WO2011101834A1

WO2011101834A1 - A method for obtaining mesenchymal stem cells, media, methods and composition thereof

Info

Publication number: WO2011101834A1
Application number: PCT/IB2011/050730
Authority: WO
Inventors: Swapnil Satish Totey; Aparna Khanna; Satish Mahadeorao Totey; Neelam Krishnan Venkataramana
Original assignee: Advanced Neuro-Science Allies Private Limited
Priority date: 2010-02-22
Filing date: 2011-02-22
Publication date: 2011-08-25

Abstract

The invention relates to a method of isolation of human mesenchymal stem cells (h MSCs) from wharton's jelly, which is the connective tissue of the umbilical cord (UC). The method comprises of deriving the cells and expansion of the cells in appropriate media for culture and maintenance of the cells. Further, the cells were induced to differentiate into oestocytes, chondrocytes and adipoctes, and pancreatic cells. The cells also displayed embryonic like characteristics as demonstrated by high expression of embryonic markers. The unique ability of these cells to form embryoid bodies like clusters (EB-LCs) having more propensity towards endoderm lineage (pancreatic lineage) as compared to undifferentiated mesenchymal stem cells. Hence from the in vitro characterization it is indicated that it can be a potential source of cells for regenerative therapies for pancreatic related disorders.

Description

A METHOD FOR OBTAINING MESENCHYMAL STEM CELLS, MEDTA, METHODS

AND COMPOSITION THEREOF"

TECH ICAL FIELD

The present disclosure relates to stem cell technology, wherein mesenchymal stem cells are isolated from a given source and cultured on a nutrient media. The mesenchymal stem cells are directed towards formation of cmbryoid body-like clusters and also depicts an inclination to differentiate into endodermal lineages or specifically pancreatic lineage. This technology can be used in regenerative medicine arid endocrine related disorders.

BACKGROUND

Wharton's jelly, discovered in the mid-l600s by Thomas Wharton, a London physician, is the gelatinous connective tissue only found in the umbilical cord. The jelly gives the cord resiliency and pliability, and protects the blood vessels in the umbilical cord from compression. The interlaced collagen fibres and small, woven bundles are arranged to form a continuous soft skeleton that encases the umbilical vessels. Wharton's Jelly has very little collagen, another indicator of the primitive state of this tissue. In Wharton's Jelly, the most abundant glycosaminoglycan is hyaluronic acid, which forms a hydrated gel around the fibroblasts and collagen fibrils and maintains the tissue architecture of the umbilical cord by protecting it from pressure. Cells in Wharton's Jelly express several stem cell genes, including telomerase. They can be extracted, cultured, and induced to differentiate into mature cell types such as neurons. Wharton's Jelly is therefore a potential source of adult stem cells. As an embryo forms, some very primitive cells migrate between the region where the umbilical cord forms and the embryo. Some primitive cells just might remain in the matrix later in gestation or stil l be there even after the baby is born.

Methods used for isolating mesenchymal stem Cells from Wharton's Jelly

Enrvmatic method: Mesenchymal stem cells have been isolated from cord vasculature wherein specifically a 15 rnin collagenase digestion from within the umbilical vein yielded a mixed population of vascular endothelial and sub-endothelial cells. Further, sparse numbers of fibroblasi-like cells appear from this cell harvest after 7 days. In another method, the extract comprises human progenitor cells and is obtained by enzymatic digestion of the Wharton's jelly proximal to the vasculature of human umbilical cord, in a region usefully termed the perivascular zone of Wharton's Jelly. The tissue within this perivascular zone, and from which the present progenitor cells are extracted, can also be referred to as perivascular tissue. The extraction procedure suitably results in an extract that is essentially free from cells of umbilical cord blood, epithelial cells or endothelial cells of the umbilical cord and cells derived from the vascular structure of the cord, where vascular structure is defined as the tunicae intima, media and adventia of arteriolar or venous vessels. The resultant extract is also distinct from other Wharton's Jelly extracts isolated from the bulk Wharton's Jelly tissue that has been separated from the vascular structures.

Modification of the single step collagenase treatment is the two step enzymatic digestion, to isolate and culture and is considered one of the most efficient way to isolate mesenchymal stem cells from umbilical cord.

Disadvantages: Enzymatic digestion might affect the viability of the Cells and the success rate of isolation, which is one of the most important factors regarding the clinical use was not described.

Non enzymatic method:

a) Explant method: Recent publications have reported methods to harvest cells from umbilical cord, rather than umbilical cord blood. A method is described wherein the umbilical vessels are discarded before harvesting the remaining tissues. The latter, which will include both the remaining Wharton's Jelly (some of which will have been discarded with the vessels, since the umbilical vessels are entirely enveloped in Wharton's Jelly) and the amniotic epithelium, is then diced to produce small tissue fragments that are transferred to tissue culture plates. These tissue fragments are then used as primary explants from which cells migrate onto the culture substratum. Disadvantages: Since it is not single cells, takes longer time to expand and results less yield.

b) Mechanical method: Friedman et al., described a novel, simple method of obtaining and cryopreserving umbilical cord-mesenchymal stem cells by extracting the Wharton's Jelly from a small piece of cord, followed by mincing the tissue and cryopreserving it in autologous cord plasma to prevent exposure to allogeneic or animal serum, thus showing that umbilical cord-mesenchymal stem cells are a reliable, easily accessible, noncontroversial source of mesenchymal stem cells. The prevalence of diabetes is rapidly rising all over the globe at an alarming rate. Over the past 30 year, the status of diabetes has changed from being considered as a mild disorder of the elderly to one of the major causes of morbidity and mortality affecting the youth and middle aged people. Type 2 Diabetes Mellitus constitutes the major chunk of diabetes and has insulin resistance as the hallmark feature in the pathogenesis. However, with the progression of the disease the insulin resistance becomes stable whereas β - cell function shows a gradual decline due to its ongoing apoptosis. This ultimately leads to inability of the β - cells to cope up with the increased demand of insulin caused due to insulin resistance and manifests as hyperglycemia. The patients with type II diabetes require insulin and it would be difficult to achieve to attain a strict glycemic control. However, with intensive insulin therapy it has been shown that glycemic control can never be perfect with patients exhibiting hyperglycemia or hypoglycemia during 24 hour glucose profile.

This led the researchers to evolve various strategies of β - cell replacement therapy e.g. pancreatic transplantation and islet cell transplantation. However, islet cell transplantation has its own limitations for example insufficient supply, being technically demanding and requirement of lifelong immunosuppressive therapy in the recipient. These shortcomings can be overcome by the use of stem cells which is an inexhaustible source of β -cells. Stem cells can be obtained from various sources like blastocyst (embryonal stem cells), umbilical cord or bone marrow. There is an evidence to suggest that stem cell transplantation can lead to improvement in pancreatic endocrine function and improvement in glycemic control in diabetic mice through various mechanisms such as transdifferentiation or regeneration of endothelial cell in the damaged islets which in turn lead to regeneration of islet cells by paracrine action.

STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to a method of obtaining mesenchymal stem cells from mammalian umbilical cord, said method comprising step of isolating the stem cells by non-enzymatic means to obtain the isolated mesenchymal stem cells; a media comprising about 50% KO-DMEM and about 50% alpha MEM media, optionally supplemented with atleast one of the supplements selected from a group comprising human serum albumin, growth factors, platelet lysate, amino acids and bioactive agents; a composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives; a method of preparing a Master Cell Bank composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives, said method comprising steps of - a) isolating and culturing the mesenchymal stem cells in nutrient media comprising about 50% Knock Out- Dulbecco's Minimal Essential Medium (KO-DMEM) and about 50% alpha Minimal Essential Medium (MEM) supplemented with the HSA, growth factors and amino acids and fractionating the culture to obtain fractionated cells, b) centrifuging the fractionated cells to obtain a pellet comprising purified cells and suspending the purified cells in the nutrient media, c) culturing the cells in the nutrient media to achieve about 80% confluency and treating the cells with Trypsin-EDTA and cryo-preserving the treated stem cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA and, d) optionally, adding pharmaceutically acceptable additives to obtain the master cell bank composition; a method of preparing a Working Cell Bank composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA) and Dimethyl Sulphoxide (DMSO) optionally along with pharmaceutically acceptable additives said method comprising steps of - a) preparing a Master Cell Bank composition, b) thawing and washing the Master Cell Bank stem cells with nutrient media comprising about 50% KO-DMEM and about 50% alpha MEM media supplemented with the HSA, growth factors and amino acids, and centrifuging the washed stem cells to obtain a cell pellet, c) re-suspending the cell pellet in the nutrient media, d) seeding and passaging cells to achieve about 80% confluency and treating the cells with Trypsin-EDTA and cryo- preserving the treated cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA and d) optionally, adding pharmaceutically acceptable additives to obtain the working cell bank composition; a method of preparing a composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA) and Dimethyl Sulphoxide (DMSO) optionally along with pharmaceutically acceptable additives , said method comprising steps of - a) preparing a Working Cell Bank composition, b) thawing and washing of the Working Cell Bank composition with nutrient media comprising about 50% KO-DMEM and about 50% alpha MEM media supplemented with the HSA, the growth factors and the amino acids, and centrifuging the washed stem cells to obtain first cell pellet, c) re-suspending the cell pellet in the nutrient media, d) seeding and passaging cells to achieve about 80% confluency, treating the cells with Trypsin-EDTA and cryo-preserving the treated cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA and d) optionally, adding pharmaceutically acceptable additives to obtain the composition; a method of inducing differentiation of mesenchymal stem cells towards endodermal lineages, said method comprising steps of - a) isolating and culturing mesenchymal stem cells in a nutrient media, b) treating the cultured cells with Trypsin-EDTA at a concentration ranging from about 0.1% to about 0.5%, preferably about 0.25% and washing the treated cells with the nutrient media to obtain embryoid body like clusters and c) inducing the embryoid body like clusters for differentiation of the cells towards the endodermal lineages; a method of managing endocrine related disorders, said method comprising step of administering a composition comprising mesenchymal stem cells and active agents, to a subject in need thereof; and a kit comprising composition, and instructions for administering the composition for managing endocrine related disorders.

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 figure 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 shows mechanical isolation of Mesenchymal Stem Cells from Umbilical cord. Umbilical cord is obtained after elective caesarean and after taking consent form. Umbilical cord is opened and blood vessel and arteries are removed. Mucosal surface is then scrapped gently in order to remove Wharton's Jelly containing mesenchymal Stem Cells. A. Umbilical cord is obtained and cleaned nicely with sterile water and ethanol. B. Umbilical cord is cut into multiple pieces. C. Cut umbilical cord pieces. D. Each piece of umbilical cord is opened longitudinally. E. Blood vessels and arteries are removed. F. opened umbilical cord with removal of blood vessels and artery. G. Scrapping of mucosal surface of umbilical cord with scrapper in order to remove Wharton Jelly. H. Wharton's Jelly is collected in 50 ml tube I. Pure population of mesenchymal stem cells is removed by fractionation three times. J. Final supernatant is centrifuge and mesenchymal stem cells are obtained. K. Collected mesenchymal stem cell pellet is re-suspended in media and L. Re-suspended mesenchymal stem cells are seeded on to 2-cell stacks for further multiplication.

Figure 2 shows morphological characteristics of umbilical cord derived mesenchymal stem cells. A & B showed typical spindle shaped fibroblastic cells.

Figure 3 shows Immunophenotypic Characterization of mesenchymal stem cells using cell surface markers by flow cytometry analysis shows that the mesenchymal stem cells isolated from umbilical cord is having homogeneous population show MSC characteristics. A. SSEA-4 (59.5%) B. CD90 (92.3%), C. CD166 (82.4%) D. CD73 (79.8%) E. CD45 (0.85%) F. CD34 (0.46%), G: HLA-DR (0.85%), H. ISOTYPE CONTROL.

Figure 4 shows Pluripotent marker expression of mesenchymal stem cells derived from umbilical cord. Figure shows that pluripotent markers are highly expressed in mesenchymal stem cells derived from umbilical cord. Immunofluorescence shows high expression of SSEA-4 A, OCT-4, B. and SOX-2 C. Differential molecular expression of pluripotent markers in mesenchymal stem cells shows that OCT-4 D, and SOX-2 E, highly expressed in mesenchymal stem cells derived from umbilical cord as compared to that of bone marrow and dental pulp stem cells.

Figure 5 shows differential expression of pluripotent and lineage specific genes in MSC derived from Wharton's Jelly and Bone marrow by DNA microarray. Some pluripotent genes and lineage specific genes are highly expressed in Wharton's Jelly as compared to the Bone marrow MSC. This suggests that Wharton's Jelly mesenchymal stem cells have propensity towards endoderm lineage.

Figure 6 shows transcription factors expressed at different stages of endoderm lineage development.

Figure 7 shows Wharton's Jelly derived mesenchymal stem cells showed typical characteristics of embryoid body structure. A. Day 4 embryoid bodies, B. Day 8 embryoid bodies, C. Day 12 embryoid bodies, D. 16 Days embryoid bodies and E. 20 days embryoid bodies.

Figure 8A-D shows Wharton's Jelly derived mesenchymal stem cells derived embryoid bodies has properties to develop pancreatic bud formation (A & B) similar to that of human embryonic stem cells (C & D).

Figure 9 shows expression of Endoderm lineage markers. Figure showed that Endoderm marker HNF3 beta and PDX is not expressing in bone marrow derived mesenchymal stem cells whereas mesenchymal stem cell derived from Wharton's Jelly showed expression of both HNF3B and PDX-1 and upon differentiation into embryoid bodies both the markers showed over expression. GAPDH is a house keeping gene.

Figure 10 shows expression of Pancreatic beta islet cells markers. Figure showed that pancreatic beta marker expression during differentiation of Wharton's Jelly mesenchymal stem cells into pancreatic beta cells.

Figure 11 shows expression of insulin, Ngn3 and Somatostatin in differentiated embryoid bodies derived from Wharton's Jelly mesenchymal stem cells.

Figure 12 shows the Sequencing data is positive for the presence of INSULIN in the Wharton's jelly MSC derived Embryoid bodies. This study confirms that Wharton's Jelly Mesenchymal Stem Cells has propensity towards pancreatic lineage. Figure 13 shows quantitative expression of pancreatic related genes during the differentiation of Wharton's Jelly mesenchymal stem cells. A: SOX- 17 promote endoderm showed increased expression during differentiation, B: GLUT-2 also shows increased expression during differentiation, C: GLUT-1 shows increased expression initially but decreases as differentiation progresses, D: NGN3 pancreatic gene showed increased expression during differentiation. Balance between GLUT-1 and GLUT-2 is therefore important in pancreatic differentiation and development. A WJ-MSC, B: BM-MSC, C: WJ-EB-4, D: WJ-EB8, E: WJ-EB 12, F: EJ-EB 16, G: WJ-EB 20.

Figure 14 shows 92.18% differentiated cells showed ISL-1 positive shows that Wharton's Jelly mesenchymal stem cells has propensity to differentiate towards pancreatic beta islet cells and hence may be excellent for Therapeutic purpose in Diabetes.

DETAILED DESCRIPTION

The present disclosure relates to a method of obtaining mesenchymal stem cells from mammalian umbilical cord, said method comprising step of isolating the stem cells by non- enzymatic means to obtain the isolated mesenchymal stem cells.

In an embodiment of the present disclosure, the mesenchymal stem cells are obtained from Wharton's Jelly within the mammalian umbilical cord.

In another embodiment of the present disclosure, the non-enzymatic means are mechanical in nature, preferably scrapping the stem cells from surface; followed by density fractionation.

The present disclosure relates to a media comprising about 50% KO-DMEM and about 50% alpha MEM media, optionally supplemented with atleast one of the supplements selected from a group comprising human serum albumin, growth factors, platelet lysate, amino acids and bioactive agents.

In an embodiment of the present disclosure, the human serum albumin is at a concentration ranging from about 1% to about 10%, preferably about 5%; the growth factors are selected from a group comprising Platelet Derived Growth Factor (PDGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 4 ng/ml; Epidermal Growth Factor (EGF) at a concentration ranging from about 1 ng/ml to about 5 ng/ml, preferably about 2 ng/ml; basic Fibroblast Growth Factor (bFGF) at a concentration ranging from about 1 to about 4 ng/ml, preferably about 2 ng/ml; Transforming Growth Factor- beta (TGF-beta) at a concentration ranging from about 1 ng/ml to about 4 ng/ml, preferably about 2 ng /ml; Insulin-like Growth Factor (IGF-1) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Hepatocyte Growth Factor (HGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Vascular Endothelial growth factor (VEGF) at a concentration ranging from about 1 ng/ml to about 100 ng/ml, preferably about 10 ng/ml; the platelet lysate is selected from blood group comprising AB and O and wherein the platelet lysate is with or without plasma, and is at a concentration ranging from about 5% to about 20%, preferably about 10%; the amino acids are selected from a group comprising L-ascorbic acid-2 phosphate at a concentration ranging from about 1 umol to about 100 umol; and L-glutamine at a concentration ranging from about 100 mM to about 400 mM, preferably about 200 mM, or combination thereof; and the bioactive agents are selected from a group comprising insulin, selenite, transferrin, IL-3 and stem cell factors or any combination thereof.

