cells are positive for cell specific markers selected from group of CD 73, CD90, CD105 and CD166 cells or any combination thereof; and less than 10% of the mesenchymal stromal cells are positive for markers selected from group of CD34, CD45, CD133, CD14, CD19 and HLA- DR or any combination thereof.

In yet another embodiment of the present disclosure, the mesenchymal stromal cells are allogenic and expanded, and wherein expansion is carried out by culturing the pooled allogeneic mesenchymal stromal cells for about 3 to about 6 passages.

In still another embodiment of the present disclosure, the pharmaceutically acceptable excipient is selected from group comprising carrier, cyropreservant, protein and pre- formulated ready to use cryopreservation mixture, or any combination thereof.

In still another embodiment of the present disclosure, the carrier is selected from a group comprising multiple electrolyte solution such as PlasmaLyte A, Hank's balanced salt solution, saline, Lactated Ringer's Injection; the cyropreservant is Dimethyl Sulfoxide; the protein is human serum albumin; and the pre-formulated ready to use cryopreservation mixture is animal protein-free defined cryopreservation medium.

In still another embodiment of the present disclosure, the liver disease is selected from group comprising alcoholic hepatitis, liver fibrosis, liver cirrhosis, conditions which could result in or result from liver fibrosis or liver cirrhosis, chronic hepatitis B, chronic hepatitis C, nonalcoholic steatohepatitis, primary biliary cirrhosis, autoimmune hepatitis, primary sclerosing cholangitis, alcoholic liver disease, hereditary hemochromatosis, Wilson's disease, alpha I antitrypsin deficiency, cardiac cirrhosis, galactosemia, cystic fibrosis, glycogen storage disease type IV and parasitic infections of the liver or any combination thereof.

In still another embodiment of the present disclosure, the subject is mammal, preferably human.

In still another embodiment of the present disclosure, the composition is administered as single dose or multiple doses at one or multiple sites through modes selected from group comprising intravenous administration, hepatic administration, pancreatic duodenal artery administration, intraperitoneal administration, hepatoportal administration, intramuscular administration, intra articular administration, subcutaneous, intradermal administration, intrahepatic administration, intraportal administration and intrasplenic administration or any combination thereof, preferably intravenous, intrahepatic or intraportal administration or any combination thereof.

In still another embodiment of the present disclosure, the composition is formulated as a formulation selected from group comprising an aqueous suspension, cream, lotion, gel, emulsion, drop, emulsion in hard or soft gel capsule, elixir, lyophilized cell powder and cell spray or any combination thereof, preferably aqueous suspension.

In still another embodiment of the present disclosure, the pooled mesenchymal stromal cells are at an amount ranging from about 15 million cells to about 800 million cells and wherein the amount of MSCs is based on the body weight of a subject in need thereof.

The present disclosure also relates to a kit for managing liver disease in a subject in need thereof comprising the composition as claimed in claim 1 optionally along with an instruction manual.

The present disclosure also relates to a method of assembling the said kit, said method comprising act of combining a composition as described above, optionally along with an instruction manual.

The present disclosure also relates to a composition comprising pooled mesenchymal stromal cells in an amount ranging from about 1 million cells to about 1000 million cells, optionally along with pharmaceutically acceptable excipient for use as a medicament for managing liver disease.

The present disclosure discloses the use of stem cell therapy for management of liver disease affecting the architecture and function of the liver.

The present disclosure relates to a composition comprising 'pooled', 'ex -vivo' expanded and 'allogeneic' mesenchymal stromal cells (MSCs) with diverse HLA genotyping representation, its effective dosage and route of administration for management of liver diseases.

In an embodiment of the present disclosure, the method of managing liver disease in a subject in need thereof comprises act of administering the cell composition comprising pooled mesenchymal stromal cells to the subject, in an amount effective to manage the liver disease. In an embodiment, effective amount/ amount effective to manage the liver disease/ effective dose is the number of cells which give the desired results.

In another embodiment of the present disclosure, the composition further comprises pharmaceutically acceptable excipient; and the pharmaceutically acceptable excipient is selected from group comprising carrier, cyropreservant, protein and pre-formulated ready to use cryopreservation solution, or any combination thereof.

In another embodiment, the present disclosure relates to use of the composition comprising pooled mesenchymal stromal cells along with excipient, for managing liver disease.

As used herein, the terms "mesenchymal stromal cell", "mesenchymal stem cell" and MSC are employed interchangeably within the instant disclosure.

As used herein, the terms "cell composition", "Investigation Medicinal product (IMP)", "final MSC composition", "final composition", "final therapeutic composition", "composition of the present disclosure" and "cell product" as used in this disclosure mean the final product comprising allogeneic pooled MSC optionally in a cryopresevation solution. As used herein, the term "cryopreservation solution" means a composition/solution used for preservation of cells for longer duration/shelf life. The alternate terms used are "freeze media", "freezing mixture", "pre-formulated ready to use preservation mixture" all of which shall mean cryopreservation solution. More specifically, in the present disclosure, MSCs are cyropreserved using a cryopreservation solution which comprises of components such as but not limiting to protein, carrier and cryoprotectant. In yet another embodiment of the present disclosure, the MSCs are cryopreserved using commercially available pre-formulated ready to use preservation mixture or solution. The cell composition is cryopreserved in liquid nitrogen at -196°C until use. On requirement, the cells are thawed and used as per the patient dosage requirement decided by the clinician.

As used herein, the term "excipient" means an inert substance used as vehicle/carrier and/or diluent for the active ingredient. It is a substance added to a formulation to provide benefit of the processing or cryo-protection of active ingredient and is intended to be present in the final product as an inactive ingredient. In an embodiment of the present disclosure, excipient includes but is not limited to physiological solution, multiple electrolyte injection, Hank's balanced salt solution (HBBS) solution, saline, Lactated Ringer's Injection, cryoprotectant, protein and so on.

As used herein, the term "pooling" or "pooled" in the context of mesenchymal stromal cells means combining or mixing the mesenchymal stromal cells of multiple donors i.e. more than one donor.

As used herein, the term "managing" or "management" includes, treating or healing of a disease condition or disorder or ill effects or side effects. The term also encompasses maintenance of the optimum state and prevention of the further progress in the disease condition or disorder or ill effects or side effects. Further, "management" or "managing" refers to decreasing the risk of death due to a disease or disorder, delaying the onset of a disease or disorder, inhibiting the progression of a disease or disorder, partial or complete cure of a disease or disorder and/or adverse effect attributable to the said disease or disorder, obtaining a desired pharmacologic and/or physiologic effect (the effect may be prophylactic in terms of completely or partially preventing a disorder or disease or condition, or a symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease or disorder and/or adverse effect attributable to the disease or disorder), relieving a disease or disorder (i.e. causing regression of the disease or disorder). In an embodiment of the present disclosure, the composition comprising pooled allogeneic Mesenchymal Stromal cells aids in managing the liver disease by regenerating the damaged/diseased liver or any part thereof. Further, in a non- limiting embodiment, the present disclosure also envisages treating the said disorder by administering therapeutically effective/efficacy dosage of the composition comprising pooled allogeneic Mesenchymal Stromal cells along with suitable carriers/excipient. The present disclosure further relates to routes of administration in order to obtain effective healing/recovery.

In an embodiment of the present disclosure, the bone marrow is obtained from a cell bank. The term "Bank" in the present disclosure means source for obtaining the bone marrow. In another embodiment of the present disclosure, such source is pre-processed and/or cryopreserved bone marrow, stored for instant or future use. In an embodiment, the source is mono-nuclear cell (MNC) bank. In another embodiment, the source is a Mesenchymal Stem Cell Bank. In embodiments of the disclosure, any depository that stores bone marrow for a pre-determined period of time is a bank. Further, any medium such as apparatus or device or vessel or container that stores bone marrow for a pre-determined period of time is considered to be a bank. In an embodiment, information about the bank is procured from bone marrow registry.

