WO2012117333A1 - Isolement et expansion de cellules souches adultes, composition thérapeutique les comprenant et utilisations associées - Google Patents

Isolement et expansion de cellules souches adultes, composition thérapeutique les comprenant et utilisations associées Download PDF

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WO2012117333A1
WO2012117333A1 PCT/IB2012/050893 IB2012050893W WO2012117333A1 WO 2012117333 A1 WO2012117333 A1 WO 2012117333A1 IB 2012050893 W IB2012050893 W IB 2012050893W WO 2012117333 A1 WO2012117333 A1 WO 2012117333A1
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concentration ranging
dpscs
cells
medium
dmem
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Vijayendran GOVINDASAMY
Ramesh R BHONDE
Satish Totey
Anjan Kumar DAS
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Stempeutics Research Malaysia Sdn Bhd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0056—Xeno-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • 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.
  • 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.
  • DPSCs dental pulp stem cells
  • Dental Pulp Stem Cells or 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.
  • MSC mesenchymal stromal/stem cells
  • 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.
  • 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 ⁇ -galactosidase (SA-P-gal) staining of DPSCs cultured in various growth media. (Arrow shows positive cells);
  • SA-P-gal Senescence associated ⁇ -galactosidase
  • 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.
  • 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
  • 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.
  • HLA- DR- Human Leukocyte Antigen -DR Figure 4 Shows the expression profile of pluripotent and lineage-specific stem cell markers of SCD and DPSCs.
  • FIG. 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.
  • Figure 8 shows karyotyping analysis and transformation marker analysis of DPSC cultures (HPL and FBS) at pre- and post-large-scale expansion.
  • 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. .
  • FIG 10 shows Expression of endoderm and pancreatic hormone genes in islet like cell aggregates (ICAs)
  • ICAs Islet-like Cell Aggregates
  • 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).
  • DMEM-KO Dulbecco's Modified Eagle's Medium- Knock Out
  • FBS Fetal Bovine Serum
  • Pen-Strep Fetal Bovine Serum
  • Glutamine Ascorbic acid
  • ITS Insulin-Transferrin-Selenium
  • 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 ⁇ g/mL to about 500 ⁇ g/mL , preferably about 100 ⁇ g/mL; and the Insulin-Transferrin-Selenium (ITS) is at concentration ranging from about 0.5X to about 2X, preferably about IX.
  • ITS Insulin-Transferrin-Selenium
  • the present disclosure further relates to a method for large-scale production of dental pulp derived stem cells (DPSCs), said method comprising acts of:
  • 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 ⁇ g/mL to about 500 ⁇ g/mL, preferably about 100 ⁇ g/mL; and Insulin-Transferrin-Selenium (ITS) at concentration ranging from about 0.5X to about 2X, preferably about IX.
  • 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
  • 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%.
  • 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.
  • 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 ⁇ g/mL penicillin/streptomycin and 1% IX Glutamine.
  • HPL Human Platelet Lysate
  • the culturing is carried out at temperature of about 37°C, at about 5% CO 2 atmosphere and at density ranging from about 800 cells/cm 2 to about 1000 cells/cm 2 , preferably about 1000 cells/cm 2 .
  • the stem cells are further subjected to cryopreserving and recovery.
  • 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:
  • DEDM Definitive Endoderm Differentiation Medium
  • the Islet-like Cell Aggregates produce human insulin.
  • 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 ⁇ to about 60 ⁇ , preferably about 50 ⁇ .
  • DMEM-KO Dulbecco's modified Eagle's medium Knock Out
  • BSA Bovine Serum Albumin
  • ITS insulin-transferrin- selenium
  • 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.
  • 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.
  • 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.
  • DPSCs Dental Pulp Stem Cells
  • Example 1 Procedure for isolation of Stem cells from Dental pulp by Explants- enzymatic technique
  • 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.
  • 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.
  • 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 ⁇ g/mL and Insulin-Transferrin-Selenium of about 0.5X to about 2X.
  • transportation medium comprises of DMEM-KO-1X, FBS- 20%, Pen-Strep-2%, Glutamine-5%, Ascorbic acid-100 ⁇ g/mL and Insulin-Transferrin- Selenium (ITS)-IX.
