WO2012117333A1

WO2012117333A1 - Isolation and expansion of adult stem cells, their therapeutic composition and uses thereof

Info

Publication number: WO2012117333A1
Application number: PCT/IB2012/050893
Authority: WO
Inventors: Vijayendran GOVINDASAMY; Ramesh R BHONDE; Satish Totey; Anjan Kumar DAS
Original assignee: Stempeutics Research Malaysia Sdn Bhd
Priority date: 2011-02-28
Filing date: 2012-02-27
Publication date: 2012-09-07
Also published as: MY177150A

Abstract

The present invention discloses highly reproducible and consistent method for large scale production of high quality clinical-grade DPSCs in a short time. The present method of DPSC production is economical and produces cells in commercial quantities for use in autologous and allogeneic transplantation therapy. It also discloses a transport medium composition for preservation and protection of the dental pulp. It further discloses a method for producing islet-like cell aggregates (ICAs) from DPSC using a 3 step protocol and using specific differentiation composition. The ICAs obtained is used as pharmaceutical composition for treating type-I diabetic conditions and also for screening novel anti-diabetic compounds.

Description

ISOLATION AND EXPANSION OF ADULT STEM CELLS, THEIR THERAPEUTIC COMPOSITION AND USES THEREOF"

TECHNICAL FIELD

The present disclosure relates to the field of stem cells in general, while in particular it relates to the isolation, culturing, expansion and characterization of adult stem cells from dental pulp. Further, it also discloses a method of generation of islet like cell aggregates and its clinical applications. BACKGROUND

Stem cells are generally defined as clonogenic cells capable of both self renewal and multi-lineage differentiation. Post-natal stem cells have been isolated from various tissues, including bone marrow, neural tissue, skin, retina, and dental epithelium. Recently, a population of putative post-natal stem cells in human dental pulp called dental pulp stem cells (DPSCs) has been reported.

Dental Pulp Stem Cells or (DPSCs) are pluripotent stem cells that have the potential to differentiate into a variety of cell types. Human tissues are different in terms of their regenerative properties. Stem cells are a promising tool for tissue regeneration, because of their characteristics of proliferation, differentiation and plasticity. DPSCs have extensive differentiation ability and several studies have been performed on DPSCs in which it was reported that these cells are multipotent stromal cells and they can be safely cryopreserved. Dental pulp is an unlimited source of human mesenchymal stromal/stem cells (MSC) for cell replacement/regeneration therapy because of their self-renewal and multi-lineage differentiation potential. Dental pulp can be isolated from deciduous and permanent teeth. However, optimal culture conditions for their long-term expansion and proliferation are necessary since large numbers of stem cells are required for therapeutic/clinical applications. The present invention provides a method for large scale expansion of high quality clinical grade DPSCs, more economical, in shorter time line compared to existing processing technology. BRIEF DESCRIPTION OF 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 Morphology, growth kinetics, and senescence in DPSCs cultured across different media at early and late passage. (A) Phase contrast microscope, 10x of DPSCs cultured in various growth media; (B) Long-term growth curves of DPSCs cultured in various growth media; (C) Senescence associated β-galactosidase (SA-P-gal) staining of DPSCs cultured in various growth media. (Arrow shows positive cells); (D) Quantification of percentage (%) of S A-pi-gal positive cells cultured in various growth media. The results represent average of four culture replicates with SD. A representative photomicrograph is given for each experiment. *p<0.05, **p<0.01 , and ***p<0.001. Abbreviations: DPSCs -Dental pulp stem cells,

SD standard deviation.

Figure 2 Effects of cell density on the proliferation of DPSCs. (A) Cell population and (B) population doubling time in hours±SD of DPSCs cultured until passage 3.

Figure 3A shows the quantification of percentage (%) of SA- -gal positive cells cultured in various growth media with ascorbic acid and without adding ascorbic acid and Figure 3B shows the immunophenotype analysis of DPSCs cultured across different media with or without ascorbic acid at early and late passage. DPSCs are tested against human antigens CD34, CD44, CD45, CD73, CD90, CD166, and HLA-DR. 7-AAD is used to check the viability of the cells.

(A) Cells cultured in ct-MEM (early and late passage);

(B) Cells cultured in a- MEM +ascorbic acid (early and late passage);

(C) Cells cultured in DMEM-KO (early and late passage);

(D) Cells cultured in DMEM-KO + ascorbic acid (early and late passage);

(E) Cells cultured in DMEM-LG (early passage),

(F) Cells cultured in DMEM-F12 + ascorbic acid (early and late passage)

Results are representative of three independent experiments,

Abbreviations: DPSCs - Dental pulp stem cells

CD - cluster of differentiations

7-AAD 7-amino-actinomycin D

HLA- DR- Human Leukocyte Antigen -DR Figure 4 Shows the expression profile of pluripotent and lineage-specific stem cell markers of SCD and DPSCs.

Abbreviations: DPSCs- Dental pulp stem cells

SCD-stem cells derived from deciduous teeth Figure 5 Shows the schematic overview of the generation of neurospheres in SCD and DPSCs and gene expression profile of selected neuron markers.

Figure 6 shows the- morphology of DPSCs cultured in different media compositions. It shows cell morphology of DPSCs expanded in the presence of HPL are smaller spindle - shaped cells than FBS (A & B). (Al , A2) Phase-contrast microscope, 10x of DPSCs expanded in HPL and FBS, respectively. While Fig (C, D) show the DPSCs expanded in HPL and FBS, respectively. Magnification insert reveals that colonies in HPL are highly dense, with cells overlapping on top of each other, compared with the only loosely connected cells in FBS cultures and it also shows gene profiling and transformation marker analysis of stem cells cultured in HPL and FBS.

Abbreviations - HPL- Human Platelet Lysate Figure 7 shows that no difference is observed in Immunophenotype analysis and chondrogenic osteogenic and adipogenic differentiation potential of DPSCs cultured in media supplemented with 10% serum and 10% HPL, indicating suitability of HPL for large scale expansion of DPSCs.

Figure 8 shows karyotyping analysis and transformation marker analysis of DPSC cultures (HPL and FBS) at pre- and post-large-scale expansion.

(A, B) DPSC cultured at pre-large-scale expansion in FBS and HPL, respectively.

(C, D) DPSC cultured at post-large-scale expansion in FBS and HPL, respectively. Show chromosomal stability.

(E) Semi-quantitative RT-PCR of selected transformation markers.

(F) Relative expression of the selected transformation markers is performed by SYBR green-based real-time RT-PCR as a confirmative study. Values are presented after being normalized to 18S mRNA levels, and DPSCs expanded under FBS in T25 culture fl asks are used as a control. The average of three replicates is displayed.

Figure 9 shows generation of islet like cell aggregates from dental pulp stem cells and differentiation of DPSCs into ICAs carried out in three stages. .

Figure 10 shows Expression of endoderm and pancreatic hormone genes in islet like cell aggregates (ICAs)

OBJECTIVES

It is the objective of the present invention to provide a method for isolation and production of high quality clinical grade DPSCs in xeno-free conditions.

It is also the objective of the present invention to provide a method for a suitable plating density to obtain large number of DPSC population in short duration.