In another embodiment of the present disclosure, the media is a serum free or a xeno-free media or a combination thereof.

The present disclosure relates to a composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives.

In an embodiment of the present disclosure, the composition has about 1 million to about 100 million mesenchymal stem cells, Dimethyl Sulfoxide at a concentration ranging from about 8% to about 15%, preferably about 10%, Human Serum Albumin at a concentration ranging from about 2% to about 10%, preferably about 5%, and the multiple electrolyte isotonic solution is at a volume ranging from about 10 ml to about 40 ml, preferably about 13.5 ml.

The present disclosure relates to a method of preparing a Master Cell Bank composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives, said method comprising steps of: a. isolating and culturing the mesenchymal stem cells in nutrient media comprising about 50% Knock Out-Dulbecco's Minimal Essential Medium (KO-DMEM) and about 50% alpha Minimal Essential Medium (MEM) supplemented with the HSA, growth factors and amino acids and fractionating the culture to obtain fractionated cells;

b. centrifuging the fractionated cells to obtain a pellet comprising purified cells and suspending the purified cells in the nutrient media; c. culturing the cells in the nutrient media to achieve about 80% confluency and treating the cells with Trypsin-EDTA and cryo-preserving the treated stem cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA; and

d. optionally, adding pharmaceutically acceptable additives to obtain the master cell bank composition.

In an embodiment of the present disclosure, the mesenchymal stem cells are isolated by non-enzymatic means, preferably mechanical isolation, more preferably by scrapping the stem cells from inner tissue surface of umbilical cord, removing Wharton's Jelly.

In another embodiment of the present disclosure, the fractionating is density fractionating to obtain pure population of stem cells and comprises steps of suspending, manually density separating and centrifuging the separated stem cells in about 50 ml of the nutrient media, at room temperature for a time duration ranging from about 5 minutes to about 20 minutes, preferably about 10 minutes.

In yet another embodiment of the present disclosure, the centrifuging is carried out at about 800 rpm to about 1800 rpm, preferably about 1200 rpm, at room temperature for time duration ranging from about 5 minutes to about 20 minutes, preferably about 10 minutes. In still another embodiment of the present disclosure, the nutrient media of the purified stem cells is changed after about 48 hours to about 72 hours, and subsequently after about 7 days, and wherein cells are removed by the treating after about 15 days to about 18 days of culturing or after the cells attain about 80% confluency; and the Trypsin-EDTA has a concentration of about 0.25%. The present disclosure relates to a method of preparing a Working Cell Bank composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA) and Dimethyl Sulphoxide (DMSO) optionally along with pharmaceutically acceptable additives said method comprising steps of:

a. preparing a Master Cell Bank composition as mentioned above; b. thawing and washing the Master Cell Bank stem cells with nutrient media comprising about 50% KO-DMEM and about 50% alpha MEM media supplemented with the HSA, growth factors and amino acids, and centrifuging the washed stem cells to obtain a cell pellet;

c. re-suspending the cell pellet in the nutrient media;

d. seeding and passaging cells to achieve about 80% confluency and treating the cells with Trypsin-EDTA and cryo-preserving the treated cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA; and

e. optionally, adding pharmaceutically acceptable additives to obtain the working cell bank composition.

In an embodiment of the present disclosure, the nutrient media of the cell pellet is changed after about 48 hours and subsequently after about every 5 days and wherein the treating is done after the cells attain about 80% confluency, and the Trypsin-EDTA is having a concentration of about 0.25%.

The present disclosure relates to a method of preparing a composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA) and Dimethyl Sulphoxide (DMSO) optionally along with pharmaceutically acceptable additives , said method comprising steps of:

a. preparing a Working Cell Bank composition according to as mentioned above;

b. thawing and washing of the Working Cell Bank composition with nutrient media comprising about 50% KO-DMEM and about 50% alpha MEM media supplemented with the HSA, the growth factors and the amino acids, and centrifuging the washed stem cells to obtain first cell pellet; c. re-suspending the cell pellet in the nutrient media;

d. seeding and passaging cells to achieve about 80% confluency, treating the cells with Trypsin-EDTA and cryo-preserving the treated cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA; and

e. optionally, adding pharmaceutically acceptable additives to obtain the composition.

In an embodiment of the present disclosure, the Master cell Bank and Working cell bank composition comprises about 1 million to about 3 million mesenchymal stem cells, Dimethyl Sulfoxide at a concentration ranging from about 8% to about 15%, preferably about 10%, Human Serum Albumin at a concentration ranging from about 2% to about 10%, preferably about 5%, and about 1 ml to 50 ml of multiple electrolyte isotonic solution in each cryopreserved vial.

In another embodiment of the present disclosure, the nutrient media of suspended working cell bank-cell pellet is changed after about 72 hours and subsequently after about 6 days. In yet another embodiment of the present disclosure, the wherein the human serum albumin is at a concentration ranging from about 1% to about 5%, preferably about 5%; the growth factors are selected from a group comprising Platelet Derived Growth Factor (PDGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 4 ng/ml; Epidermal Growth Factor (EGF) at a concentration ranging from about 1 ng/ml to about 5 ng/ml, preferably about 2 ng/ml; basic Fibroblast Growth Factor (bFGF) at a concentration ranging from about 1 to about 4 ng/ml, preferably about 2 ng/ml; Transforming Growth Factor- beta (TGF-beta) at a concentration ranging from about 1 ng/ml to about 4 ng/ml, preferably about 2 ng/ml; Insulin- like Growth Factor (IGF-1) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Hepatocyte Growth Factor (HGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Vascular Endothelial growth factor (VEGF) at a concentration ranging from about 1 ng/ml to about 100 ng/ml, preferably about 10 ng/ml; the platelet lysate is selected from blood group comprising AB and O and wherein the platelet lysate is with or without plasma, and is at a concentration ranging from about 5% to about 20%, preferably about 10%; the amino acids are selected from a group comprising L-ascorbic acid-2 phosphate at a concentration ranging from about 1 umol to about 100 umol; and the L-glutamine at a concentration ranging from about 100 mM to about 400 mM, preferably about 200 mM, or combination thereof; and the bioactive agents are selected from a group comprising insulin, selenite, transferrin, IL-3 and stem cell factors or any combination thereof.

In still another embodiment of the present disclosure, the pharmaceutically acceptable additives are selected from a group comprising heparin, penicillin and streptomycin.

The present disclosure relates to a method of inducing differentiation of mesenchymal stem cells towards endodermal lineages, said method comprising steps of:

a. isolating and culturing mesenchymal stem cells in a nutrient media;

b. treating the cultured cells with Trypsin-EDTA at a concentration ranging from about 0.1% to about 0.5%, preferably about 0.25% and washing the treated cells with the nutrient media to obtain embryoid body like clusters; and

c. inducing the embryoid body like clusters for differentiation of the cells towards the endodermal lineages.

In an embodiment of the present disclosure, the nutrient media comprises about 50% KO- DMEM and about 50% alpha MEM media, optionally supplemented with atleast one of the supplements selected from a group comprising Human Serum Albumin, growth factors, L- ascorbic acid-2 phosphate, L-glutamine and bioactive agents.

In another embodiment of the present disclosure, the bioactive agents are are selected from a group comprising insulin, selenite, transferrin, IL-3 and stem cell factors or any combination thereof.

In yet another embodiment of the present disclosure, the isolating is from mammalian umbilical cord, preferably Wharton's Jelly, by non-enzymatic means.

In still another embodiment of the present disclosure, the endodermal lineage is preferably pancreatic lineage, more preferably beta-pancreatic lineage.