In an embodiment of the present disclosure, bone marrow derived mesenchymal stem cells (BM-MSCs) are obtained by processing of bone-marrow which do not involve any surgical process. In another embodiment, stem cells (BM-MSCs) are obtained from sources including but non-limiting to stem cell banks (a repository of stem cells), as gifts from collaborating laboratories or a scientific person in this field of technology, and so on, and the same does not include any surgical/invasive step for obtaining said stem cells. Further, all the aforementioned approaches or sources of isolating/obtaining stem cells are within the scope of the present disclosure.

In an embodiment of the instant disclosure, the term liver disease includes but is not limited to any damage to the liver or disease of the liver. There are various factors which could damage the liver such as drugs, poisons, drinking too much alcohol, or various causes of diseases to the liver such as viruses like hepatitis A, hepatitis B and hepatitis C; etc. Damage or disease to the liver affects its architecture and function. Chronic liver diseases may lead to fibrosis, which leads to derangement of the architecture, portal hypertension and may produce an irreversible rearrangement of the circulation as to cause cirrhosis. In another embodiment of the present disclosure, the term liver disease includes late stage liver diseases.

In yet another embodiment of the present disclosure, the liver disease is selected from a group comprising but not limiting to alcoholic hepatitis, liver fibrosis, liver cirrhosis, idiopathic liver cirrhosis, chronic hepatitis B or C, non-alcoholic steatohepatitis, primary biliary cirrhosis, autoimmune hepatitis, primary sclerosing cholangitis, alcoholic liver disease, hereditary hemochromatosis, Wilson's disease, alpha I antitrypsin deficiency, cardiac cirrhosis, galactosemia, cystic fibrosis, glycogen storage disease type IV and parasitic infections of the liver or any combination thereof. In another embodiment of the present disclosure, the term liver disease includes but is not limited to conditions which could result in or result from liver fibrosis. In another embodiment of the present disclosure, the term liver disease includes any manifestation of liver cirrhosis or conditions which could result in or result from liver cirrhosis.

In an exemplary embodiment of the present disclosure, the liver disease is liver fibrosis. Liver fibrosis is the process that represents the liver's response to injury. Liver fibrosis is inflammation followed by death of hepatocytes which can lead to liver cirrhosis. It may be triggered due to loss of hepatocyte due to any liver disease. In an embodiment of the present disclosure, the term liver fibrosis also includes but is not limited to conditions which could result in or result from liver fibrosis.

In another exemplary embodiment of the present disclosure, the liver disease is liver cirrhosis. In another embodiment of the present disclosure, the term liver cirrhosis also includes any manifestation of liver cirrhosis.

In another embodiment of the present disclosure, the term liver cirrhosis also includes any manifestation of liver cirrhosis.

In a preferred embodiment of the present disclosure, the liver disease is selected from a group comprising liver fibrosis, conditions which could result in or result from liver cirrhosis and any manifestation of liver cirrhosis. In an embodiment of the present disclosure, liver cirrhosis represents a late stage of progressive hepatic fibrosis characterized by distortion of hepatic architecture and formation of regenerative nodules. Cirrhosis occurs when normal liver cells are replaced by scar tissue, such as a result of chronic liver disease. Cirrhosis has many possible causes; sometimes more than one cause is present in the same patient. Some of the non-limiting causes are:

• Alcoholic liver disease (ALD): Alcohol seems to injure the liver by blocking the normal metabolism of protein, fats, and carbohydrates.

• Chronic hepatitis C: Infection with this virus causes inflammation and low grade damage to the liver that over several decades could lead to cirrhosis.

• Chronic hepatitis B: The hepatitis B virus is probably the most common cause of cirrhosis. Hepatitis B causes liver inflammation and injury that over several decades could lead to cirrhosis.

• Non-alcoholic steatohepatitis (NASH): In NASH fat builds up in the liver and eventually causes scar tissue. This type of hepatitis appears to be associated with diabetes, protein malnutrition, obesity, coronary artery disease and treatment with corticosteroid medications.

• Primary Biliary Cirrhosis (PBC) and Primary Sclerosing Cholangitis (PSC): Patients may be asymptomatic or complain of fatigue, pruritus, skin hyperpigmentation with hepatomegaly. PSC is a progressive cholestatic disorder presenting with pruritus, steatorrhea, fat soluble vitamin deficiencies, and metabolic bone disease. There is a strong association with inflammatory bowel disease (IBD), especially ulcerative colitis.

• Some of the less common causes of cirrhosis are Autoimmune hepatitis, hereditary hemochromatosis, Wilson's disease, alpha I antitrypsin deficiency, cardiac cirrhosis, Galactosemia, cystic fibrosis, glycogen storage disease type IV, drugs or toxins and some parasitic infections.

Out of all the above mentioned causes of liver cirrhosis, alcohol is the most frequent and fully established cause of both acute and chronic liver disease. The disease spectrum of alcoholic liver disease ranges from asymptomatic hepatomegaly (fatty liver) to profound hepatocellular failure and portal hypertension. Pathologically, it is manifested by fatty infiltration of hepatocytes at the stage of transition of asymptomatic hepatomegaly to frank cirrhosis, with decompensation at the end stage. In an exemplary embodiment of the present disclosure, the liver disease is alcoholic liver cirrhosis.

In an embodiment of the present disclosure, fibrosis is a part of the inflammation activities and is preceded by cirrhosis. In the fibrosis stage, there is no lobular regeneration and this distinguishes it from liver cirrhosis. Advancement of fibrosis to cause fibrostic separations (or bridging) between the portal areas or between the portal area, the center vein, and the formation of pseudo-lobule, implies that the fibrosis has entered the final stage, which is liver cirrhosis.

Human MSCs are present as a rare population of cells in bone marrow, representing 0.001 to 0.01% of the nucleated cells, but they rapidly grow in culture without losing their sternness. MSCs can be expanded in vitro >2 million fold and retain their ability to differentiate into several mesenchymal lineages. MSCs differentiate not only into mesodermal lineage such as bone, cartilage and adipose tissue but also transdifferentiate into other lineages like neurons and endothelial cells.

Adult MSCs do not express human leukocyte antigen (HLA) class II antigens on the cell surface and do not elicit a proliferative response from allogeneic lymphocytes, thus proving that the cells are not inherently immunogenic. The MSCs also do not express co- stimulatory molecules CD80, CD86, CD40, CD31/Platelet Endothelial Cell Adhesion Molecule- 1 (PECAM-1), CD18/ Leucocyte Cell Adhesion Molecule B (LCAM) and CD56/ Neural Cell Adhesion Molecule (NCAM-1), and hence do not activate allo-reactive T cells. The cell composition of the present invention comprises allogeneic MSCs which do not induce lymphocyte proliferation when used in a mixed lymphocyte reaction (MLR), which is an invito) model of immune cell activation.

In an embodiment of the present disclosure, the MSCs are derived from various sources including but not limited to Bone marrow, Adipose tissue, Dental pulp and so on. In a preferred embodiment, the source of MSCs is Bone marrow. In another embodiment of the present disclosure, the MSCs are allogeneic and pooled in nature. Bone marrow derived MSCs have several advantages over other type of stem cells such as:

a. Non embryonic source,

b. Reduced likelihood of neoplasia, c. Ease of isolation,

d. High expansion potential,

e. Immune privileged,

f. Genetic stability,

h. Reproducible characteristics,

i. Compatibility with tissue engineering principles, and

j. Potential to enhance repair in many vital tissues.