It is still another objective of the present invention to provide a transport medium for preserving the dental pulp and the stem cells until further processing. It is still further objective of the of the present invention to provide a method for obtaining Islet-like Cell Aggregates (ICAs) from the DPSCs using different differentiation medium. DETAILED DESCRIPTION

The present disclosure relates to a transport medium comprising Dulbecco's Modified Eagle's Medium- Knock Out (DMEM-KO), Fetal Bovine Serum (FBS), Pen-Strep, Glutamine, Ascorbic acid and Insulin-Transferrin-Selenium (ITS). In an embodiment of the present disclosure, the transport medium is also used as preserving medium.

In another embodiment of the present disclosure, DMEM-KO is at concentration ranging from about 0.5X to about 5X, preferably about IX; the FBS is at concentration ranging from about 10% to about 30%, preferably about 20%; the Pen-Strep is at concentration ranging from about 1% to about 5%, preferably about 2%; the Glutamine is at concentration ranging from about 1% to about 7%, preferably about 5%; the Ascorbic acid is at concentration ranging from about 50 μg/mL to about 500 μg/mL , preferably about 100μg/mL; and the Insulin-Transferrin-Selenium (ITS) is at concentration ranging from about 0.5X to about 2X, preferably about IX.

The present disclosure further relates to a method for large-scale production of dental pulp derived stem cells (DPSCs), said method comprising acts of:

a) obtaining dental pulp tissue and optionally storing in Transport medium;

b) mincing the tissue and incubating with collagenase to obtain digested cell tissue; c) neutralizing the digested tissue with FBS and centrifuging to obtain cell pellet; and d) culturing and maintaining the cells in xeno-free culture medium to obtain DPSCs.

In an embodiment of the present disclosure, the transport medium comprises DMEM-KO at concentration ranging from about 0.5X to about 5X, preferably about IX; FBS at concentration ranging from about 10% to about 30%, preferably about 20%; Pen-Strep at concentration ranging from about 1% to about 5%, preferably about 2%; Glutamine at concentration ranging from about 1% to about 7%, preferably about 5%; Ascorbic acid at concentration ranging from about 50 μg/mL to about 500 μg/mL, preferably about 100μg/mL; and Insulin-Transferrin-Selenium (ITS) at concentration ranging from about 0.5X to about 2X, preferably about IX.

In another embodiment of the present disclosure, the incubating is carried out at temperature of about 37°C for time duration ranging from about 10 to about 30 minutes, preferably about 20 minutes and the collagenase is at concentration ranging from about 0.2% to about 0.5%, preferably about 0.3%.

In yet another embodiment of the present disclosure ,the FBS is at concentration ranging from about 5% to about 15%, preferably about 10%; and the centrifuging is carried out at about 800 rpm to about 1200 rpm preferably about 1000 rpm; for time duration ranging from about 5 minutes to about 15 minutes, preferably about 10 minutes.

In still another embodiment of the present disclosure, the xeno-free culture medium comprises of DMEM-KO at concentration ranging from about 85% to about 95%, preferably at about 90%; Human Platelet Lysate (HPL) at concentration ranging from about 5% to about 20%, preferably at about 10%; 0.5% 10000 μg/mL penicillin/streptomycin and 1% IX Glutamine.

In still another embodiment of the present disclosure, the culturing is carried out at temperature of about 37°C, at about 5% CO₂ atmosphere and at density ranging from about 800 cells/cm² to about 1000 cells/cm², preferably about 1000 cells/cm².

In still another embodiment of the present disclosure, the stem cells are further subjected to cryopreserving and recovery. In an embodiment of the present disclosure, the dental tissue is obtained from teeth selected from group comprising permanent teeth, deciduous teeth and periodontal ligament; or any combinations thereof. The present disclosure further relates to a method of obtaining Islet-like Cell Aggregates (ICAs) from Dental-Pulp Stem Cell, said method comprising acts of:

a) culturing the DPSC in Definitive Endoderm Differentiation Medium (DEDM) to obtain cultured stem cells;

b) culturing and maturing the cultured stem cells in Pancreatic Endoderm Differentiation Medium (PEDM) and Islet Hormone Maturation Medium (IHMM) to obtain ICAs.

In an embodiment of the present disclosure, the Islet-like Cell Aggregates (ICAs) produce human insulin. In another embodiment of the present disclosure, the DEDM comprises Dulbecco's modified Eagle's medium Knock Out (DMEM-KO) at concentration ranging from about 95% to about 99%, preferably about 99%; Bovine Serum Albumin(BSA) at concentration ranging from about 5% to about 2%, preferably about 1%; insulin-transferrin- selenium(ITS) at concentration ranging from about 5X to about 2X, preferably about IX; activin A at concentration ranging from about 2 nM to about 5 nM, preferably about 4nM; 1 sodium butyrate at concentration ranging from about 0.5nM to about 2 nM, preferably about InM; and 2-mercaptoethanol at concentration ranging from about 20 μΜ to about 60 μΜ , preferably about 50 μΜ. In still another embodiment of the present disclosure, the PEDM comprises DMEM-KO at concentration ranging from about 95% to about 99%, preferably about 99%; BSA at concentration ranging from about 0.5% to about 2%, preferably about 1%; ITS at concentration ranging from about 0.5X to about 2X, preferably about IX; and taurine at concentration ranging from about 0.1 mM to about 0.5mM, preferably about 0.3mM. In yet another embodiment of the present disclosure, the IHMM comprises DMEM-KO at concentration ranging from about 95% to about 99%, preferably about 99%; BSA at concentration ranging from about 1% to about 2%, preferably about 1.5%; ITS at concentration ranging from about 0.5X to about 2X, preferably about IX; taurine at concentration ranging from about 2mM to about 4mM, preferably about 3 mM; glucagon-like peptide at concentration ranging from about 50nM to about 150nM, preferably about 100 nM; nicotinamide at concentration ranging from about 2.5mM to about 2mM, preferably about 1 mM; and nonessential amino acids at concentration ranging from about 2X to about IX, preferably about IX.

The Present invention provides a method for isolation and processing of stem cells from an easily accessible source of dental pulp and also discloses a transport medium which protects the dental pulp from deteriorating when compared to the existing medium. It also discloses a method of isolation for autologous DPSCs from dental pulp and banking by cryopreservation. Further, it provides a method for large scale production of allogeneic MSCs for clinical applications.

The present invention discloses a method of isolation, expansion and characterization of stem cells from dental origin. The term dental origin in the present invention includes but not limited to permanent teeth, deciduous teeth; periodontal ligament etc. further it also discloses which type of stem cell source is more potent and best for targeted clinical application to get better results. The stem cells isolated from different source can have specific lineage and specific propensity, identification of this can help in providing a better targeted clinical application of stem cells.

The Present method is used for autologous and allogeneic therapy. Dental pulp is a good source for autologous therapy for patient suffering from type 1 diabetes especially in young children. The teeth are extracted/ collected and the stem cell harvested is used immediately or cyropreserved for later use. The present invention also discloses a transport medium composition which is used for preserving the dental pulp tissue without damage so that the cell is preserved until isolated for further processing. The transport media used in the art are not very effective and transporting the dental pulp to the lab for extraction of the tissue has been an issue. The present invention further discloses a method for up-scaling the Dental Pulp Stem Cells (DPSCs) isolated in xeno free medium under current manufacturing practice grade for therapeutic use.