In still another embodiment of the present disclosure, the treating is carried out after the cultured cells attain about 80% confluency. The present disclosure relates to a method of managing endocrine related disorders, said method comprising step of administering a composition comprising mesenchymal stem cells and active agents, to a subject in need thereof.

In an embodiment of the present disclosure, the active agents are selected from a group comprising multiple electrolyte isotonic solution, Human Serum Albumin, Dimethyl Sulphoxide or any combination thereof, optionally along with pharmaceutically acceptable additives.

In another embodiment of the present disclosure, the endocrine related disorder is selected from a group comprising pancreatic disorders, liver diseases and metabolic disorders. In yet another embodiment of the present disclosure, the composition is diluted with multiple electrolyte isotonic solution in a cryocyte bage and is administered parenterally.

The present disclosure relates to a kit comprising composition, and instructions for administering the composition for managing endocrine related disorders.

In an embodiment of the present disclosure, the composition comprise active agents selected from a group comprising multiple electrolyte isotonic solution, Human Serum

Albumin, Dimethyl Sulphoxide or any combination thereof, optionally along with pharmaceutically acceptable additives.

In another embodiment of the present disclosure, the pharmaceutically acceptable additives are selected from a group comprising heparin, penicillin and streptomycin.

The present disclosure relates to a method of obtaining and culturing Mesenchymal stem cells. These mesenchymal stem cells are multipotent stem cells and can be induced to differentiate into specific cell types. The present disclosure elaborates a unique method to obtain mesenchymal stem cells from mammalian umbilical cords. Particularly the cells are obtained from the Wharton's Jelly, present within the umbilical cord by non-enzymatic- mechanical means. These mesenchymal stem cells are cultured on a special nutrient media with bio-active agents. These methods are further elaborated in the example below, however the example should not be construed to limit the scope of the invention. The present disclosure not only relates to a method of obtaining the mesenchymal stem cells from the Wharton's Jelly but also elaborates on a method of culturing the same on specific media with bio-active agents which induce differentiation of the stem cells, towards endodermal lineages. Initially formation of cell aggregates is observed, followed by the formation of embryoid body like structures in low attachment tissue culture plate. These embryoid body like structures are further induced to differentiate into the endodermal lineage cells, specifically beta pancreatic lineage cells.

In an embodiment of the present disclosure, the obtained mesenchymal stem cells depict exclusive propensity or tendency towards endoderm lineages. The cultured mesenchymal stem cells from the Wharton's Jelly show high expression of pluripotent markers such as OCT-4, Nanog, Rex-1 etc. The mesenchymal stem cells cultured can be cryopreserved using the freezing media or by the method of slow freezing using crypreservatives like DMSO.

In an embodiment of the present disclosure, the composition comprising the mesenchymal stem cells along with active agents is formulated and administered for endocrinic disorders like pancreatic disorders, liver diseases and metabolic disorders. In another embodiment of the present disclosure fresh human umbilical cords (normal cesarean section) are obtained after birth. These are thereafter rinsed in normal saline and blood clots are cleared. The umbilical cord is cut into small pieces and rinsed with normal saline containing antibiotic-antimycotic and subsequently with DPBS to remove the traces of ethanol. The washed cord pieces are taken and are slit apart to expose the Wharton's Jelly. Further, the blood vessels are removed and the surrounding jelly is scraped off with a cell scraper. The Wharton's Jelly is collected in a separate falcon and subjected to centrifugation. The media changes are done regularly. The mesenchymal stem cells grow on the nutrient media and a fibroblast like morphology characteristic of mesenchymal stem cells are observed after continued culturing of the cells. In an embodiment of the present disclosure, the Wharton's Jelly derived mesenchymal stem cells are further analysed for various markers like the pluripotency markers, stromal specific markers etc. The present invention is further elaborated by the following examples and figures. However, these examples should not be construed to limit the scope of the invention.

Example 1:

ISOLATION AND CULTURE OF WJ-MSCS FROM WHARTON'S JELLY

Isolation of WJ-MSC

After getting consent and Ethics committee approval human umbilical cords (Normal/ Cesarean section) are obtained immediately after birth and brought to the laboratory. Umbilical cord is rinsed in sterile water at least 5-6 times and final two washings are given by the normal saline. Ethanol is sprayed on the cord so as to disinfect outer surface and then they are mobbed dry. All the blood clots are cleared off. Cord is cut into 2-3 cms pieces and rinsed again with normal saline containing Antibiotic-Antimycotic (IX) for 5-6 times and with DPBS for 4 times to remove the traces of ethanol. The washed cord pieces are taken on 10cm dish and are slit apart to open up the Wharton's jelly. All the blood vessels are removed. Wharton's jelly is then scrapped off using scrapper from one end to other (Fig-1). Wharton's jelly removed from the umbilical cord after scrapping the surface are then collected and suspended in nutrient medium consisting of about 50% KO-DMEM and about 50% alpha MEM media supplemented with about 1% to about 5% human serum albumin, about 1 ng/ml to about 10 ng/ml Platelet derived growth factor (PDGF), about 1 ng/ml to about 5 ng/ml Epidermal growth factor (EGF), about 1 ng/ml to about 4 ng/ml basic fibroblast growth factor (bFGF), about 1 ng/ml to about 4 ng/ml Transforming growth factor- beta (TGF-beta), about lng/ml to about 10 ng/ml Insulin-like growth factor (IGF-1), about 1 ng/ml to about 10 ng/ml Hepatocyte growth factor (HGF), about 1 ng/ml to about 100 ng/ml Vascular Endothelial growth factor (VEGF), about 1 umol to about 100 umol L-ascorbic acid-2 phosphate and about 200 mM L-glutamine. Mesenchymal stem cells are then fractionated by suspending Wharton's jelly cells in 50 ml of the nutrient media and allow it stand at room temperature for 5 minutes. Supernatant are removed leaving all the big tissue pieces at the bottom of the tubes. Place the supernatant in another 50 ml capacity tube containing media and again allow it stand at room temperature for 5 minutes. Thus, fractionation is done at least three times. Finally cells are removed by centrifugation at a speed of 1800 rpm for 20 minutes. The pellet contains pure population of mesenchymal stem cells from Wharton's jelly (Figure-1 A-L). Preparation of Master Cell Bank

The cells of the previous steps are collected and re-suspended in media consisting of about 50% KO-DMEM and about 50% alpha MEM media supplemented with about 1% to about 5% human serum albumin, about lng/ml to about lOng/ml Platelet derived growth factor (PDGF), about lng/ml to about 5ng/ml Epidermal growth factor (EGF), about lng/ml to about 4ng/ml basic fibroblast growth factor (bFGF), about lng/ml to about 4ng/ml Transforming growth factor- beta (TGF-beta), about lng/ml to about 10 ng/ml Insulin-like growth factor (IGF-1), about lng/ml to about lOng/ml Hepatocyte growth factor (HGF), about lng/ml to about lOOng/ml Vascular Endothelial growth factor (VEGF), about lumol to about 100 umol L-ascorbic acid-2 phosphate and about 200 mM L-glutamine. The cells are plated directly in the two 2- cell stack. The first media change is done after about 48 to about 72 hours and subsequently every week. After about 15 to about 18 days of culture or after the cells are about 80% confluent, the cells are removed with the help of trypsin- EDTA about 0.25% and cryopreserved as a master cell batch (MCB) at P-0 passage. Each frozen vial contain 3 million cells with about 10% dimethyl sulfoxide (DMSO), about 5% human serum albumin and Naclor or Denilyte-E or Plasmalyte-A. Naclor or Denilyte-E or Plasmalyte-A are Multiple Electrolyte Solutions Type I either IP grade or USP grade. The cells of master cell bank (MCB) are at P-0. Each MCB represent each umbilical cord. Similarly MCB is prepared from several cord and established individual cord seed bank.