Under physiological conditions, resting hepatocytes in mammalian liver have the ability to re-enter the cell cycle rapidly and efficiently after an injury has occurred. However, in cases of persistent liver injury, the sustained proliferative stress exhausts their ability to replicate. In this situation, hepatic progenitor cells or Oval cells' which normally reside in smallest of the biliary channel 'the ducts of Herring' appear as a rich population of small round cells spreading from the peri-portal area to the parenchyma and support liver regeneration. These oval cells have been demonstrated to be bi-potential progenitor cells which are capable of differentiating into both hepatocytes and cholangiocytes.

Hepatocyte growth factor (HGF) is important for hepatocyte proliferation and regeneration in liver. Also, MMP secretion plays an important role in degradation of excess collagen. BM- MSCs express MMP1, MMP2, MMP3, MMP 14, MMP 15 and MMP 16 at high level that help in degradation of ECM proteins and hence in reduction of liver cirrhosis and scar size in cardiac ischemia. MMP1, MMP2 and MMP3 are secretory matrix mettaloproteinases (MMPs) which can be measured in conditioned media. MMP14, MMP15 and MMP16 are membrane associated matrix mettaloproteinases that help in matrix degradation and activation of secretory MMPs. Hence, MMP 1, 2 and 3 are good candidates for LC potency assay development in BM-MSCs.

In an embodiment of the present disclosure, MSCs facilitate hepatocyte proliferation and repair by increasing HGF secretion. Hepatocyte regeneration helps in improvement of liver function. Further, secretion of MMPs helps in degrading the excess collagen and reversing the fibrosis in the cirrhotic liver. Hence, there is overall lobular architectural, histological and function improvement in fibrotic or cirrhotic liver. In embodiment of the present disclosure, mesenchymal stem cells are used in cases of end stage liver disorders to prevent further degeneration of hepatocytes through their (a) immunomodulatory properties that turn off T cell surveillance and chronic inflammatory process (b) ability to generate new hepatocytes and possibly to reverse the process of degeneration by virtue of their regenerative capacities in the diseased livers (c) fibrolytic effects of stem cells that may be related to over-expression of matrix metalloproteinases (MMPs) (d) hepatic stellate cells apoptosis and (e) secrete paracrine factors which can stimulate endogenous stem cells (oval cells). Furthermore, increments in the liver volume are explained by the fact that bone marrow stem cells increase hepatocyte proliferation by supplying cytokines and growth factors such as HGF, VEGF, MMP and PDGF, critical for the recovery process.

Allogeneic mesenchymal stem cells are used to initiate a regressive process upon the inflammation in cirrhotic liver and then to mount a regenerative process in the liver to replenish all types of damaged cells.

The cell composition of the present disclosure shows low immunogenicity and has been demonstrated in in-vivo in animal models using infusion of allogeneic MSCs. The injections of allogeneic MSCs do not stimulate the formation of allo-specific antibodies and do not lead to a T cell sensitization of the recipient to alloantigen in different animal models. Studies have also shown that the MSCs possess the ability to engraft, persist and function in an unrelated mismatched allogeneic host.

In an embodiment of the present disclsoure, the mesenchymal Stromal cells of the instant disclosure are allogeneic. In another embodiment, the allogeneic MSCs refer to MSCs which are derived from individual(s)/donor(s) other than the recipient, however belonging to the same species. The MSC are derived from multiple healthy donors with proper informed consent and approval.

In another embodiment of the present disclosure, the mesenchymal stromal cells of the instant disclosure are xenogeneic when employed for recipient belonging to a different species than that of the donor. In an embodiment, the composition of the present disclosure is obtained from processing bone marrow collected from multiple healthy donors. The cell composition comprises of pooled Mesenchymal stem cells from multiple donors optionally along with vehicle/ pharmaceutical acceptable excipient/carriers. In an exemplary embodiment, the MSCs from at least two or more donors are pooled to prepare the cell composition of the present invention.

In an embodiment of the present disclosure, the MSCs are derived from human donors selected from male, female, or a combination thereof. In another embodiment of the present disclosure, the number of donors is at least two. In yet another embodiment of the present disclosure, the number of donors is three, four, five, six, seven, eight, nine or ten, and so on, as suitable.

In an exemplary embodiment of the present disclosure, the number of donors of MSCs is but not limiting to preferably 3 donors.

In a specific embodiment of the present disclosure, the allogeneic pooled Mesenchymal Stromal Cells (MSCs) possess numerous advantages when compared to single donor derived MSCs. Pooled MSC composition of the present invention have significantly improved immunomodulatory characteristics, thus ensuring that there is minimal biological variability/immunological reaction in the recipient post administration of pooled MSC composition.

Therefore, the various advantages of using the cell composition comprising pooled MSC along with the carriers/excipient are as follows:

a. Individual variability minimized;

b. Consistent non-immunogenic & immunosuppressive properties;

c. Broader Cytokine/Growth Factor (GF) array;

d. Donor specific advantage;

e. Increased potential for various disease indications; and

f. Large product doses compared to single donor.

In a non-limiting embodiment of the present disclosure, the composition targets the fibrosis to manage the liver disease. In an embodiment of the present disclosure, the pooled MSC's of the cell composition are characterised by HLA typing and markers of MSCs obtained from individual donors versus pooled cells.

In another embodiment of the present disclosure, the mesenchymal stromal cells are obtained by ex-vivo cell culturing; and wherein at least 80% of the mesenchymal stromal cells are positive for cell specific markers selected from group comprising CD44, CD73, CD90, CD105 and CD166 cells or any combination thereof; and less than 10% of the mesenchymal stromal cells are positive for markers selected from group comprising CD14, CD19, CD34, CD45, and HLA-DR or any combination thereof, in other words at least 90% of the cell are negative for CD14, CD19, CD34, CD45, and HLA-DR; and also negative for co- stimulatory molecules including but not limiting to CD markers like CD40, CD80 and CD86.

In an exemplary embodiment, the primary aim of the present disclosure is to provide for compositions comprising allogeneic, expanded, ex-vivo pooled Mesenchymal Stromal Cells, wherein said compositions are formulated with specific dosage ranges and administration routes to provide improve therapeutic effects in the liver. Further, the MSCs as mentioned above can be derived from sources including but not limited to bone marrow, adipose tissue and dental pulp from any mammal including but not limiting to humans or from cell bank or any other source. in an embodiment, the formulation or composition of the present disclosure employed towards management of liver disease comprises pooled MSCs optionally along with carrier/excipients. In an embodiment of the present disclosure, the excipient is selected from a group comprising carrier, protein, cyropreservant and pre-formulated ready to use cryopreservation solution, or any combination thereof. In an embodiment of the present disclosure, the excipient is carrier such as Multiple Electrolytes Injection/ PlasmaLyte-A, cryoprotectant such as DMSO, and protein like albumin such as human serum albumin (HSA), or any combination thereof, wherein said composition/formulation is optionally further diluted with carrier/vehicle selected from a group comprising PlasmaLyte-A, Hank's balanced salt solution (HBBS), saline, Lactated Ringer's Injection, or any combination thereof, and employed for administration purpose. Alternatively, the formulation or composition comprises pooled MSCs optionally along with pre-formulated ready to use commercial cyropreservation mixture selected from a group comprising CryoStor^® family- a commercially available animal protein-free defined cryopreservation medium from Biolife Solutions: such as: CyroStor5 (CS5)- an optimized freeze media pre-formulated with 5% DMSO, CyroStorlO (CS IO)- an optimized freeze media pre-formulated with 10% DMSO, CyroStor2 (CS2)- an optimized freeze media pre-formulated with 2% DMSO] and hypothermosol wherein said composition/formulation is further diluted with carrier/vehicle such asmultiple electrolyte injection/solution like PlasmaLyte-A, Hank's balanced salt solution (HBBS), saline, Lactated Ringer's Injection, etc., and employed for administration purpose.