In view of the potential application of DPSCs for clinical medicine, there is growing interest in optimizing their expansion protocols so as to produce large quantities of cells for therapeutic applications, which is cost-effective and yet maintains their phenotype and functional capabilities. To date, there is inconsistency among laboratories concerning the types of media and supplementary factors for the successful isolation and expansion of DPSCs, resulting in heterogeneous cell populations both in ex vivo experiments and in clinical trials. The Present invention, overcomes the inconsistency by setting optimal culture conditions for the effective clinical-grade production of large number of DPSCs in a short time, economizing on cost and time to serve for better cellular therapy. The results of the study are highly reproducible and consistent, making them useful for in vivo as well as in vitro manipulation without the DPSCs losing their vigor and chromosomal stability.

The present disclosure is further elaborated with the help of following examples and associated figures. However, these examples should not be construed to limit the scope of the present disclosure.

Isolation and culturing of Stem cells

Example 1: Procedure for isolation of Stem cells from Dental pulp by Explants- enzymatic technique

(The technique of isolation of dental pulp from the extracted tooth is followed as described by Gronthos et al., 2000).

The Present invention provides a method of preserving, culturing and large scale expansion of stem cells isolated from dental pulp. The dental pulp used is from a single individual or pooled from multiple donors. The individual dental pulp is best used in case of autologous stem cell therapy and regenerative medicine; while individual or pooled Dental pulp derived MSC is used for allogeneic cell therapies. The dental tissues/pulp are collected as per Gronthos et al technique and preserved in a transporting medium before being transported to lab for further processing and expansion. The term transporting medium for this application shall mean a medium comprising of Dulbecco's Modified Eagle's Medium- Knock Out (DMEM-KO), Fetal Bovine Serum (FBS), Pen-Strep, Glutamine, Ascorbic acid and Insulin-Transferrin- Selenium used for transferring dental pulp after isolation to the lab with no or minimal deterioration of the cells. The transporting medium of present invention preserves the dental pulp (DP) tissue along with the cells associated with DP for subsequent isolation of Dental Pulp derived Mesenchymal Stem Cells (DP-MS C) for expansion, characterization and determination of differentiation potential in-vitro. Thus collected tissue is washed in a washing medium by immersing the tissue in the medium for about 20-30 seconds, preferably for 30 seconds in a 1.5mL centrifuge tube. The transporting medium comprises of DMEM-KO in the range of IX to 5X, FBS in the range of 10-20%, Pen-Strep in the range of 1-5%, Glutamine in the range of 1-7%, Ascorbic acid in the range of 50-500 μg/mL and Insulin-Transferrin-Selenium of about 0.5X to about 2X.

In one of the embodiments, transportation medium comprises of DMEM-KO-1X, FBS- 20%, Pen-Strep-2%, Glutamine-5%, Ascorbic acid-100μg/mL and Insulin-Transferrin- Selenium (ITS)-IX. In general, transport media in the prior art use antibiotic and BSA or even Balanced Salt Solution (BSS) which have no nutritional value. In the transport medium of the present disclosure, use of basal medium (DMEM-KO) provides the nutrition and keeps the cell viable until processing, thus utilizing maximum cell density available from the dental pulp. Gronthos et all does not disclose the transport medium nor the exact step as mentioned in present invention. As compared to the enzymatic Gronthos protocol, the present invention uses explants-enzymatic method.

The washing medium mainly comprises of 5% Pen-Strep and basal medium [DMEM- KO]. Transfer the tissue to a clean, sterile 1.5 mL centrifuge tube containing 200 \L of 0.2 % collagenase type I and 200 \L of basal media (DMEM-KO). Mince the dental pulp tissue into tiny pieces with a sterile scissor. Incubate at 37°C for up to 20 min and mix well intermittently. Add another 500 μΐ_^ of 0.2 % collagenase( range 0.2- 05%) and incubate for 10 min. Transfer the digested tissue into a 15 mL tube and inactivate the collagenase by diluting it with 8 mL of DP-MSCs primary culture medium. Centrifuge at 1000 rpm for 10 min at room temperature. Discard the supernatant and wash the digested tissue again with 3 mL of DP-MSCs primary culture medium. Culture cells in DP-MSCs primary culture medium (2 mL of medium per T25-flask) at 37°C and 5% C0₂ in the incubator. The flask is left undisturbed for at least 48 hours. Add 1 mL of medium on day 3. Seven days after the cell isolation, wash the culture vessels with IX PBS and change the DP- MSCs Primary culture medium. After that, the medium changed every 48 hrs until cell confluence is reached. This method is used for isolation of stem cells from Deciduous and Permanent teeth. The same method is used for autologous and allogeneic therapy.

Table 1: Different types of medium and their component used in present invention Different types of culture medium compositions used in the present invention are disclosed in the table 1. Tissue Transportation medium or transporting medium is used for transporting the extracted dental pulp to the lab for further processing. This medium protects the cells and minimizes the cell damage during the transport. Transporting medium comprises of DMEM-KO, FBS, Pen-Strep, glutamine, ascorbic acid and ITS, while DP-MSCs primary culture medium is a medium used initially for boosting the Dental Pulp Derived stem cells (DPSCs)/(DP-MSCs) growth and; wherein said DP- MSCs primary culture comprises DMEM-KO, FBS, Pen-Strep and glutamine. (See table 1). After the initial step of culturing, the DPSCs are further maintained on DP-MSCs routine culture medium (See table 1). Fourth type of medium used is the washing medium comprising of DMEM-KO and Pen-Strep. Example 2: Sub-culture of DPSCs

Replenish the DP- MSC s primary culture medium depending upon the presence of floaters and background, if necessary every 48 hrs. Take two representative microphotographs of the flask each time during observation. Culture the cells until the flask is 80% confluent. Remove the spent media using a pipette. Wash twice by adding DPBS to confluent flask Stack for 1 min and discard the DPBS using a pipette. Care should be taken not to touch any external surface of the flask or the bottle.

Trypsinize the flasks by adding 0.05% trypsin-EDTA and keep in the incubator at 37° C for 1 -2 min till the cells round up and float (check under microscope).

Add DP-MSCs primary culture medium to neutralize 0.05% trypsin-EDTA. Pipette media twice without any froth.

The trypsinization should not be carried out for a long time. If it exceeds more than 2-3 minutes, then transfer the cell suspension to a centrifuge tube. Centrifuge the MSC suspension at 1000 rpm for 10 min at room temperature (18-25°C). Discard the supernatant and add DP-MSCs primary culture medium to re-suspend the pellet.