Multiple electrolyte isotonic solution Type-I USP of each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C₆HnNa0₇); 368 mg of Sodium Acetate Trihydrate, USP (Γ₂Η₃^0₂·3Η₂0); 37 mg of Potassium Chloride, USP (KC1); and 30 mg of Magnesium Chloride, USP (MgCl₂ ^»6H₂0). The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

Preparation of Working Cell Bank

One vial of each MCB is thawed at 37 degree centigrade water bath and mixed together. Mixture of cells from minimum of 5 different cords is recommended in order to standardize the Investigational product and in order to avoid batch to batch inconsistency. Thawed cells are washed with basal media consisting of about 50% KO-DMEM and about 50% alpha MEM media supplemented with about 1% to about 5% human serum albumin, about lng/ml to about lOng/ml Platelet derived growth factor (PDGF), about 1 ng/ml to about 5ng/ml Epidermal growth factor (EGF), about lng/ml to about 4ng/ml basic fibroblast growth factor (bFGF), about lng/ml to about 4ng/ml Transforming growth factor- beta (TGF-beta), about lng/ml to about lOng/ml Insulin-like growth factor (IGF-1), about lng/ml to about lOng/ml Hepatocyte growth factor (HGF), about lng/ml to about lOOng/ml Vascular Endothelial growth factor (VEGF), about 1 umol to about 100 umol L- ascorbic acid-2 phosphate and about 200 mM L-glutamine and centrifuged at about 1800 rpm for about 10 minutes. Supernatant is removed and the cell pellate is resuspended again with basal nutrient media consisting of about 50% KO-DMEM and about 50% alpha MEM media supplemented with about 1% to about 5% human serum albumin, about lng/ml to about lOng/ml Platelet derived growth factor (PDGF), about 1 ng/ml to about 5ng/ml Epidermal growth factor (EGF), about lng/ml to about 4 ng/ml basic fibroblast growth factor (bFGF), about lng/ml to about 4ng/ml Transforming growth factor- beta (TGF- beta), about lng/ml to about lOng/ml Insulin-like growth factor (IGF-1), about lng/ml to about lOng/ml Hepatocyte growth factor (HGF), about lng/ml to about lOOng/ml Vascular Endothelial growth factor (VEGF), about lumol to about 100 umol L-ascorbic acid-2 phosphate and about 200 mM L-glutamine. The cells are seeded onto 5 cell stack at the seeding density of about 1000 cells per sq cm. 1^st media change is done after about 48 hours and subsequently every 5 days. Once the cells get confluent to about 80%, the cells are removed using trypsin-EDTA about 0.25% and cryopreserved as a working cell batch (MCB) at P-l passage. Each frozen vial contain about 3 million cells with about 10% dimethyl sulfoxide (DMSO), about 5% human serum albumin, about 10% human platelet lysate, plasma free human platelet lysate and bioactive agents suspended in IX Naclor or Denilyte-E or Plasmalyte-A, B or C. The bioactive agents include IGF-I, EGF, PDGF, bFGF, VEGF, insulin, selenite, transferring, IL-3 and stem cell factors. The cells of working cell bank (WCB) are at P-l . Each WCB represent mixture of cells from five different umbilical cords.

In master cell bank and working cell bank, cells at concentration of about 1 to about 3 million cells are cryopreserved, as these are the major stock banks for the production. Master cell bank is usually individual healthy donor cells and they are stocked at the concentration of about 1 to about 3 million cells per vial. In working cell bank, mix 3-4 vials of master cell bank of various donors are mixed in order to avoid inconsistency of individual donor factor. About three-four donors are mixed. Hence working cells banks have mixed population of stem cells of 3-4 donors. This is also cryopreserved at the rate of about 1 to about 3 million cells per vial. From this working cell bank, individual vials are taken for production. Since this directly goes for human infusion, cells are cryopreserved at concentration of about 50 to about 100 million cells in cryocyte bags of about 50 ml total suspension. Cryopreservation is done in about 15 ml suspension containing about 100 million cells. After thawing, about 35 ml of multiple electrolyte isotonic solution is added so that DMSO is diluted from about 10% to about 3.5%. This entire 50 ml suspension is ready for infusion.

In an embodiment, the master cell bank and working cell bank are cryopreserved at the concentration of 3 million cells in vial, each vial containing 10% DMSO, 5% human serum albumin and 1 ml of plasmalyte-A. Each vial is then kept in programmable freezer (kryo- 10 from planner inc) and frozen at the rate of -l°c per minute till -80°c and finally stored in liquid nitrogen having temperature -196°c. For thawing each vial is rapidly thawed at 37°c water bath and diluted with media and centrifuged in order to remove DMSO. After washing the cells at least 2 times the cells are seeded for culturing. Preparation of Investigational product Production

One vial of WCB is removed from liquid nitrogen container and thawed at about 37 degree centigrade in a water bath. The cells are washed with basal media consisting of about 50% KO-DMEM and about 50% alpha MEM media supplemented with about 1% to about 5% human serum albumin, about lng/ml to about lOng/ml Platelet derived growth factor (PDGF), about lng/ml to about 5ng/ml Epidermal growth factor (EGF), about lng/ml to about 4ng/ml basic fibroblast growth factor (bFGF), about lng/ml to about 4ng/ml Transforming growth factor- beta (TGF-beta), about lng/ml to about 10 ng/ml Insulin-like growth factor (IGF-1), about lng/ml to about lOng/ml Hepatocyte growth factor (HGF), about lng/ml to about lOOng/ml Vascular Endothelial growth factor (VEGF), about lumol to about lOOumol L-ascorbic acid-2 phosphate and about 200 mM L-glutamine and centrifuged at about 1800 rpm for about 10 minutes. Supernatant is removed and the cell pellate is resuspended again with basal nutrient media consisting of about 50% KO- DMEM and about 50% alpha MEM media supplemented with about 1% to about 5% human serum albumin, about lng/ml to about lOng/ml Platelet derived growth factor (PDGF), about lng/ml to about 5ng/ml Epidermal growth factor (EGF), about lng/ml to about 4ng/ml basic fibroblast growth factor (bFGF), about lng/ml to about 4ng/ml Transforming growth factor- beta (TGF-beta), about lng/ml to aboutlO ng/ml Insulin-like growth factor (IGF-1), about lng/ml to about lOng/ml Hepatocyte growth factor (HGF), about lng/ml to about lOOng/ml Vascular Endothelial growth factor (VEGF), about lumol to aboutlOO umol L-ascorbic acid-2 phosphate and about 200 mM L-glutamine. The cells are seeded onto 10 cell stack at the seeding density of about 1000 cells per sq cm. 1^st media change is done after about 72 hours and subsequently every 6 days. Once the cells get confluent to about 80%, the cells are removed using trypsin-EDTA about 0.25% and washed three times with Naclor or Denilyte-E IX or plasmalyte A, B and C. Cell pellate obtained is re-suspended with IX Naclor or Denilyte-E or Plasmalyte A and used as investigational product or stem cell product for clinical trial or stem cell therapy. In an embodiment of the present disclosure, a composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution Type-I USP selected from a group comprising Naclor, Denilyte-E or Plasmalyte-A, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives. The pharmaceutically acceptable additives are heparin, pencillin and streptomycin. The heparin is Sodium heparin IP grade heparin.

In an embodiment of the instant disclosure, the investigational product is frozen and contains 100 million cells. Each frozen product contains 100 million isolated MSC, 13.5 ml plasmalyte-A, 5% human serum albumin and 10% dimythyl sulphoxide (DMSO). This product after thawing needs to be diluted with 35 ml plasmalyte-A so that final volume will be 50 ml. Hence, the final concentration of 1% human serum albumin and 3.5% DMSO is obtained. Conclusion: The method of isolation of umbilical cord mesenchymal stem cells is unique, novel, easy and free from enzymatic digestion. Method of isolation and culture is also free from animal source material and eligible for therapeutic use. Method of isolation and culture also do not cross more than passage 2 for clinical purpose and hence extremely safe and avoid risk of karyotypic abnormality and tumor transformation.

Example 2:

Cryop reservation of WJ-MSCs from Wharton's Jelly as an Investigational Product

The mesenchymal stem cells isolated from Wharton's Jelly, using the method as described in Example- 1 is further cryo-preserved using cryopreservatives. Maximum 100 million of the mesenchymal stem cells derived from Wharton's Jelly are suspended slowly in about 15 ml of IX Naclor or Denilyte-E or Plasmalyte-A, B and C containing about 10% DMSO and about 5% human serum albumin and then placed in about 50 ml of cryocyte bag (Baxter Inc). Cryocyte bag is then placed in programmable freezer (Planner Inc) and gradually frozen at the rate of -1°C per minute until the temperature reaches at -7°C. Thereafter temperature is reduced at the rate of -0.5°C per minute until -70°C. Once the temperature reaches at -70° C, the cryocyte bag is removed from the programmable freezer and store it liquid nitrogen container for long-term storage.

Conclusion: The method of cryopreservation is a unique method and can be directly used for therapeutic purposes and as an "off the shelf product, free from animal serum.