In a preferred embodiment, the cell composition is prepared by employing pooled mesenchymal stromal cells, optionally along with human serum albumin, plasmaLyte-A and DMSO. Wherein the said cell composition is further diluted using a carrier/vehicle such as PlasmaLyte-A to obtain the final cell composition for administration. In another preferred embodiment, pooled Mesenchymal stromal cells are cryopreserved in commercially available pre-formulated ready to use preservation mixture such as cyrostor-5 (CS5), CyroStorlO (CS IO), CyroStor2 (CS2) and/or hypothermosol, wherein the said cell composition is further diluted using a carrier/vehicle for administration.

As described above, the pooled bone marrow derived Mesenchymal Stromal Cells of the present disclosure are either in combination with excipients selected from a group comprising carrier, protein and cryopreservant, optionally along with other pharmaceutically acceptable excipients/additives or any combination thereof OR the cell composition comprises Mesenchymal Stromal Cells in a pre-formulated ready to use preservation mixture such as cyrostor-5 (CS5), CyroStorlO (CS IO), CyroStor2 (CS2) and/or hypothermosol OR the cell composition comprises Mesenchymal Stromal Cells alone or in combination with a carrier.

In an embodiment of the present disclosure, cell dosage to be injected to the patient is decided by the clinician depending on various parameters such as weight, age, manifestation of disease condition etc. of the patient preferably based on the weight of the patient. Further, the cell composition before administration is diluted in a physiological compatible carrier. For the above mentioned cell product dosage, the carrier/excipient range is about 1 ml to about 100 ml, preferably about 5 ml to about 50 ml. In an embodiment of the present disclosure, the cell product dosage ranges from about 0.5 to 8 million cells per kg human bodyweight in about 5 to about 50 ml of the excipient. In an embodiment, the excipient is selected from a group comprising protein, carrier, cryopreservant, and pre-formulated ready to use cryopreservation solution, or any combination thereof.

In an embodiment of the present disclosure, the Human Equivalent Dose is calculated based on the guidelines issued by the FDA, the Center for Drug Evaluation and Research (CDER), July 2005, Pharmacology and Toxicology.

In an embodiment, the present disclosure discloses that in the final MSC composition which is to be administered for management of liver disease, the dosage of MSCs ranges from t 0.5 million cells / kg body weight to about 8 million cells / kg body weight. Preferably, the dosage of MSCs in the final MSC composition is 0.5 million cells/kg body weight, 1 million cells/kg body weight, 1.5 million cells/kg body weight, 2 million cells / kg body weight, t 2.5 million cells/kg body weight, 3 million cells/kg body weight, 3.5 million cells/kg body weight, 4 million cells /kg body weight, 4.5 million cells/kg body weight, 5 million cells/kg body weight, 5.5 million cells/kg body weight, 6 million cells/kg body weight, 6.5 million cells/kg body weight, 7 million cells/kg body weight, 7.5 million cells/kg body weight or 8 million cells/kg body weight. In a most preferred embodiment, the dosage of MSCs in the final MSC composition is 2-5 million cells / kg body weight.

In an embodiment of the present disclosure, the effective therapeutic dose of the pooled and expanded mesenchymal stromal cells ranges from about 1 million cells to about 3.5 billion cells.

In an embodiment of the present disclosure, the effective therapeutic dose of the pooled and expanded mesenchymal stromal cells ranges from about 1 million cells to about 1000 million cells, preferably about 15 million cells to about 800 million cells, more preferably about 20 million cells to about 640 million cells, based on the body weight of the subject in need thereof to which the composition is being administered to.

In a non-limiting embodiment of the present disclosure, the body weight of the subject is ranging from about 30 kg to about 100 kg, preferably about 40 kg to about 80 kg. In an embodiment of the present disclosure, the amount of MSCs to be administered is arrived at using dosage ranging from about 0.5 to 8 million cells per kg human bodyweight. The exact dosage of mesenchymal stem cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, the inflammatory response being treated, and the extent and severity thereof.

In accordance with the above embodiments, an exemplary embodiment of the present disclosure relates to the amount of MSCs, wherein when the body weight of the subject is ranging from about 30 kg to about 100 kg, the amount of MSCs administered is ranging from about 15 million cells to about 800 million cells, respectively.

In another exemplary embodiment of the present disclosure, when the body weight of the subject is ranging from about 40 kg to about 80 kg, the amount of MSCs administered is ranging from about 20 million cells to about 640 million cells, respectively.

In an embodiment of the present disclosure, the MSC composition is cryopreserved in either a cryobag or vial or other means available for storing and packing of cell product. The bag or vial comprises 25 to 200 million cells in about 4 to about 15 ml of the excipient (i.e. protein + carrier + cryopreservant OR pre-formulated ready to use preservation mixture OR carrier OR cyropreservation solution). Depending on the manufacturing requirement the cell number for cryopreservation will vary.

In an embodiment of the present disclosure, the mesenchymal stromal cells are in an amount ranging from 1 million cells to 200 million cells. In another embodiment of the present disclosure, the mesenchymal stromal cells are in an amount ranging from 1 million cells to 100 million cells. In another embodiment of the present disclosure, the MSCs in the final MSC composition is at concentration of 1 million cells, 5 million cells, about 10 million cells, 15 million cells, 20 million cells, 25 million cells, 30 million cells, 35 million cells, 40 million cells, 45 million cells, 50 million cells, 55 million cells, 60 million cells, 65 million cells, 70 million cells, 75 million cells, 80 million cells, 85 million cells, 90 million cells, 95 million cells, 100 million cells, 105 million cells, 110 million cells, 115 million cells, 120 million cells, 125 million cells, 130 million cells, 135 million cells, 140 million cells, 145 million cells, 150 million cells, 155 million cells, 160 million cells, 165 million cells, 170 million cells, 175 million cells, 180 million cells, 185 million cells, 190 million cells, 195 million cells, 200 million cells, 225 million cells, 250 million cells, 275 million cells, million cells, 300 million cells, 350 million cells, 400 million cells, 450 million cells, 500 million cells, 550 million cells, 600 million cells, 650 million cells, 700 million cells, 750 million cells, 800 million cells, 850 million cells, 900 million cells, 950 million cells, 1000 million cells.

In an embodiment of the present disclosure, the safe and efficacy drug dose is estimate by preclinical study.

In an embodiment of the present disclosure, MSCs upto a single dose of about 20 xlO⁶ cells/kg body weight are used which is found to be non-toxic in preclinical studies to the subject without infusional or other toxicities. In another embodiment, the effective dose range of cells used is 0.5 - 8 million cells/kg body weight in humans and is non-toxic to the subject.

From a preclinical and clinical perspective, several parameters need to be optimized for efficacious outcome of stem cell transplantation, including cell quality, cell quantity and the route of cell administration. In an embodiment of the present disclosure, the MSC compositions are administered through modes selected from a group comprising intramuscular administration, intravenous administration, intra articular administration, pancreatico duodenal artery administration, hepatoportal, subcutaneous, intradermal administration, intrahepatic administration, intra portal administration or injection in any other appropriate part or any combination thereof. In a preferred embodiment, the MSCs are delivered to injured liver by directional (intrahepatic, intra portal), and systemic (intravenous) routes. MSCs have been administered to the injured liver by intrahepatic injection, which is considered directional delivery; and intravenous routes which is example of systemic delivery.