Count the cells on Haemocytometer and check the viability by trypan blue dye exclusion principle. Split the cells at a density of 1 x 106/ mL. Example 3: Cryopreservation and Recovery of DPSCs

Wash the flasks with DPBS three times, add 2mL of 0.05% trypsin-EDTA solution and incubate at 37°C for 2 min, confirm that 80% of the attached cells have become detached from the surface, then add DP-MSCs routine medium (1 : 10 ratio of trypsin and media). Centrifuge at 1000 rpm at 10 min, remove supernatant and add 90% FBS and 10% DMSO to the pellet. Add aliquotes to cryopreservation vials and place in a programmed freezer. A controlled freezing container at -80°C freezer may also be used to achieve a slow cooling rate. Place the vials in liquid nitrogen for long-term storage so as to make a DPSC bank. Example 4: Recovery of DPSCs from frozen storage

Place the vials in a warm water bath (37°C). Quick thawing (1-2 min) is important for best recovery. Add 10ml of DP-MSC Primary culture medium and mix well and centrifuge at 500 rpm for 6 min. Remove the supernatant and resuspend cells in MSC medium. Viable cells are counted with Trypan Blue solution and then seeded and cultured in MSC medium at 37°C and 5% C0₂. . The volume of MSC medium added is according to the type of culture dishes: 2ml/35mm dish; 6ml/T25 flask; 20ml/T75 flask. The seeding concentration of DPSCs ranges from 1000-5000 cells/cm .

The culture media used for expansion of DPSCs in the present invention is found to support long term expansion of the cells without losing their phenotype, cell morphology or differentiation potential. Plating density of DPSCs has been shown to affect the overall yield of the stem cells. In the present invention, dental pulp tissue is used as alternative source for isolation of Mesenchymal Stem Cells (DP-MSCs). Together with optimum culture medium supplemented with anti-oxidant agent (L- ascorbic acid) and plating density, this significantly improves DP-MSC yield and reduces expansion time by 30-24 hrs compared to the prior art, thus reaching the necessary yield for therapeutic/clinical applications in short time duration.

MSCs cultured in culture medium disclosed in present invention when compared to other type of culture medium and method used in prior art have many advantages such as: maintain cell morphology of the cultured cells, show better growth kinetics while preserving the phenotypic and differentiation potential during prolonged culture and are less susceptible to senescence.

Figure 1 shows the morphology, growth kinetics, and senescence in DPSCs cultured across different media at early and late passage. Isolated DPSC is cultured using different basal medium selected from a-MEM, DMEM-KO, DMEM-F12 and DMEM-LG to see if there is any change in the cell characteristics. (A) Phase contrast microscope, 10x of DPSCs cultured in various growth media; (B) Long-term growth curves of DPSCs cultured in various growth media; (C) Senescence associated β-gaiactosidase (SA- -gal) staining of DPSCs cultured in various growth media. (Arrow shows positive ceils); (D) Quantification of percentage (%) of SA- -gal positive cells cultured in various growth media. The results represent average of four culture replicates with SD. A representative photomicrograph is given for each experiment. *p<0.05, **p<0.01 , and ***p<0.001. Abbreviations: DPSCs - Dental pulp stem cells

SD - standard deviation.

The DPSC cells are fibroblast-like ceils appearing thin, slender with tapering ends. The DPSC are mononucleated cells. They forms typical whirlpool like pattern after attain confluences and are plastic/glass adherence. Further characterization has been carried out by immunophenotype analysis in example 5 and figure 3B. The karyotyping of the DPSC cells show the cells are normal. Example 5:

In the present invention, different basal media and growth supplements are compared in order to identify optimal culture conditions for proliferation of dental pulp stem cells (DPSCs). Cell morphology, growth kinetics, senescence pattern, cell surface marker expression, pluripotent marker expression and differentiation capacity of DPSCs among all the media compositions in the presence and absence of L-ascorbic acid 2- phosphate, is compared. Different media used for the study include a-MEM, DMEM-KO, DMEM- F12 and DMEM-LG in presence of 10% Fetal bovine serum (FBS) with and without L- ascorbic acid 2- phosphate. Conclusion of the study shows a-MEM, DMEM-KO media to be better compared to media tested.

Figure IB shows that among the various basal media tested, a-MEM and DMEM-KO supplemented with 10% fetal bovine serum (FBS) are the optimum growth media as they support the ex-vivo expansion of DPSCs while preserving the phenotypic and differentiation potential in prolonged cultures while addition of L-ascorbic acid 2 phosphate in the basal medium, did not show any significant effect on proliferation and expansion. Plating density of DPSCs has been shown to affect overall yield. Use of L- ascorbic acid 2 phosphate in the basal medium is to modulate the potency of DPSCs. Ascorbic acid is found to prolong the life of cell by delaying the cellular senescence. Thus addition of L-ascorbic acid 2 phosphate to the culture medium depends on the indication to be treated. For treating osteoarthritis, addition of L-ascorbic acid 2 phosphates can enhance the cell potency thus improving the efficacy of the treatment. Ascorbic acid is used for cell differentiation to oestocytes. Hence the DPSCs are cultured in a tailor made fashion for suitable disease to be treated.

Flow cytometric analysis

The immunophenotypes of DPSCs cultured at different types of media are examined using flow cytometery at passage 1 (early) and passage 9 (late). On reaching 90% confluency, the cells are harvested with 0.05% trypsin (Invitrogen) and resuspended in PBS at a cell density of 1.5X 106 cells/ml. Two hundred microliters of the cell suspension (1 x 105 cells) is incubated with the labeled antibodies in dark for 1 h at 37°C. The following antibodies are used to mark the cell surface epitopes-CD90-phycoerythrin (PE), CD44-PE, CD73-PE, CD166-PE and CD34-PE, CD45-fluoroisothyocyanate (FITC), and HLA-DR-FITC (all from BD Pharmmgen, San Diego, CA). All analyses are standardized against negative control cells incubated with isotypespecific IgGl-PE and IgGl-FITC (BD Pharmmgen). At least 10,000 events are acquired on Guava Technologies flow cytometer, and the results are analyzed using Cytosoft, Version 5.2, Guava Technologies, Hayward, CA. Figure 3B shows the immunophenotype analysis of DPSCs cultured across different media with or without ascorbic acid at early and late passage. DPSCs are tested against human antigens CD34, CD44, CD45, CD73, CD90, CD 266, and HLA-DR. 7-AAD is used to check the viability of the cells. (A) Cells cultured in a-MEM (early and late passage);(B) Cells cultured in a- MEM + ascorbic acid (early and late passage); (C) Cells cultured in DMEM-KO (early and late passage); (D) Cells cultured in DMEM-KO + Ascorbic acid (early and late passage); (E) Cells cultured in DMEM-LG (early passage); (F) Ceils cultured in DMEM-F12 + Ascorbic acid (early and late passage). Results are representative of three independent experiments. Abbreviations: DPSCs -Dental pulp stem cells,

CD -cluster of differentiations.

Proliferation rate of DPSCs is significantly higher in a-MEM and DMEM-KO compared with that of DMEM-F12 and DMEM-LG (p<0.001). After the end of the passage 9, overall cell yield is significantly higher in a-MEM and DMEM-KO (35x 106 and 35x 106, respectively) in T25 cm2 flask as compared with DMEM-F12 and DMEM-LG (24x 106 and 5.2x 106, respectively; p<0.05; Fig. IB).