Example 3:

Thawing of WJ-MSCs from Wharton's Jelly as an Investigational Product for clinical trial or therapy

Cryocyte bag containing frozen mesenchymal stem cells is removed from the liquid nitrogen and immediately thawed at about 37°C clean water bath. Slowly about 35 ml of IX Plasmalyte A is added to the cryocyte bag (already containing about 15 ml of IX Naclor or Denilyte-E or Plasmalyte A, about 10% DMSO and about 5% human serum albumin) in order to make total volume about 50 ml. Addition of about 35 ml of IX Naclor or Denilyte-E or Plasmalyte as a diluent in the bag will ensure the dilution of about 10% DMSO to around about 4%, which is recommended by the US-FDA. This 50 ml of investigational product of mesenchymal stem cell derived from Wharton's Jelly is now ready for infusion intravenously.

Conclusion: The method of cryopreservation and thawing is a unique method and can be directly used for therapeutic purposes and as an "off the shelf product and can be directly used without exposing to the environment and need of further processing.

Example 4:

Morphological analysis of Wharton's Jelly derived Mesenchymal stem Cells

The Wharton's Jelly derived mesenchymal stem cells when cultured in basal medium comprising 50% KO-DMEM and 50% alpha-MEM medium, maintain the typical morphology (Figure 2). The MSC maintained the typical spindle-shaped morphology. Population doubling (PD) time of MSC was 18.7 ± 1.12 hours.

Conclusion: The isolated umbilical cord mesenchymal stem cells shows homogeneous stromal cell population.

Example 5:

Immunophenotyping of the isolated WJ-MSCs using stromal and stem cells specific markers

The surface markers of Wharton's Jelly derived mesenchymal stem cells are analyzed by FACS antibodies after dissociation. Cells are stained with fluorescein or phycoerythrin coupled antibodies, including Clustered of Differentiation, CD90, CD73, CD166, CD45, CD34 (all antibodies purchased from Becton-Dickinson, San Jose, CA, USA). Stained samples and unstained control cells are analyzed with Guava EasyCyte 5HT (Millipore, USA). For nuclear antibodies, cells are permeabilized and analysed (Figure 3 A-B). Immunophenotyping of the WJ-MSCs shows high expression of stromal specific markers specific for MSCs and negligible expression of endothelial and hematopoietic markers, which is a characteristic of pure MSCs population (Figure 3 C-H). Conclusion: The expression of CD markers confirm that the cells isolated from umbilical cord are truly mesenchymal stem cells

Example 6:

Comparison of Real time PCR analysis of mesenchymal stem cells obtained from various sources

Pluripotent markers such as SSEA-4, OCT-4, SOX-2 by immunofluorescence microscopy (Figure- 4 A-C) and Real time PCR analysis from mesenchymal stem cells isolated from various sources are carried out (Figure 4 D-E). The mesenchymal stem cells for this analysis are obtained from 4 different sources, namely Bone Marrow, Dental Pulp, Wharton's Jelly and Adipose Tissue. To carry out the analysis pre-designed Assays on Demand TaqMan probes and primers are procured from Applied BioSystems. qRT-PCR analysis is conducted using ABI PRISM 7900 Sequence Detection System (Applied BioSystems). After an initial denaturation for about 10 mins at about 95°C, the reaction was run for about 40 cycles of PCR (about 95°C for about 15 sec, about 60°C for about 1 min). Changes in gene expression (in triplicates) are normalized to 18S rRNA levels in terms of fold change. Figure 4 showed high expression of pluripotent markers (Oct 4 and Sox 2) in Wharton's jelly derived mesenchymal stem cells as compared to bone marrow and dental pulp derived mesenchymal stem cells. However, the expression is 100 times lower than human embryonic stem cells, suggesting that the cells are non-tumorogenic. The overall results show the presence of high pluripotency in WJ-MSCs indicating that they are better than other MSC derived from other sources.

On comparing the detail transcriptome profiling of mesenchymal stem cells derived from Wharton's Jelly and Bone Marrow using DNA microarray with the panel of known pluripotent markers and other markers. It shows that transcriptome expression in Wharton's Jelly MSCs are different from Bone Marrow MSC. Most of high expressions are from endodermal lineages and pancreatic lineages (Figure-5). Conclusion: The gene expression from different MSc sources reflects their tissue of origin, indicating that the MSc tissue heterogenety is biologically relevant. Thus different tissue source may generate MSc products with different cytokine expression. Hence, different MSc source may be especially suited for specific clinical applications. Example 8:

Immunohistochemical staining of WJ-MSCs

Cultures are permeabilized with 0.1% Triton X-100 (Gibco) after fixation with 4% Paraformaldehyde. Rabbit anti -human OCT-4 (Abeam, USA) and mouse anti -human Nestin (Chemicon, USA) are used as primary antibody (1 :200) at room temperature for about 30 min. Green - Fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG (Abeam, USA) and fluorescence conjugated goat anti-mouse IgG (Chemicon, USA) are applied as second antibody. Figure 4 A-C shows high expression of pluripotent marker SSEA-4, OCT 4, SOX-2 and ectodermal marker (nestin) in the Wharton's Jelly derived Mesenchymal Stem Cells.

Conclusion: Immunohistochemical staining of Wharton's Jelly derived mesenchymal stem cells demonstrates high expression of Pluripotent markers (OCT-4) which is in congruent with the real time data. The expression ectodermal marker Nestin is also very high in Wharton's Jelly derived mesenchymal stem cells. For mesenchymal stem cells to differentiate to pancreatic endodermal cells it is imperative to have a high Nestin positive population.

Example 9:

Morphological evaluation of embryoid body-like clusters (EB-LC) from WJ-MSCs

Mesenchymal stem cells are isolated and cultured from Wharton's jelly till it reaches about 80% confiuency. Cells are dissociated by the trypsin-EDTA about 0.25% and wash two times with basal media. Dissociated cells are then seeded with a density of 25000 - 40000 cells/cm2 in a ultra low attachment 60mm bacteriocidal low adherent culture dish. Normal Mesenchymal stem cells media which is comprised of basal media DMEM-KO with 10% FBS is used for the formation and culture of embryoid body like clusters. (Note: No additional growth factors are added in the media). Media change is done every 3 to 4 days. The Embryoid body like clusters (EB-LC) are formed 1 to 2 days after seeding and can then be collected after desired time intervals. Specifically, EBs were collected on day 4, 8, 12, 16, 18 and 20 days. For induction of embryoid bodies (EB) formation, the WJ-MSCs are seeded on ultra low attachment 60mm bacteriological petri dishes (Nunc) in DMEM- KO media supplemented with 10% FBS. The EBs formed is screened for their typical round morphology under an inverted microscope (NIKON). The morphological images can be seen in figure 7. Embryoid bodies also show pancreatic bud formation after about 8 days, as usually seen in human embryonic stem cells developed embryoid bodies Figure-8. Conclusion: The embryoid body like clusters are observed after 1 to 2 days after seeding the Wharton's Jelly derived mesenchymal stem cells on a low adherent culture dish. This shows that Mesenchymal stem cells derived from Wharton's Jelly is close to human embryonic stem cells and are more primitive than other mesenchymal stem cells yet they are not tumorogenic and safe for clinical purpose. Mesenchymal stem cells derived from other sources did not show any formation of embryoid body like structure.

Example 10:

Gene expression profiling of WJ-MSCs towards the endoderm lineage during different days in culture of differentiation, using endoderm specific markers

Normal endoderm lineage pathways towards pancreatic beta islet cell gene expression profile are given in Figure-6. Cells are rinsed once with ice cold PBS. Cells are lysed directly by adding about 1 ml of TRIZOL Reagent. Pass the cell lysate several times through a pipette and vortex thoroughly. The amount of TRIZOL reagent added is based on the number of cells. An insufficient amount of TRIZOL Reagent may result in DNA contamination of the isolated RNA. About 0.2 ml of chloroform is added per 1 ml of TRIZOL and mixed thoroughly. Tube is centrifuged and two phases separated out. Lower layer is phenol-chloroform phase, middle as interphase and top as clear aqueous phase. RNA is present in aqueous phase. RNA is precipitated by adding isopropyl alcohol. Total RNA from cells is isolated. Complementary DNA is synthesized using Superscript II First Strand Synthesis system (IN VITRO GEN).. PCR is carried out using IU Taq DNA Polymerase (Sigma) and MgCl₂ to a final concentration of 1.5 mM in a total volume of 25 μΐ /reaction. GAPDH is used as the housekeeping control. PCR cycles consisted of initial denaturation at 95°C for 5 minutes followed by 35 amplification cycles of denaturation at 94°C for 45 seconds, annealing for 45 seconds, and extension at 72°C for 45 seconds and final extension at 72°C for 10 minutes. Results from Figure 9 and Figure- 10 show that the expression of early endodermal marker (AFP) is absent throughout except for day 4 EB-LC and the expression of late endodermal marker (Albumin) is absent throughout all the time points. There is a consistent expression of BMP4 throughout all the time points. Faint expression of HNF 3β and PDX 1 is observed in MSCs and early day point EB-LC. But prominent expression is observed from day 8 WJ derived EB-LCs. Differentiated cells also show expression of Insulin and Somatostatin (Figure-11) and sequencing insulin gene fragment shows correct gene sequence on conformation (Figure- 12).