MSCs are known to be selectively recruited to injured tissue directed by chemokine gradient and engraft into the tissue by crossing the blood vessels through transendothelial migration. Chemokine signals released from the injured area and corresponding receptors expressed on MSC are critical determinants for homing. Interestingly, it is reported in rats with CC1₄- induced liver injury that the timing and numbers of MSC homing to the liver are closely related to the presence of liver injury but not to the route of MSC infusion, e.g., through the tail vein or the portal vein. In an embodiment, the route of MSC administration includes intravenous and hepatic artery routes for preclinical and clinical application respectively. In an embodiment of the present disclosure, the bone marrow MSCs are obtained by ex-vivo culturing.

In another embodiment of the instant disclosure, the method of cell composition preparation comprises of isolating the mesenchymal Stromal cells (MSCs) from bone marrow of multiple donors and subjected to various processes to store or cryopreserve said cells. In an embodiment of the present disclosure, the pooled mesenchymal stromal cells employed for the management of liver disease are derived from such stored or cryopreserved forms. The MSCs isolated from bone marrow of each individual donor are passaged and cultured to obtain/establish master cell bank composition (MCB). Said MCB comprising MSCs of individual donors is cyropreserved in Fetal Bovine Serum (FBS) and DMSO. Preferably, the MCB comprises MSCs of individual donors, cyropreserved in FBS at a concentration of about 90% (v/v) and DMSO at a concentration of about 10% (v/v). The MSCs from multiple MCBs are 'pooled' and subsequently passaged/expanded to arrive at working cell bank composition (WCB) comprising pooled MSCs, cyropreserved in FBS and DMSO. Further, the said WCB preferably comprises pooled MSCs, cyropreserved in FBS at a concentration of about 90% (v/v) and DMSO at a concentration of about 10% (v/v).

The aforesaid WCB is further subjected to passaging/culturing and washing process to remove presence of xeno component or other impurities to obtain a final therapeutic composition/cell composition/Investigational medicinal product(IMP)/Investigational product(IP)/final MSC composition comprising bone marrow derived pooled allogeneic mesenchymal Stromal cells with diverse HLA genotyping representation optionally along with excipient. In an embodiment of the present disclosure, the excipient is a commercially available cyropreservation solution. In another embodiment of the present disclosure, the excipient is selected from group comprising Plasmalyte-A, multiple electrolyte injection, Hank's balanced salt solution, saline, Lactated Ringer's Injection, HSA and DMSO or any combination thereof, preferably Plasmalyte-A, HSA and DMSO. Optionally, other pharmaceutically acceptable excipients/additives can be added for administration. In an embodiment of the present disclosure, the cell composition employed for the management of liver disease is derived from such compositions. In an embodiment of the present disclosure the Investigational medicinal product (IMP) composition is cryopreserved until further use.

The composition or formulation comprises of MSCs optionally along with a cryopreservation solution. In an embodiment of the present disclosure, the pooled mesenchymal stem cells employed for the management of liver diseases are derived from master cell bank compositions (MCB) or working cell bank compositions (WCB) or IMP compositions or MSCs isolated from WCB/IMP compositions or pooled allogeneic MSCs optionally along with other excipients or cry opreserv ants. In a preferred embodiment of the present disclosure, the pooled mesenchymal stem cells employed for the management of liver diseases are derived from IMP compositions optionally along with other excipients or cry opreserv ants.

In an embodiment of the present disclosure, the IMP composition is cryopreserved in freezing media. Thereafter, at the time of administration, the cryobags are thawed and mixed with physiologically compatible carrier to make up the total volume of the composition. In an exemplary embodiment of the present disclosure, the IMP composition is cryopreserved in about 15 ml of freezing media. Thereafter, at the time of administration, the cryobags are thawed and mixed with physiologically compatible carrier (volume of about 35 ml) to make a total volume of 50 ml.

In an embodiment of the present disclosure, 'pooling' is mixing of MSCs from more than one donor at the stage of preparing master cell bank (MCB) composition in the manufacturing process.

In an embodiment of the present disclosure, the composition comprising pooled population of BM-MSCs prepared from different (batch of) Working cell bank elicits protection against hepatic diseases. In an embodiment of the present disclosure, hepatic inflammation is reduced by administration of the composition due to anti-inflammatory property of BM-MSC. In another embodiment of the present disclosure, the BM-MSCs, generate new hepatocytes and reverse the process of degeneration by virtue of their regenerative capacities in the diseased liver. In another embodiment of the present disclosure, the BM-MSCs facilitate hepatocyte proliferation and repair by increasing secretion of growth factors such as HGF, VEGF, etc. Hepatocyte regeneration helps in improvement of liver function. Further, secretion of MMPs helps in degrading the excess collagen and reversing the fibrosis. In another embodiment of the present disclosure, the BM-MSCs regenerate the hepatocyte and restore the architecture and function of the diseased liver.

Thus, the present disclosure aims at providing bone marrow derived pooled and allogeneic MSC compositions with specific dosage ranges and a combination of modes of administration for management of liver disease. Said dosage ranges and administration routes result in improved efficacy in managing liver disease by inducing regeneration of liver or any part thereof. In exemplary embodiments, said compositions are employed for managing liver disease such as liver fibrosis, liver cirrhosis, or any condition which could result in or result from liver fibrosis or liver cirrhosis in a subject in need thereof.

In an embodiment, it has to be understood that though considerable emphasis has been placed herein on the particular features of this disclosure, various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein and the various features and advantageous details thereof are explained with reference to the non- limiting embodiments in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLE 1

EXAMPLE 1A: PREPARATION OF POOLED ALLOGENEIC MESENCHYMAL STROMAL CELLS COMPOSITION

The Investigational Medicinal Product (IMP) or the composition comprising of pooled BM- MSCs (along with the vehicle/excipient) is prepared, wherein the pooled MSCs are obtained from bone marrow aspirates of healthy donors who are not HLA matched to the recipients. The volunteers for bone marrow donation are tested according to 21 Code of Federal Regulations (CFR) 640, FDA donor suitability & ICMR guidelines for healthy bone marrow donor screening.

Mesenchymal stromal cells are isolated/obtained from the donor's bone marrow mononuclear stem cells using density gradient separation method and thereafter cultured. The cells are expanded in-vitro to manufacture the required number of cells and to establish a donor specific master cell bank (MCB) at Passage (PI). MSC isolated from each donor is cryopreserved separately in individual vials and is labeled with the donor number of each donor. MCB comprising of cells so cryopreserved serve as a source of MSCs for future manufacturing of working cell bank (WCB).

The MCB vials of individual donors are thawed (at about 37°C in a water bath) for further culturing and pooled in equal proportion to establish working cell bank composition at passage 3 (P3). The working cell banks are maintained for routine upscaling and quality control purposes. The working cell banks are upscaled further to produce the cell composition at about passage 4 (P4) to passage 6 (P6). Once, the desired numbers of cells are produced, aliquots of samples are provided for quality control testing purposes. These include complete characterization of MSCs by flow cytometry and differentiation capacity of these cells to osteocytes, chondrocytes and adipocytes. In addition, sterility, mycoplasma, and endotoxin testing are performed at the level of MCB, WCB and IMP to confirm that the cells are devoid of any microbial contaminants and are sterile. Aliquots of the cells are transferred into liquid nitrogen storage vials for quality testing. Release criteria for pooled MSC cell composition used for administration in the in-vivo study are based on the following:

a) negative results for microbiological testing,

b) endotoxin content of < 0.06 EU/ml,

c) cell viability (trypan blue exclusion test) of > 80%,

d) pH between 7.2 to 7.4,

e) exhibiting normal DNA ploidy,

f) normal Karyotype,

g) phenotypic marker analysis by flow cytometry as above, and

h) confirmation of differentiation of cells to osteocyte, chrondocyte and adipocyte. Table 1; Investigational Medicinal Product (IMP) specification

Similarly, the flow cytometry analysis of molecular marker expression of positive markers and low expression markers, for MSCs from Wharton Jelly is depicted in figures 2(c) and 2(d).