Example 6:

Senescence phenomenon is monitored by β-galactosidase activity in early and late passages. Percent senescence activity in the cells cultured in DMEM-KO (12.26±3.92) and a-MEM (10±3.21) is significantly lower in prolonged passages as compared with the cells cultured in DMEM-F12 (34.6±0.92) and DMEM-LG (79.8±3.92; p< 0.05). (Fig. 1C, D). Figure 3A shows the Quantification of percentage (%) of SA- -gal positive cells cultured in various growth media with and without adding ascorbic acid. The addition of ascorbic acid shows delayed cell senescence and Figure 3B shows the immunophenotype analysis of DPSCs cultured across different media with or without ascorbic acid at early and late passage. Population doubling time (PDT) of DPSCs in all the four media is analyzed. The cells cultured in DMEM-KO, a-MEM, and DMEM-F12 showed average PDT 29,6+0,8, 29,2+0,9, and 30.6±2.25, respectively. On the contrary, DPSCs grown in DMEM-LG did not support the growth of the cells beyond passage 5. The average PDT at PI and P5 for DMEM-LG is 33.2±

1.2 and 50.8+1.2, respectively, which is significantly higher than the cells grown in DMEM-KO, a-MEM, and DMEM-F12 (Table 1).

Figure 2 shows the cell expansion against the time for three (3) passage levels and effects of cell density on the proliferation of DPSCs. Fig 2(A) shows cell population and Fig 2(B) shows population doubling time (PDT) in hour ± SD of DPSCs cultured until passage 3. DPSCs are plated on BD falcon tissue culture flasks at 200, 400, 600, 800, 1000, 200, 5000 or 10000 cells/cm² from P-l until P-3. The data presented in Figure 2B shows that initial seeding densities at 800 and 1000 cells/cm provided the best starting conditions for cell proliferation. Overall yield is better when DPSC is seeded at 800-1000 cells/cm² than higher or lower seeding densities (p<0.05). Morphology of DPSCs by phase contrast microscopy shows that DPSCs cultured at the densities of 800 - 1000 cells/ cm² have thin spindle- shaped cells whereas cell morphology at 200-600 cells/ cm² density showed wider, spindle shaped cells. On the contrary cell morphology at higher densities such as 5000- 10000 cells/ cm showed flat; very large cluster of cells. Cells cultured under 5000 and 10000 cells/ cm never reached 80 % confluency. This result clearly showed that seeding density critically affects DPSCs proliferation rates and overall yield.

The above disclosed method and culture medium is used with different types of dental tissue including permanent teeth, deciduous teeth; periodontal ligament etc Example 7: Proliferation, gene expression profile and lineage specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth also referred to as (DPSC)

Lack of understanding of nature of the new stem cell sources and their lineage specific propensity may hinder their full potential. Therefore, understanding the gene expression profile which indicating their lineage specific proclivity is fundamental to the development of successful cell-based therapies.

In one embodiment, proliferation rate, gene expression profile and lineage specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over five passages is compared. The proliferation rate of SCD is higher (cell number (cells/ml) 25 x 106; percent CFUs: 151.67 ± 10.5; percent cells in S/G2 phase: 12.4 ± 1.48) than DPSCs (cell number (cells/ml) 21 x 106; percent CFUs: 133 ± 17.62; percent cells in S/G2 phase: 10.4 ± 1.18. It is observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG and REX1 are higher (> 2) in SCD as compared to DPSCs. However DPSCs showed higher expression of neuro-ectodermal markers PAX6, GBX2, NESTIN (fold expression >100). Similarly, higher neurospheres formation and neuronal marker expression (NF, GFAP) are found in the differentiated DPSCs into neuron like cells as compared to SCD. In figure 4, the expression profile of pluripotent and lineage-specific stem cell markers of SCD and DPSCs is shown. (A) Semiquantitative RT-PCR of selected pluripotent/stem cells and lineage markers. (B) Relative levels of selected pluripotent/stem cells and lineage markers are performed by Taqman-based assay qRT-PCR. (C) Relative levels of selected pluripotent/stem cells and lineage markers are performed by SYBR green-based qRT-PCR. The lower the CT value, the more copies are present in the specific sample. Values are presented after normalized to 18s R A level. The average of 2 replicates is displayed.

In figure 5 an overview of the generation of neurospheres in SCD and DPSCs and gene expression profile of selected neuron markers is shown. Figure 5(A, B)-Formation of neurosphere in non-coated dish for 15 days under neuron media in SCD and DPSCs, respectively; (C) the neurospheres formed under neuron-condition media at day 15. The number of neurospheres are divided into small (61-80 mmol/L), medium (81-100 mmol/L), and large diameters (>100 mmol/L). (D, E) Neurospheres are transferred into a coated dish at day 16 and are migrated radially out of the sphere in SCD and DPSCs, respectively. (F, G) After 2 days of maturation, cells had morphologic features typical of neuron in SCD and DPSCs, respectively. (H, I) After 5 days of maturation, dendrite-like outgrowth from SCD and DPSCs, respectively, showing complex neuronal processess (arrow and insert); (J) gene expression profile of OCT4, nestin, b-III tubulin, GFAP, and NF at day 20 in SCD and DPSCs. (K-N) Specific co-immunocytochemical staining of neurospheres indicated the presence of b-III tubulin and GFAP at day 20 in SCD and DPSCs, respectively. In both co-immunocytochemical pictures, nuclei are stained with DAPI (blue) and OCT4 (green); *P < .05, **P < .01 , and ***P < .001. This clearly indicates that SCD retained their plasticity over the passages whereas DPSCs lost their plasticity and are shown to be more committed toward neuronal lineage. It clearly demonstrates that both SCD and DPSCs act as useful candidates for regenerative medicine in various diseases, emphasizing the usage of DPSCs for neurologic diseases.

Example 8: Large scale expansion of DPSC for therapeutic/clinical application in xeno-free culture medium

The present invention provides a system/method for efficient large scale production of DPSCs in undifferentiated state in xeno-free culture conditions suitable for clinical application. DPSCs are obtained from single donor or multiple donors and pooled. Dental Pulp stem cells (DPSCs) are considered as a promising tool in regenerative therapy. However, for clinical applications, a large scale production of DPSCs is required without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS), an undesirable xeno component as supplement risking the chance of contamination. The Present invention, in one of its embodiments, discloses Xeno-Free culture medium comprising DMEM-KO, and 10% Human Platelet Lysate (HPL) for large scale expansion of DPSCs, isolated as disclosed in example 1 without compromising on current Good Manufacturing Practice (GMP) in a T25 culture flask. DPSCs are obtained from single or multiple donors.

HPL preparation

HPL is prepared from 30 - 40 donors who had donated at the University of Malaya Blood Bank (Kuala Lumpur, Malaysia). Briefly, whole blood (WB) is collected into a quadruple blood-bag system (Baxter Health Care Corporation, Deerfield, IL, USA) and centrifuged at 4250 g for 13 min at 22 ° C to separate the plasma and buffy coat. Platelet-rich plasma (PRP) is prepared by mixing 4 units buffy coat (from donor group O) and 1 unit plasma (from donor group AB). Immediately after preparation, the PRP is frozen down to - 80 ° C and subsequently thawed at 37 ° C to obtain platelet-released growth factors. 10 to 12 thawed PRP units, now called HPL, are pooled to prepare a standardized pooled HPL (pHPL). The pHPL is centrifuged at 4000 g for 15 min at 4 ° C to remove the platelet fragments. The supernatant plasma is filtered further using a 40- μΜ filter (BD Biosciences, Franklin Lakes, NJ, USA) and transferred into 50-mL vials (BD Biosciences). Sterility tests, such as endotoxin and mycoplasma, areconducted, and 2 U/mL heparin (Heparinol; Ain Medicare Sdn Bhd, Kota Bahru, Malaysia) are added into the pooled HPL before releasing it for experimental use.