Conclusion: The gene expression data of Wharton's Jelly derived mesenchymal stem cells differentiation and Wharton's Jelly derived embryoid body like clusters demonstrates high expression of endoderm markers. This suggests that only Wharton's Jelly derived mesenchymal stem cells has propensity towards endoderm lineage and especially towards pancreatic beta cell and hence may be beneficial for diabetes. This also suggests that Mesenchymal stem cells derived from other source may have different propensity character and may be beneficial for other diseases. Example 11:

Real time PCR analysis of WJ-MSCs at different days in culture with markers indicative of pancreatic differentiation

Pre-designed Assays on Demand TaqMan^® probes and primers are procured from Applied BioSystems. qRT-PCR analysis is conducted using ABI PRISM 7900 Sequence Detection System (Applied BioSystems). After an initial denaturation for 10 mins at 95°C, the reaction is run for 40 cycles of PCR (95°C for 15 sec, 60°C for 1 min). Changes in gene expression (in triplicates) are normalized to 18S rR A levels in terms of fold change. See figure 13 for results. NGN3 (neurogenin 3): The expression of NGN3 is significantly increased in the wharton's jelly derived Embryoid bodies. Day 16 Embryoid bodies show reduced NGN3 expression. Very low expression of NGN3 is observed in Wharton Jelly and Bone Marrow derived MSCs. SOX 17: The expression of Sox 17 is significantly higher in Wharton's Jelly Embryoid bodies compared to Wharton's Jelly MSc and BM MScs. Day 16 Embryoid bodies show reduced Sox 17 expression. GLUT 2: There is high expression of GLUT 2 observed in EB-LC on day 20 whereas there is consistent expression on other day points. The expression in BM and WJ-MSCs is low when compared to the EB-LCs at different day points

GLUT 1: There is high expression of GLUT 2 observed in WJ-MSCs compared with EB- LCs derived from WJ-MSCs

Conclusion: Wharton's Jelly derived embryoid bodies show increased expression of pancreatic beta markers compared to bone marrow derived mesenchymal stem cells.

Example 12:

Immunophenotyping of WJ-MSCs at P2 using undifferentiated markers and markers indicative of endoderm specific differentiation

The surface markers of WJ-MSCs are analysed by FACS antibodies after dissociation. Cells are stained with fluorescein or phycoerythrin coupled antibodies, (all antibodies purchased from Becton-Dickinson, San Jose, CA, USA). Stained samples and unstained control cells are analyzed with Guava EasyCyte 5HT (Millipore, USA). For nuclear antibodies, cells are permeabilized and analysed. Figure 8 indicates that there is a negligible expression of mature markers like PDX 1, Sox 17 and c-peptide in undifferentiated WJ-MSCs. Low and moderate expressions of NGN3 and Glut 2 is observed. There is a very high expression of ectodermal marker Isl 1 (Islet 1) Figure-14.

Conclusion: Wharton's Jelly derived mesenchymal stem cells show high expression of ISL1 which is a terminally differentiated marker in the differential pathways as shown in figure 6. Similarly Wharton's Jelly derived mesenchymal stem cells have propensity to differentiate into pancreatic beta islet cells and hence showed significantly high expression of PDX1 , SOX- 17, Glut-2, in matured differentiated cells but negligible to moderate expression in undifferentiated cells. This shows that Wharton's Jelly cells has highest propensity towards pancreatic beta islet cells.

Wharton's Jelly derived mesenchymal stem cells show negligible expression for PDX1 , Sox 17, and cPeptide, partial expression for GLUT 2, and very high expression for the ectodermal marker Isl 1. Very small percentage population is positive for NGN3.

Example 13:

A pre-clinical study on diabetic animals is undertaken in the instant example. Sprague- dawley rats are injected with streptozotocin at the dose of about 140 mg/kg body weight intraperitonially. Blood glucose is monitored every week and once blood glucose level reachs the level of about 300 mg/dl, MSC derived from umbilical cord is injected intravenously at the dose of 4 million cells per kg body weight. 50% animals are injected saline intravenously as placebo control. Diabetic model injected with MSC showed reduction in glucose level from about 300 mg/dl to about 80 mg/dl in about 3 weeks time and also showed regeneration of pancreatic cells in histology sections. Whereas in saline control the destruction of pancreatic cells is observed along with the level of glucose reaching about 400 mg/dl.

Conclusion: Infusion of MSC intravenously not only decreases the glucose level to the normal level but also regenerate pancreatic cells within 3 weeks. For clinical trial in human therefore intravenous route may be an appropriate route of infusion for diabetic treatment.

Advantages

Some of the advantages of the instant disclosure are:

1. The viability of cells is increased as the enzymatic digestion (using collagenase and trypsin) step is eliminated from the isolation protocol.

2. Since the viability is more, the yield is also increased significantly. Wharton's jelly has maximum number of stem cells through out the umbilical cord. Since only the jelly portion of the cord is being taken, we get pure population of MSCs at P0 unlike the other isolation protocols.

The time taken for the isolation of stem cells from umbilical cord by this method is considerably reduced.

This protocol is less expensive and more economical. (The cost of collagenase and trypsin is eliminated).

Claims

We claim:

1. A method of obtaining mesenchymal stem cells from mammalian umbilical cord, said method comprising step of isolating the stem cells by non-enzymatic means to obtain the isolated mesenchymal stem cells.

2. The method as claimed in claim 1, wherein the mesenchymal stem cells are obtained from Wharton's Jelly within the mammalian umbilical cord.

3. The method as claimed in claim 1, wherein the non-enzymatic means are mechanical in nature, preferably scrapping the stem cells from surface; followed by density fractionation.

4. A media comprising about 50% KO-DMEM and about 50% alpha MEM media, optionally supplemented with at least one of the supplements selected from a group comprising human serum albumin, growth factors, platelet lysate, amino acids and bioactive agents.

5. The media as claimed in claim 4, wherein the human serum albumin is at a concentration ranging from about 1% to about 10%, preferably about 5%; the growth factors are selected from a group comprising Platelet Derived Growth Factor (PDGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 4 ng/ml; Epidermal Growth Factor (EGF) at a concentration ranging from about 1 ng/ml to about 5 ng/ml, preferably about 2 ng/ml; basic Fibroblast Growth Factor (bFGF) at a concentration ranging from about 1 to about 4 ng/ml, preferably about 2 ng/ml; Transforming Growth Factor- beta (TGF-beta) at a concentration ranging from about 1 ng/ml to about 4 ng/ml, preferably about 2 ng /ml; Insulin-like Growth Factor (IGF-1) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Hepatocyte Growth Factor (HGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Vascular Endothelial growth factor (VEGF) at a concentration ranging from about 1 ng/ml to about 100 ng/ml, preferably about 10 ng/ml; the platelet lysate is selected from blood group comprising AB and O and wherein the platelet lysate is with or without plasma, and is at a concentration ranging from about 5% to about 20%, preferably about 10%; the amino acids are selected from a group comprising L-ascorbic acid-2 phosphate at a concentration ranging from about 1 umol to about 100 umol; and L-glutamine at a concentration ranging from about 100 mM to about 400 mM, preferably about 200 mM, or combination thereof; and the bioactive agents are selected from a group comprising insulin, selenite, transferring, IL-3 and stem cell factors or any combination thereof.