EXAMPLE IB: MSCs CULTURED FROM BONE MARROW AND THEIR LARGE SCALE EXPANSION

MSCs are obtained from cell banks or bone marrow of healthy donors after obtaining informed consent. The protocol is approved by the institutional ethics committee (Manipal Hospital, Bangalore, India). After isolation, MSCs are expanded in large numbers. Briefly, the isolated cells are plated in T-75 cm culture flasks (Becton- Dickinson) and cultured in Knockout Dulbecco's Modified Eagle Medium (KO-DMEM) supplemented with about 10% fetal bovine serum (FBS; HyClone), about 2mM glutamax, pencillin-streptomycin at about 100 units of penicillin and about 100 microgram of streptomycin/ml (Life technologies) with about 2ng/ml bFGF and incubated at about 37°C and about 5% humidified C0₂. Upon confluency, the cells are harvested with about 0.25% trypsin-EDTA (Life technologies) and replated in one-cell stack at a density of about 1,000 BM-MSCs cells/cm (Corning Life Sciences). Pooled MSCs from three different individual donors are used. For large scale expansion, BM- MSCs are expanded in about 10 cell stacks up to passage 5. EXAMPLE 1C: IMMUNOPHENOTYPING BY FLOW CYTOMETRY ANALYSIS

MSCs are harvested upon reaching about 80-90% confluency and resuspended in Dulbecco s Phosphate Buffered Saline (DPBS) at a cell density of about 1 million cells/ml. About two hundred microliters of the cell suspension is incubated with the labelled antibodies for about 30 min at about 4°C in dark. The concentration of antibody used for the immunophenotyping is indicated in table 2. Both positive and negative marker antibodies are used to detect the positive cell surface epitopes CD44- phycoerythrin (PE), CD73-PE, CD90-PE, CD105-PE, CD166-PE and negative markers CD 14 fluoroisothiocyanate (FITC), CD19-PECy-7, CD34-FITC, CD45-FITC, and HLA-DR-FITC (all from BD Pharmingen, San Diego, CA) respectively. MSCs incubated with isotype specific IgGl-PE and IgGl-FITC (BD Pharmingen) are used for analyzing the data. At least about 10,000 events are acquired on Guava Technologies flow cytometer, and the results are analyzed using Cytosoft, Version 5.2 (Guava Technologies, Hayward, CA).

Table 2: Concentration of antibody used for the immunophenotyping

Flow cytometric analysis showed that BM-MSCs expressed high levels of positive markers such as CD44, CD73, CD90, CD 105, CD166 (Figure 2a) and are negative for hematopoietic lineage markers such as CD 14, CD 19, CD34, CD45 and HLA DR (Figure 2b).

EXAMPLE 2:

EXAMPLE 2A: ASSAY FOR QUALITY CHECK AND STUDY OF EFFICACY OF MSC COMPOSITIONS

VEGF is a potent pro-angiogenic factor and consistently gets expressed over the passages. Hence, VEGF is selected as a surrogate potency marker for consistent quality check on cells manufactured and released for treating liver diseases. The levels of VEGF in the conditioned medium of the present cell composition/IMP (BM MSCs) at P5 cultures are tested in 13 production batches. On an average, the amount of VEGF present per million cells of the IMP ranges from 2-5 ng/ million cells/ 72hrs (Figure 3) which shows that the MSCs of the IMP are of high quality. Human VEGF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) is used for the experiments according to the directions of the manufacturer.

EXAMPLE 2B: COMPARISON OF GROWTH FACTOR SECRETION PROFILE BETWEEN MSCs OF INDIVIDUAL DONORS AND POOLED COMPOSITIONS & THEIR EFFECT IN MANAGEMENT OF LIVER DISEASES

Since it is observed that there is considerable variation in the growth factor secretion profile of the individual in the growth (GF) array, a few growth factors are selected which are relevant for regeneration of liver and quantified by ELISA for both the individual and pooled cells. The secretion profile of individual donors varies for each growth factor considerably except for TGF- β at both passage 3 and passage 5. On the other hand, consistent secretion of the factors is observed for the pooled cells for most of the growth factors tested. Concentration of the factors secreted by the pooled cells averages out compared to the individual donors except for Angl and TGF-β (Figure 4).

The pooled MSCs secrete higher levels of these factors when compared to the individual donors as seen in Figure 4. Further, it is known that basic cytokine and growth hormone prolife of the individual donors vary for some of these factors and not for all. The present data (Figure 4) clearly establishes that pooling enhances the growth factor secretion profile and thus significantly increases the ability of pooled MSC compositions for the management of liver disease.

As per Figure 4, a considerable variability in the secretion profile of the individual donors is observed. On the contrary, a minor variation is observed with the pooled cells. Further, pooled BM-MSCs produce consistent level of growth factors such as VEGF, TGF-β and Ang-1 at passage 5. Overall results obtained from quantifying the paracrine secretion clearly establishes that pooling of individual donors produces consistency in terms of factors such as cytokines and growth factors like HGF, VEGF, Ang-1, TGF-β and PDGF, that are known to play an important role in regeneration of hepatocyte, reversing inflammation and replenish all types of damaged cells; thus suggesting the improved efficacy of pooled and expanded allogeneic MSCs in the management of liver disease.

Also, potential reasons for the inconsistent results in MSC clinical trials of individual MSCs is due to their variability in the secretion profile of various growth hormones which are overcome by the pooling technology of the instant disclosure, and thereby enhancing the efficacy in the treatment of liver disease.

EXAMPLE 3

EXAMPLE 3A: IN-VIVO EVALUATION OF THERAPEUTIC POTENTIAL OF MESENCHYMAL STROMAL CELLS IN ANIMAL MODEL

Experiments to evaluate efficacy and the effective therapeutic dosage of pooled human bone marrow derived Mesenchymal Stromal Cells (MSCs) in the treatment of liver cirrhosis is carried out in an animal model of CC1₄ induced liver fibrosis.

Briefly, clinical grade MSCs are isolated from human bone marrow and characterized by molecular phenotyping and differentiation potential. Rats are injected with CC1₄ for about 8 weeks to induce liver fibrosis, followed by intravenous injection of bone marrow MSCs and plasmalyte A (placebo) in three separate cirrhotic animal groups. After ab o u t 8 weeks of treatment, rats with bone marrow-MSC exhibited significant reduction in liver fibrosis, as evaluated by Masson trichrome and Sirus red staining. There is marked improvement in lobular architecture and gross pathology of liver in MSCs treated group as compared to placebo. The results suggest that MSCs transplantation is effective in the treatment of liver fibrosis.