Sound, intact, human third molars from multiple adults in age group 24 - 35 years of age are collected with informed consent from patients undergoing extraction at the Department of Oral and Maxillofacial Surgery, University of Malaya, under approved guidelines set by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [medical ethics clearance number DF CD0907/0042(L)]. Under sterilized conditions, the root surface of the tooth is cleaned with 100% povi done-iodine (Sigma- Aldrich, St Louis, MO, USA) and the pulp extirpated within 2 h post extraction. Thereafter, the tissue is kept in a 1.5-mL tube containing IX Dulbecco ' s modified Eagle 's medium - knock-out (DMEM-KO), 10% FBS, 2% Pen-Strep, 5% GlutaMax, 100 μ g/mL ascorbic acid and 1 x insulin-transferrin-selenium (ITS). Then it is transported to a nearby current good manufacturing practice (cGMP) certificated laboratory for isolation of the cells.

Isolation of primary cells DPSC to establish primary cultures

The pulp tissue is minced into small fragments prior to digestion in a solution of 0.2% to 0.5% preferably 0.3% collagenase type I (Gibco, Grand Island, NY, USA; http://www.invitrogen.com) for 20 min at 37 ° C. After neutralization with 5% to 15% preferably 10% FBS (lot number ATJ33090; Hyclone, South Logan, UT, USA; www.thermo.com/hyclone), the cells are centrifuged and seeded in a T75 culture flask (BD Biosciences) with culture medium containing DMEM-KO (Invitrogen, Carlsbad, CA, USA; www. invitrogen.com), 0.5% and 10000 μg/mL penicillin/ streptomycin (Invitrogen), 1% 1 X Glutamine (Invitrogen) and 10% FBS, in a humidified atmosphere of 95% air and 5% CO 2 at 37 ° C. Non-adherent cells are removed 48 h after initial plating. The medium is replaced every 3 days until the cells reached 80 - 90% confluency.

In vitro expansion of human dental stem cells from single and pooled donors

The large scale expansion of DPSCs in presence of DMEM-KO and 10% FBS and DMEM-KO and 10% HPL in T25 culture flask is compared for the efficacy of HPL as a substitute for FBS. The DPSCs are cultured in DMEM-KO either with 10% FBS or 10% HPL and characteristics of DPSCs at pre (T25 culture flask) and post (5-STACK- chamber) large scale expansion in terms of their identity, quality, functional ability, molecular signatures and cytogenetic stability is compared. In both pre and post large scale expansion, DPSCs expanded in HPL showed extensive proliferation of cells (~2- fold) as compared with FBS as well as the purity, immune phenotype, colony forming unit potential and differentiation are comparable. In one of the embodiments, clinical-scale expansion procedure is provided wherein the pooled DPSC are used for the large-scale expansion. These cells are cultured at 1000 cells/cm² in either 10% HPL or 10% FBS supplemented with DMEM-KO (Invitrogen), 0.5% and 10000 μg/mL penicillin/streptomycin (Invitrogen) and 1% 1 X Glutamax (Invitrogen) using a T25 flask (designated as pre-large-scale expansion; total surface area 25 cm² ; BD Biosciences) and 5-STACK chamber (designated as post-large-scale expansion; total surface area 3180 cm² ; Corning Life Sciences, Chelmsford St Lowell, MA, USA; www.corning.com/lifesciences).

Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetic characteristics of DPSCs expanded under HPL and FBS are compared, revealing similar expression profile. Hence present invention discloses a highly economized expansion process of DPSCs in HPL yielding double the amount of cells while retaining their basic characteristics in shorter time duration under cGMP conditions, making it suitable for therapeutic applications. The process is highly economized because costly FBS is replaced with HPL and yield obtained by using the present method is higher.

Figure 6 shows morphology, of DPSCs expanded in the presence of different media compositions. It also shows cells morphology of DPSCs expanded in the presence of HPL are smaller spindle-shaped cells than FBS (A & B). (Al , A2) Phase-contrast microscope, 10 X of DPSC expanded in HPL and FBS, respectively. Fig (C, D) shows the DPSC expanded in HPL and FBS, respectively. Magnification insert reveals that colonies in HPL are highly dense, with cells overlapping on top of each other, compared with only loosely connected cells in FBS cultures. The colony-forming properties of DPSCs expanded in HPL and FBS are assessed. The CFUs are highly packed in cells expanded in HPL as compared to cells expanded in FBS. This result is reflected at the growth kinetics. In the large scale expansion experiment (5-STACK chamber), DPSCs cultured in HPL produced higher yield (4.98 ± 0.8 x 10⁸ cells/ cm²; p<0.05) with an approximate population doubling of 7.23 and population doubling time of 35.80 ± 3.5 h as compared to FBS (3.20 ± 1.1 x 10⁸ cells/cm²) with approximate population doubling of 6.65 and population doubling time of 39.19 ± 4.1 h.

While comparing immunophenotype of the final populations obtained from passage 2 it is found that the cells expanded in HPL or FBS did not diverge from each other either at the pre or post large scale expansion of DPSCs. The cells consistently shared the same "mesenchymal stem cell phenotype" as recommended by the International Society for Cellular Therapy (ISCT),that is more than 80% positivity for CD44, CD73 and CD90 and negativity for CD45 and CD34. The HLA-DR expression at pre and post large scale expansion obtained from DPSCs expanded under HPL and FBS is compared, which expressed less than 2% and hence it is concluded that DPSCs are poorly immunogenic in vitro. The viability of the harvested DPSCs at pre and post large scale expansion are checked by 7-amino-actinomycin D (7-AAD) and is found to be more than 80%. The DPSCs cultured under HPL are assessed to see if there is any difference compared to cells cultured in FBS. A series of pluripotent gene analyses on cells at pre and post large scale expansion using BM-MSCs as a calibrator (control) are carried out. In order to evaluate the transcriptional changes between the different cultured cells, focus is on genes which have >1.5 fold change. As shown in Figure 6, an increased expression of stem cell markers such as OCT4, SOX2 and NANOG in DPSCs cultured in HPL or FBS at pre or post large scale expansion as compared to BM-MSCs is observed. Importantly DPSCs expressed higher neuron/ectoderm markers than BM-MSCs. Nevertheless the expression of the neuronal/ectoderm makers such as NES, GBX2, PAX6, and TH is retained in DPSCs cultures (HPL and FBS) in pre large scale expansion as well at the post large scale expansion. Quantitative RT-PCR confirmed the fidelity of the array data, where selected genes expressed in accordance with their differential expression pattern in the array (Figure 6). The expression of OCT4, SOX2 and NANOG is almost similar in all the tested samples (Figure 6). This also is confirmed at the protein level (Figure 6)

No difference is observed in Immunophenotype analysis and chondrogenic osteogenic and adipogenic differentiation potential of DPSCs cultured in media supplemented with 10% serum and 10% HPL, indicating suitability of HPL for large scale expansion of DPSCs ( see figure 7).