6. The media as claimed in claim 4, where in the media is a serum free or a xeno-free media or a combination thereof.

7. A composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives.

8. The composition as claimed in claim 7, wherein the composition has about 1 million to about 100 million mesenchymal stem cells, Dimethyl Sulfoxide at a concentration ranging from about 8% to about 15%, preferably about 10%, Human Serum Albumin at a concentration ranging from about 2% to about 10%, preferably about 5%, and the multiple electrolyte isotonic solution is at a volume ranging from about 10 ml to about 40 ml, preferably about 13.5 ml.

9. A method of preparing a Master Cell Bank composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA), Dimethyl Sulphoxide (DMSO), optionally along with pharmaceutically acceptable additives, said method comprising steps of:

a. isolating and culturing the mesenchymal stem cells in nutrient media comprising about 50% Knock Out-Dulbecco's Minimal Essential Medium (KO-DMEM) and about 50% alpha Minimal Essential Medium (MEM) supplemented with the HSA, growth factors and amino acids and fractionating the culture to obtain fractionated cells;

b. centrifuging the fractionated cells to obtain a pellet comprising purified cells and suspending the purified cells in the nutrient media;

c. culturing the cells in the nutrient media to achieve about 80% confluency and treating the cells with Trypsin-EDTA and cryo-preserving the treated stem cells with the multiple electrolyte isotonic solution along with the DMSO and the HSA; and

10. The method as claimed in claim 9, wherein the mesenchymal stem cells are isolated by non-enzymatic means, preferably mechanical isolation, more preferably by scrapping the stem cells from inner tissue surface of umbilical cord, removing Wharton's Jelly.

11. The method as claimed in claim 9, wherein the fractionating is density fractionating to obtain pure population of stem cells and comprises steps of suspending, manually density separating and centrifuging the separated stem cells in about 50 ml of the nutrient media, at room temperature for a time duration ranging from about 5 minutes to about 20 minutes, preferably about 10 minutes.

12. The method as claimed in claim 9, wherein the centrifuging is carried out at about 800 rpm to about 1800 rpm, preferably about 1200 rpm, at room temperature for time duration ranging from about 5 minutes to about 20 minutes, preferably about 10 minutes.

13. The method as claimed in claim 9, wherein the nutrient media of the purified stem cells is changed after about 48 hours to about 72 hours, and subsequently after about 7 days, and wherein cells are removed by the treating after about 15 days to about 18 days of culturing or after the cells attain about 80% confluency; and the Trypsin-EDTA has a concentration of about 0.25%.

14. A method of preparing a Working Cell Bank composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA) and Dimethyl Sulphoxide (DMSO) optionally along with pharmaceutically acceptable additives said method comprising steps of:

a. preparing a Master Cell Bank composition according to claims 9 to 13; b. thawing and washing the Master Cell Bank stem cells with nutrient media comprising about 50% KO-DMEM and about 50% alpha MEM media supplemented with the HSA, growth factors and amino acids, and centrifuging the washed stem cells to obtain a cell pellet;

c. re-suspending the cell pellet in the nutrient media;

15. The method as claimed in claim 14, wherein the nutrient media of the cell pellet is changed after about 48 hours and subsequently after about every 5 days and wherein the treating is done after the cells attain about 80% confluency, and the Trypsin-EDTA is having a concentration of about 0.25%.

16. A method of preparing a composition comprising Mesenchymal stem cells, multiple electrolyte isotonic solution, Human Serum Albumin (HSA) and Dimethyl

Sulphoxide (DMSO) optionally along with pharmaceutically acceptable additives , said method comprising steps of:

a. preparing a Working Cell Bank composition according to claims 14 to 16; b. thawing and washing of the Working Cell Bank composition with nutrient media comprising about 50% KO-DMEM and about 50% alpha MEM media supplemented with the HSA, the growth factors and the amino acids, and centrifuging the washed stem cells to obtain first cell pellet; c. re-suspending the cell pellet in the nutrient media;

17. The method as claimed in claims 9, 14 and 16, wherein the Master cell Bank and Working cell bank composition comprises about 1 million to about 3 million mesenchymal stem cells, Dimethyl Sulfoxide at a concentration ranging from about 8% to about 15%, preferably about 10%, Human Serum Albumin at a concentration ranging from about 2% to about 10%, preferably about 5%, and about 1 ml to 50 ml of multiple electrolyte isotonic solution in each cryopreserved vial.

18. The method as claimed in claim 16, wherein the nutrient media of suspended working cell bank-cell pellet is changed after about 72 hours and subsequently after about 6 days.

19. The methods as claimed in claims 9, 14 and 16, wherein the wherein the human serum albumin is at a concentration ranging from about 1% to about 5%, preferably about 5%; the growth factors are selected from a group comprising Platelet Derived Growth Factor (PDGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 4 ng/ml; Epidermal Growth Factor (EGF) at a concentration ranging from about 1 ng/ml to about 5 ng/ml, preferably about 2 ng/ml; basic Fibroblast Growth Factor (bFGF) at a concentration ranging from about 1 to about 4 ng/ml, preferably about 2 ng/ml; Transforming Growth Factor- beta (TGF-beta) at a concentration ranging from about 1 ng/ml to about 4 ng/ml, preferably about 2 ng/ml; Insulin-like Growth Factor (IGF-1) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Hepatocyte Growth Factor (HGF) at a concentration ranging from about 1 ng/ml to about 10 ng/ml, preferably about 2 ng/ml; Vascular Endothelial growth factor (VEGF) at a concentration ranging from about 1 ng/ml to about 100 ng/ml, preferably about 10 ng/ml; the platelet lysate is selected from blood group comprising AB and O and wherein the platelet lysate is with or without plasma, and is at a concentration ranging from about 5% to about 20%, preferably about 10%; the amino acids are selected from a group comprising L-ascorbic acid-2 phosphate at a concentration ranging from about 1 umol to about 100 umol; and the L- glutamine at a concentration ranging from about 100 mM to about 400 mM, preferably about 200 mM, or combination thereof; and the bioactive agents are selected from a group comprising insulin, selenite, transferrin, IL-3 and stem cell factors or any combination thereof.

20. The compositions as claimed in claim 7, and the methods as claimed in claims 9, 14 and 16, wherein the pharmaceutically acceptable additives are selected from a group comprising heparin, penicillin and streptomycin.

21. A method of inducing differentiation of mesenchymal stem cells towards endodermal lineages, said method comprising steps of

a. isolating and culturing mesenchymal stem cells in a nutrient media;

22. The method as claimed in claim 21, wherein the nutrient media comprises about 50%) KO-DMEM and about 50% alpha MEM media, optionally supplemented with atleast one of the supplements selected from a group comprising Human Serum Albumin, growth factors, L-ascorbic acid-2 phosphate, L-glutamine and bioactive agents.

23. The method as claimed in claim 21 , wherein the bioactive agents are are selected from a group comprising insulin, selenite, transferrin, IL-3 and stem cell factors or any combination thereof.

24. The method as claimed in claim 21, wherein the isolating is from mammalian umbilical cord, preferably Wharton's Jelly, by non-enzymatic means.

25. The method as claimed in claim 21 , wherein the endodermal lineage is preferably pancreatic lineage, more preferably beta-pancreatic lineage.

26. The method as claimed in claim 21, wherein the treating is carried out after the cultured cells attain about 80% confluency.

27. A method of managing endocrine related disorders, said method comprising step of administering a composition comprising mesenchymal stem cells and active agents, to a subject in need thereof.

28. The method as claimed in claim 27, wherein the active agents are selected from a group comprising multiple electrolyte isotonic solution, Human Serum Albumin, Dimethyl Sulphoxide or any combination thereof, optionally along with pharmaceutically acceptable additives.

29. The method as claimed in claim 27, wherein the endocrine related disorder is selected from a group comprising pancreatic disorders, liver diseases and metabolic disorders.

30. The method as claimed in claim 27, wherein the composition is diluted with multiple electrolyte isotonic solution in a cryocyte bage and is administered parenterally.

31. A Kit comprising composition of claim 7, and instructions for administering the composition for managing endocrine related disorders.

32. The kit as claimed in claim 31 , wherein the composition comprise active agents selected from a group comprising multiple electrolyte isotonic solution, Human Serum Albumin, Dimethyl Sulphoxide or any combination thereof, optionally along with pharmaceutically acceptable additives.

33. The method as claimed in claim 27 and the kit as claimed in claim 31, wherein the pharmaceutically acceptable additives are selected from a group comprising heparin, penicillin and streptomycin.