EXPERIMENTAL PROCEDURE

Induction of Liver Fibrosis in rat to create an animal model:

Male Sprague-Dawley rats (body weight of about 250-300 g) purchased from Harlan Laboratory, USA, are used as animal models of CC1₄ induced liver fibrosis. The experiments are approved in accordance with the guidelines for the care and use of laboratory animals by the animal research ethics committee of Institute for Medical Research, Ministry of health, Malaysia. The rats are treated with a mixture of CC1₄ and olive oil (in a ratio of about 1:1 vol/vol), at a dose of about 2 mL of mixture/kg of body weight for about 8 weeks. Before cell treatment, some of the rats are sacrificed and the liver is removed for histological examination with Masson' s Trichrome staining to confirm the fibrosis. Transplantation of BM - MSCs:

MSCs are thawed at about 37°C in water bath and resuspended in culture media containing about 10% FBS, centrifuged at about 1200 rpm for about 10 minutes and resuspended in about 500 microliter of Multiple Electrolyte Solution (MES) such as Plasmalyte A. Animals are divided into plasmalyte A (control) and BM-MSCs (test) groups; about 4 animals in each group. The MSC Cells are injected via tail vein. Tails are cleaned with xylene and the cells resuspended in about 500 microliter of plasmalyte A are injected slowly. About 5 million human equivalent dose is used in this study. Animals are sacrificed at about 4 week, about 8 week and about 12 week. The liver is harvested, photographed and subjected to histological analysis by using Masson's Trichrome staining and Sinus (Picrosirius) red staining.

Picrosirius Red Staining:

Liver sections (about 5 μιη) are deparaffinized, rehydrated and incubated for about one hour with about 0.1% sirius red (Direct Red, Sigma Aldrich, USA) dissolved in saturated solution of picric acid (Sigma Aldrich, USA). The sections are then rinsed in about two changes of acidified water (about 0.5% glacial acetic acid in water) and dehydrated in about 3 changes of about 100% ethanol for about one minute, cleared in xylene and mounted in a resinous medium.

Masson's Trichrome Staining:

Masson's trichrome staining is performed according to a protocol suggested by the manufacturer (American Master Tech Scientific Inc., Lodi, CA). All Images are observed under bright field using an Olympus 1X71 inverted microscope, and images are captured with cell D, version 3.4

A schematic diagram showing liver fibrosis induction in the animal models, grouping of the animals, cell dosages and route of delivery is depicted in Figure 5.

Results and Observation:

Characterization of Mesenchymal Stromal Cells from Bone Marrow:

To determine whether stromal cells from bone marrow express MSC markers, cells from bone marrow are isolated and cultured in KO-DMEM supplemented with about 10% FBS with about 2 ng/ml bFGF. Microscopic observation of the cells demonstrated a fibroblast-like phenotype as illustrated in Figure 1. Flow cytometric analysis, done as per Example 1C, showed that BM-MSCs expressed high levels of positive markers (CD44, CD73, CD90, CD 105, CD166) (Figure 2a) and are negative for hematopoietic lineage markers (CD14, CD19, CD34, CD45 and HLA DR) (Figure 2b).

CCLt-Induced Liver Fibrosis in Rats:

To establish the CC1₄ induced liver cirrhosis model, the following protocol is done. After about 8 week of CC1₄ injection, animals are sacrificed and subjected to histopathology by using Masson's Trichome staining. Liver fibrosis is clearly revealed by the significant increase in extra cellular matrix proteins in CC1₄ treated animals as compared to normal control group (Figure 6).

Effect of MSCs on CC - Induced Liver Fibrosis:

To see the effect of MSCs in treatment of CC1₄ induced liver fibrosis, BM-MSCs are transfused in the animal models via tail vein and the animals are sacrificed at various time points (about week 4, 8 and 12) as shown in (Figure 7). The Livers are subjected to gross pathology and histology. a) Gross Pathological Analysis of Liver:

For gross pathological examination, animals are sacrificed at various time points (about week 4, 8 and 12) post MSCs transplantation. Livers are dissected out, washed with phosphate buffer and photographed. Better lobular architecture is observed in both the MSCs treated groups as compared to plasmalyte A at about week 8 and 12 (Figure 8). b) Histo Pathological Analysis of Liver:

The livers are subjected to histopatholgy by Masson's Trichome staining and Sirus red staining. It is observed that fibrosis is reduced after about 8 weeks of treatment in group administered with MSCs as compared to the control group administered with Plasmalyte A (Figures 9).

Hence, from this example, it can be observed that administration of MSCs is found to be effective in reducing liver fibrosis in the animal models at about 4, 8 and 12 weeks posttransplantation of MSCs. EXAMPLE 3B: IN-VIVO EVALUATION OF THERAPEUTIC POTENTIAL OF

MESENCHYMAL STROMAL CELLS IN ANIMAL MODEL

Experiments to evaluate efficacy and the effective therapeutic dosage of pooled human bone marrow derived Mesenchymal Stromal Cells (BM-MSCs) in the treatment of liver cirrhosis is carried out in animal model of CC1₄ induced liver cirrhosis.

Rats are injected with CC for about 8 weeks to induce liver cirrhosis, followed by intravenous injection of BM-MSCs and Plasmalyte A (placebo). After ab o u t 30 d ay s and ab o u t 70 day s of treatment, it is observed that rats with BM-MSCs exhibited significant reduction in liver fibrosis and cirrhosis, as evaluated by liver hydroxylproline content, Masson's tri chrome and Sirius red staining. The results suggest that BM-MSCs transplantation is effective in the treatment of liver cirrhosis.

EXPERIMENTAL PROCEDURE

Induction of Liver Cirrhosis in rat to create an animal model:

Male Sprague-Dawley rats (body weight of about 150 - 200 g) at about 3-4 weeks of age are used as animal model of CC14 induced liver cirrhosis. The experiment is approved in accordance with the guidelines for the care and use of laboratory animals by the animal research ethics committee of Institutional Animal Ethics Committee (IAEC), India. In the experiment, about twenty five rats received intraperitoneal injection of CC14:01ive oil (1: 1 ratio), twice a week with about 2 ml/kg for initial 2 weeks, followed by about 1 ml/kg twice weekly for next 6 weeks and about 15 rats received only olive oil (vehicle). After about 8 weeks of CC14 injection, the disease onset is confirmed by liver injury and functional markers, gross and histopathology examination.

Transplantation of BM - MSCs:

Before the BM-MSCs treatment, diseased animals are randomized based on the liver functional parameters into Plasmalyte A disease control (n=12) and BM-MSCs treated group (n=10).The olive oil injected animals served as sham control group.

BM-MSCs are thawed at about 37°C in water bath and re-suspended in culture media containing about 10% FBS, centrifuged at about 1200 rpm for about 10 minutes and re- suspended in Plasmalyte A. BM-MSCs groups received transplantation of about 5 x 10⁶ human equivalent dose of BM-MSCs re-suspended in about 500 microliter of Plasmalyte A, injected slowly via tail vein. Plasmalyte A disease control and sham control group are injected with about 500 microliter of Plasmalyte A. Animals are sacrificed at about day 30 (about 6 animals in each group) and about day 70 (about 4 in BM-MSCs group, about 6 in Plasmalyte A and sham control group each). Before scarification retro-orbital blood collection is made for biochemistry analysis. At the time of scarification, the liver is harvested, photographed and subjected to hydroxyproline assay and histopathological analysis.

Results and Observation:

Liver Hydroxyproline Content:

As compared to sham control, the liver hydroxyproline content is significantly increased in disease control at about day 30 and about day 70, signifying the breakdown of excess collagen. The BM-MSCs treated animals showed statistically significant decrease when compared to the Plasmalyte A disease control animals at both about 30 days and about 70 days as shown in figure 10. The hydroxyproline levels in liver tissues of sham control, vehicle, BM-MSCs and WJ-MSCs treated groups on day 30 and 70. We observed statistically significant differences between following groups. At day 30 (A): vehicle vs. BM-MSCs, *p<0.05; vehicle vs. WJ-MSCs, ns; vehicle vs. sham control, **p<0.01. At day 70 (B): vehicle vs. BM-MSCs, *p<0.05; vehicle vs. WJ-MSCs, ns; vehicle vs. sham control, ***p<0.001. ns, not significant. The observation indicates that BMMSCs cells are significantly effective compared to the WJ-MSCs in the therapeutic effects. Even though the MSC derived from BM-MSC and WJ-MSC appear to be similar from their cytometry analysis of molecular marker, the hydroxyproline analysis show that functionally they there are not same.