Numerical and structural chromosomal abnormalities in DPSCs after large scale expansion under different media compositions (HPL and FBS), samples is analyzed by GTG-banding.

The cytogenetic stability of DPSC is evaluated using conventional karyotyping. After pre- and post large- scale expansion, cells are incubated with 0.1 μ g/mL colcemid (Gibco) for 2 h in 5% CO 2 at 37 °C. The chromosome profile is assessed by trypsin/ Giemsa staining. A minimum of 25 metaphases from each sample is counted. None of the samples are found to be abnormal, indicating stable karyotypes (Figure 8 (A, B, C and D) ). There are reports which suggest that MSCs when cultured in-vitro for a long time can undergo spontaneous transformation and immortalize at a high frequency. Cells are observed for any signs of transformation when cultured under HPL and FBS in 5-STACK chamber (large scale expansion) by comparing to those in T25 culture flask (pre large scale expansion). There are no significant differences (p>0.05) in the mRNA level of some tumor suppressor genes such as p21 and p53 with almost no expression of pi 6 in all the tested cultures. This indicates normal genetypic stability despite multifold expansion in all culture conditions. Notably, the mRNA expression of other group, DNA repairs enzymes such as RAD51, ERCC3 and XRCC4 are also not significantly changed (p>0.05) between pre and post large scale expansion cultures further documenting normal molecular signatures/cytogentic stability ( see Figure 8 E and F).

Quality control of up-scaled DPSC: All DPSC products obtained at pre- and post-large scale expansion are tested for all standard quality controls required for cell therapy products produced in cGMP conditions, including sterility, endotoxin and mycoplasma testing. The cell products are found to fall within the acceptable ranges in all cases and, as such, are notionally releasable for clinical use (Table 2).

Example 9:

Type 1 diabetes or Insulin-dependent Diabetes Mellitus (IDDM) is a disorder characterized by total loss of pancreatic β-cells as a result of autoimmune destruction. The only remedy for IDDM is islet transplantation, which is hampered by the lack of availability of donor pancreas coupled with lifelong immune suppression. In this scenario, the only source of autologous stem cells is bone marrow, the acquisition of which is invasive and painful. Hence, there is a need for suitable alternative sources of stem cells for possible autologous transplantation of islets, eliminating the need for immune suppression.

The intact deciduous molars are extracted from children around 7-11 years with medical approval number DFCD0907/0042[L]). DPSCs are isolated and propagated as disclosed in this invention above. The DPSCs are differentiated into pancreatic cell lineage resembling islet-like cell aggregates (ICAs).

The figure9B shows generation of islet like aggregates from dental pulp stem cells. In one of the embodiments, potential of DPSCs to differentiate into insulin producing, pancreatic cell lineage resembling, islet like cell aggregates (ICAs) is disclosed. DPSCs are isolated and propagated as disclosed in the earlier paragraphs here. Cell characterized demonstrated that it could be differentiated into adipogenic, chondrogenic and osteogenic lineage upon exposure to appropriate cocktail of differentiating agents. Employing a three step protocol, reported ICAs are obtained from DPSCs. The identity of ICAs is confirmed as islets by diathiozone positive staining as well by expression of C-peptide, Pdx-1, Pax4, Pax6 Ngn3 and Isl-1 (see figure 10). There is several -folds up regulation of these transcription factors proportional to days of differentiation as compared to undifferentiated DPSCs. Results obtained demonstrate for the first time that DPSCs could be differentiated into pancreatic cell lineage and offer an easily available, non- controversial source of human tissue that could be used for autologous stem cell therapy in diabetes type 1.

Protocol- generation of Islet-like cell aggregates from DP-MSCs

Differentiation of DPSCs into ICAs is carried out in three stages (see figure 9A). Undifferentiated DPSCs are resuspended in Serum Free Medium (SFM)-A referred to as Definitive Endoderm Differentiation Medium (DEDM) and plated in petri-dish [1 x 10⁶ cells/cm²]. DEDM contained Dulbecco's modified Eagle's medium Knock Out (DMEM- KO), 1% BSA Cohn fraction V fatty acid free ( Sigma- Adrich), 1 X insulin-transferrin- selenium (ITS), 4 nM activin A, 1 mM sodium butyrate and 50 μΜ 2-mercaptoethanol. The cells are cultured in this medium for 2 days. On the third day, the medium is changed to SFM-B referred to as Pancreatic Endoderm Differentiation Medium (PEDM), which contains DMEM-KO, 1% BSA, ITS and 0.3 mM taurine and finally shifted to SFM-C on the fifth day. SFM-C containes DMEM-KO, 1.5% BSA, ITS, 3 mM taurine, 100 nM glucagon-like peptide (GLP)-l (ambide fragment 7-36; Sigma Aldrich), 1 mM nicotinamide and IX nonessential amino acids ( EAAs). The cells are fed with fresh SFM-C referred to as Islet Hormone Maturation Medium (IHMM) every 2 days for another 5 days. All chemicals are purchased from Sigma Aldrich unless otherwise indicated. After 5 days of incubation in (IHMM) , most of the day- 10 ICAs stained positive for dithiazone (DTZ), a zinc-chelating agent known to selectively stain pancreatic beta-cells (figure 9B).

Figure 10, shows the expression of endoderm and pancreatic hormone genes in ICAs. (A) Immunofluorescence analysis is performed on control (undifferentiated) DPSCs on day 10 and day 10 ICAs for the expression of definitive endoderm genes like Soxl7, early pancreatic genes like PDX-1 and Ngn3, and pancreatic hormone genes like Isl-1, C- peptide, and Glut2. Sox 17 is a transcription factor gene and hence displayed nuclear localization, while PDX-1 , Ngn-3, C-peptide, Isl-1, and Glut2 demonstrated cytoskeletal localization. DAPI is used as a counter stain; green and red represent FITC and rhodamine conjugates, respectively. Except for Soxl7, there is no expression of PDX-1 , Ngn3, Isl-1 , C-peptide, and Glut2 in the control (undifferentiated) DPSCs as compared with day 10 ICAs. (B) Flow cytometric analysis is performed to determine the expression of PDX-1, C-peptide, and Isl-1 in day 10 ICAs. Isotype-matched antibody control is also used to eliminate background.Staining. Scale bar: 200 μηι. (C) ICAs release insulin in response to glucose in vitro. The amount of insulin in the culture media is measured by the use of an Immulite 1000 Insulin Kit (LKINl), after the cells are exposed to different concentrations of glucose. (D) ICAs release C-peptide in response to glucose in vitro. The amount of C-peptide in the culture media is measured by the use of an Immulite 1000 C-peptide Kit (LKPEPl) after the cells are exposed to different concentrations of glucose.

The present invention discloses a method for differentiation of multipotent DPSCs into ICAs. The in vitro testing of ICAs by static stimulation assays showed that ICAs responded to the addition of glucose, as shown by measurable increase in increased insulin and C-peptide in a dose dependent manner. There are several alternative, well documented sources of stem cells for generating insulin-producing ICAs in the prior art. However, all seem to be suitable only for allogeneic islet transplantation, which unfortunately requires immuno-suppression. Furthermore, adult stem cells have yielded controversial results with regard to their ability to secrete insulin in vitro and normalize hyperglycemia in vivo. The ICAs obtained in this invention are used for cell therapy in type 1 diabetic conditions. The ICAs are formulated into a pharmaceutical composition, comprising ICAs, isotonic excipient such as plamalyte A or other suitable excipient to maintain the cells viable, optional may also include growth factor suitable for enchaining the the effect of insulin production. Further, the ICAs are used for screening novel anti-cancer diabetic compounds and also in research.