Histopathological Analysis of Liver:

The livers are subjected to histopatholgy by H&E staining, Masson's Trichome staining and Sirius red staining. It is observed that fibrosis is reduced in BM-MSCs treated group as compared to the disease control group administered with Plasmalyte A (Figures 11- 16-).

EXAMPLE 3C: EFFICACY OF BONE MARROW DERIVED MESENCHYMAL STROMAL CELLS IN TREATING LIVER CIRRHOSIS

Efficacy of bone marrow derived mesenchymal stromal cells (BM-MSCs) in treating CC1₄ induced liver cirrhosis in Wistar rats, purchased from The Wistar Institute, Philadelphia, is evaluated. CC1₄ is administered at about 1 ml/kg body weight, for about 3 days a week for about 28 days, intra peritoneally. Very high levels of liver enzymes used in CC1₄ treated animal model groups, when compared to control group, indicates that liver cirrhosis is induced in the rat, and the histology also showed the disarrangement of hepatocytes, vacuole formation, increased space among sinusoids, and disarrangement of central vein as is observed during liver cirrhosis.

Amount of drugs administered to the rats depends on their body weights. About 30 healthy female Wistar rats are used which are randomly allotted into one of the five experimental groups: Group A (Control), Group B (CC1₄ treated group which are administered with about 0.1 ml/kg body weight of CC1₄ to induce liver cirrhosis), Group C (CC1₄ treated group +silymarin at about lOOmg/ kg body weight), Group D (CC1₄ treated group + MSCs at about 5.8 million cells per animal)) and Group E (CC1₄ treated group + MSCs at about 9.75 million cells/kg body wt+ Silymarin at about lOOmg/ kg body weight, orally), each group containing 6 animals, wherein the MSCs are administrated intravenously through tail vein.

At the end of about 5, 10 and 15 days, blood samples are collected and sera is used for liver enzyme estimations. On the 16^th day, animals are sacrificed by injecting ketamine; liver tissue is used for histopathology study.

Liver enzyme levels are increased in the CC1₄ treated rats (Table 3). Administration of silymarin and BM-MSCs showed significant hepatoprotective activity (p<0.05) when compared to the CC1₄ treated group. The results indicate that MSCs treatment is more effective when compared to silymarin treatment (p<0.05). Histhopatological examination of liver tissue of CC1₄ treated animals showed disarrangement of normal hepatic cells with centrilobular necrosis, vacuolization of cytoplasm (Figure 17). Liver sections of the rats treated with MSCs and silymarin showed a sign of protection as it is evident by the absence of necrosis and vacuoles.

CONCLUSION: Results indicate that BM-MSCs treatment is effective in treating liver cirrhosis in Wistar Rats. Table 3: Effect of BM-MSCs and Silymarin on the biochemical parameters against CC induced hepatotoxicity in rats at the end of 5 , 10 and 15 day

Values are expressed as mean+SD of 6 samples.

Thus, the present disclosure provides for but is not limited to bone marrow/Wharton's Jelly derived allogeneic pooled MSC compositions with specific dosage ranges and modes of administration. In an important aspect of the disclosure, said MSC compositions at specified dosages and route of administration are employed for managing Liver diseases such as Liver Cirrhosis in a subject in need thereof.

Claims

We Claim:

1. A composition for managing liver disease comprising pooled mesenchymal stromal cells in an amount ranging from about 1 million cells to about 1000 million cells, optionally along with pharmaceutically acceptable excipient.

2. A method of managing liver disease in a subject having or suspected of having the liver disease, said method comprising act of administering a composition comprising pooled mesenchymal stromal cells at a dose ranging from 0.5 million cells per kg of body weight of the subject to 8 million cells per kg of body weight of the subject, optionally along with pharmaceutically acceptable excipient to the subject.

3. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the mesenchymal stromal cells are derived from source selected from group comprising bone marrow, adipose tissue and dental pulp, or any combination thereof, preferably bone marrow.

4. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the mesenchymal stromal cells are obtained by ex-vivo cell culturing; and wherein at least 80% of the mesenchymal stromal cells are positive for cell specific markers selected from group of CD 73, CD90, CD105 and CD166 cells or any combination thereof; and less than 10% of the mesenchymal stromal cells are positive for markers selected from group of CD34, CD45, CD133, CD14, CD19 and HLA-DR or any combination thereof.

5. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the mesenchymal stromal cells are allogenic and expanded, and wherein expansion is carried out by culturing the pooled allogeneic mesenchymal stromal cells for about 3 to about 6 passages.

6. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the pharmaceutically acceptable excipient is selected from group comprising carrier, cyropreservant, protein and pre-formulated ready to use cryopreservation mixture, or any combination thereof.

7. The composition or the method as claimed in claim 1 or the method as claimed in claim 2, wherein the carrier is selected from a group comprising multiple electrolyte solution such as PlasmaLyte A, Hank's balanced salt solution, saline, Lactated Ringer's Injection; the cyropreservant is Dimethyl Sulfoxide; the protein is human serum albumin; and the pre-formulated ready to use cryopreservation mixture is animal protein-free defined cryopreservation medium.

8. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the liver disease is selected from group comprising alcoholic hepatitis, liver fibrosis, liver cirrhosis, conditions which could result in or result from liver fibrosis or liver cirrhosis, chronic hepatitis B, chronic hepatitis C, non-alcoholic steatohepatitis, primary biliary cirrhosis, autoimmune hepatitis, primary sclerosing cholangitis, alcoholic liver disease, hereditary hemochromatosis, Wilson's disease, alpha I antitrypsin deficiency, cardiac cirrhosis, galactosemia, cystic fibrosis, glycogen storage disease type IV and parasitic infections of the liver or any combination thereof, preferably liver cirrhosis or liver fibrosis.

9. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the subject is mammal, preferably human.

10. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the composition is administered as single dose or multiple doses at one or multiple sites through modes selected from group comprising intravenous administration, hepatic administration, pancreatic duodenal artery administration, intraperitoneal administration, hepatoportal administration, intramuscular administration, intra articular administration, subcutaneous, intradermal administration, intrahepatic administration, intraportal administration and intrasplenic administration or any combination thereof, preferably intravenous, intrahepatic or intraportal administration or any combination thereof.

11. The composition as claimed in claim 1 or the method as claimed in claim 2, wherein the composition is formulated as a formulation selected from group comprising an aqueous suspension, cream, lotion, gel, emulsion, drop, emulsion in hard or soft gel capsule, elixir, lyophilized cell powder and cell spray or any combination thereof, preferably aqueous suspension.

12. The composition as claimed in claim 1, wherein the pooled mesenchymal stromal cells are at an amount ranging from about 15 million cells to about 800 million cells and wherein the amount of MSCs is based on the body weight of a subject in need thereof.

13. A kit for managing liver disease in a subject in need thereof comprising the composition as claimed in claim 1 optionally along with an instruction manual.

14. A method of assembling a kit as claimed in claim 13, said method comprising act of combining a composition as claimed in claim 1, optionally along with an instruction manual.

15. A composition comprising pooled mesenchymal stromal cells in an amount ranging from about 1 million cells to about 1000 million cells, optionally along with pharmaceutically acceptable excipient for use as a medicament for managing liver disease.