Advantages of the present method of processing and culturing of dental pulp compared to the known technique in art are as follows:

-Present method uses a combination of explants and enzymatic dissociation technique for dental pulp processing.

-It also discloses an improved transport medium for preserving the dental pulp during transportation.

-It overcomes the inconsistency seen in the prior art on the types of media and supplementary factor for isolation and expansion of DPSCs resulting in heterogeneous cell population.

-It discloses methods of using optimal culture conditions for the effective clinical-grade production of the large number of DPSCs in a short time, economizing on cost and time to serve for better cellular therapy.

-The results of the study are highly reproducible and consistent, making them useful for in vivo as well as in vitro manipulation without losing their vigor and chromosomal stability.

-The large-scale expansion of cells in xeno-free medium having HPL instead of FBS is highly economical as the usage of enzymes is reduced and it provides a method of isolating and expansion of cells that are free of bovine antigen, for clinical application. -It is less time consuming as large scale expansion of cells is performed in short duration. The cell so obtained have >80% viability.

The Present invention provides a technique for processing and culturing of DPSC. In addition it also discloses cryopreservation of DPSCs at -196°C so as to make DPSC bank for preserving DPSCs for future use.

Claims

WE CLAIM:

1. A transport medium comprising Dulbecco's Modified Eagle's Medium- Knock Out (DMEM-KO), Fetal Bovine Serum (FBS), Pen-Strep, Glutamine, Ascorbic acid and Insulin-Transferrin-Selenium (ITS).

2. The transport medium as claimed in claim 1 , wherein the transport medium is also used as preserving medium.

3. The transport medium as claimed in claim 1, wherein the DMEM-KO is at concentration ranging from about 0.5X to about 5X, preferably about IX; the FBS is at concentration ranging from about 10% to about 30%, preferably about 20%; the Pen- Strep is at concentration ranging from about 1% to about 5%, preferably about 2%; the Glutamine is at concentration ranging from about 1% to about 7%, preferably about 5%; the Ascorbic acid is at concentration ranging from about 50 μg/mL to about 500 μg/mL , preferably about 100μg/mL; and the Insulin-Transferrin-Selenium (ITS) is at concentration ranging from about 0.5X to about 2X, preferably about IX.

4. A method for large-scale production of dental pulp derived stem cells (DPSCs), said method comprising acts of:

e) obtaining dental pulp tissue and optionally storing in Transport medium;

f) mincing the tissue and incubating with collagenase to obtain digested cell tissue; g) neutralizing the digested tissue with FBS and centrifuging to obtain cell pellet; and h) culturing and maintaining the cells in xeno-free culture medium to obtain DPSCs.

5. The method as claimed in claim 4, wherein the transport medium comprises DMEM-KO at concentration ranging from about 0.5X to about 5X, preferably about IX; FBS at concentration ranging from about 10% to about 30%, preferably about 20%; Pen- Strep at concentration ranging from about 1% to about 5%, preferably about 2%; Glutamine at concentration ranging from about 1% to about 7%, preferably about 5%; Ascorbic acid at concentration ranging from about 50 μg/mL to about 500 μg/mL, preferably about 10(^g/mL; and Insulin-Transferrin-Selenium (ITS) at concentration ranging from about 0.5X to about 2X, preferably about IX.

6. The method as claimed in claim 4, wherein the incubating is carried out at temperature of about 37°C for time duration ranging from about 10 to about 30 minutes, preferably about 20 minutes and the collagenase is at concentration ranging from about 0.2% to about 0.5%, preferably about 0.3%.

7. The method as claimed in claim 4, wherein the FBS is at concentration ranging from about 5% to about 15%, preferably about 10%; and the centrifuging is carried out at about 800 rpm to about 1200 rpm preferably about 1000 rpm; for time duration ranging from about 5 minutes to about 15 minutes, preferably about 10 minutes.

8. The method as claimed in claim 4, wherein the xeno-free culture medium comprises of DMEM-KO at concentration ranging from about 85% to about 95%, preferably at about 90%; Human Platelet Lysate (HPL) at concentration ranging from about 5% to about 20%, preferably at about 10%; 0.5% 10000 μg/mL penicillin/streptomycin and 1% IX Glutamine.

9. The method as claimed in claim 4, wherein the culturing is carried out at temperature of about 37°C, at about 5% CO₂ atmosphere and at density ranging from about 800 cells/cm 2 to about 1000 cells/cm 2 , preferably about 1000 cells/cm 2.

10. The method as claimed in claim 4, wherein the stem cells are further subjected to cryopreserving and recovery.

11. The method as claimed in claim 4, wherein the dental tissue is obtained from teeth selected from group comprising permanent teeth, deciduous teeth and periodontal ligament; or any combinations thereof.

12. A method of obtaining Islet-like Cell Aggregates (ICAs) from Dental-Pulp Stem Cells, said method comprising acts of:

a) culturing the DPSCs in Definitive Endoderm Differentiation Medium (DEDM) to obtain cultured stem cells;

13. The method as claimed in claim 12, wherein the Islet-like Cell Aggregates (ICAs) produce human insulin.

14. The method as claimed in claim 12, wherein the DEDM comprises Dulbecco's modified Eagle's medium Knock Out (DMEM-KO) at concentration ranging from about 95% to about 99%, preferably about 99%; Bovine Serum Albumin(BSA) at concentration ranging from about 5% to about 2%, preferably about 1%; insulin-transferrin- selenium(ITS) at concentration ranging from about 5X to about 2X, preferably about IX; activin A at concentration ranging from about 2 nM to about 5 nM, preferably about 4nM; 1 sodium butyrate at concentration ranging from about 0.5nM to about 2 nM, preferably about InM; and 2-mercaptoethanol at concentration ranging from about 20 μΜ to about 60 μΜ , preferably about 50 μΜ.

15. The method as claimed in claim 12, wherein the PEDM comprises DMEM-KO at concentration ranging from about 95% to about 99%, preferably about 99%; BSA at concentration ranging from about 0.5% to about 2%, preferably about 1%; ITS at concentration ranging from about 0.5X to about 2X, preferably about IX; and taurine at concentration ranging from about 0.1 mM to about 0.5mM, preferably about 0.3mM.

16. The method as claimed in claim 12, wherein the IHMM comprises DMEM-KO at concentration ranging from about 95% to about 99%, preferably about 99%; BSA at concentration ranging from about 1% to about 2%, preferably about 1.5%; ITS at concentration ranging from about 0.5X to about 2X, preferably about IX; taurine at concentration ranging from about 2mM to about 4mM, preferably about 3 mM; glucagon-like peptide at concentration ranging from about 50nM to about 150nM, preferably about 100 nM; nicotinamide at concentration ranging from about 2.5mM to about 2mM, preferably about 1 mM; and nonessential amino acids at concentration ranging from about 2X to about 1X, preferably about 1X.