  • transport media in the prior art use antibiotic and BSA or even Balanced Salt Solution (BSS) which have no nutritional value.
  • BSS Balanced Salt Solution
  • DMEM-KO basal medium
  • Gronthos et all does not disclose the transport medium nor the exact step as mentioned in present invention.
  • the present invention uses explants-enzymatic method.
  • the washing medium mainly comprises of 5% Pen-Strep and basal medium [DMEM- KO].
  • DMEM-KO basal medium
  • 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).
  • 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
  • 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.
  • 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.
  • dental pulp tissue is used as alternative source for isolation of Mesenchymal Stem Cells (DP-MSCs).
  • DP-MSCs Mesenchymal Stem Cells
  • anti-oxidant agent L- ascorbic acid
  • 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 ⁇ -gaiactosidase (SA- -gal) staining of DPSCs cultured in various growth media.
  • SA- -gal Senescence associated ⁇ -gaiactosidase
  • 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 The karyotyping of the DPSC cells show the cells are normal.
  • DPSCs dental pulp stem cells
  • 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.
  • FBS Fetal bovine serum
  • 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.
  • L-ascorbic acid 2 phosphate to the culture medium depends on the indication to be treated.
  • 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.
  • the DPSCs are cultured in a tailor made fashion for suitable disease to be treated.
  • 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.
  • 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).
  • 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).
  • Senescence phenomenon is monitored by ⁇ -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.
  • FIG. 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.
  • 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 ⁇
  • 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/cm 2 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/cm 2 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/ cm 2 have thin spindle- shaped cells whereas cell morphology at 200-600 cells/ cm 2 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.
  • 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)
  • 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.
  • FIG 5 an overview of the generation of neurospheres in SCD and DPSCs and gene expression profile of selected neuron markers is shown.
  • 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.
  • 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.
  • cGMP current good manufacturing practice
  • Existing protocols for cell culturing make use of fetal bovine serum (FBS), an undesirable xeno component as supplement risking the chance of contamination.
  • FBS fetal bovine serum
  • 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 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.
  • HPL 10 to 12 thawed PRP units, now called HPL
  • 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- ⁇ 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.
  • 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 ⁇ 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.
  • DMEM-KO IX Dulbecco ' s modified Eagle 's medium - knock-out
  • FBS FBS
  • Pen-Strep 5% GlutaMax
  • GlutaMax 100 ⁇ g/mL ascorbic acid
  • ITS insulin-transferrin-selenium
  • 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.
  • 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.
  • 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.
  • clinical-scale expansion procedure wherein the pooled DPSC are used for the large-scale expansion.
  • These cells are cultured at 1000 cells/cm 2 in either 10% HPL or 10% FBS supplemented with DMEM-KO (Invitrogen), 0.5% and 10000 ⁇ g/mL penicillin/streptomycin (Invitrogen) and 1% 1 X Glutamax (Invitrogen) using a T25 flask (designated as pre-large-scale expansion; total surface area 25 cm 2 ; BD Biosciences) and 5-STACK chamber (designated as post-large-scale expansion; total surface area 3180 cm 2 ; Corning Life Sciences, Chelmsford St Lowell, MA, USA; www.corning.com/lifesciences).
  • the transcriptomes and cytogenetic characteristics of DPSCs expanded under HPL and FBS are compared, revealing similar expression profile.
  • 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.
  • DPSCs cultured in HPL produced higher yield (4.98 ⁇ 0.8 x 10 8 cells/ cm 2 ; 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 10 8 cells/cm 2 ) with approximate population doubling of 6.65 and population doubling time of 39.19 ⁇ 4.1 h.
  • 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.
  • cytogenetic stability of DPSC is evaluated using conventional karyotyping. After pre- and post large- scale expansion, cells are incubated with 0.1 ⁇ 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.
  • Type 1 diabetes or Insulin-dependent Diabetes Mellitus is a disorder characterized by total loss of pancreatic ⁇ -cells as a result of autoimmune destruction.
  • IDDM Insulin-dependent Diabetes Mellitus
  • the only remedy for IDDM is islet transplantation, which is hampered by the lack of availability of donor pancreas coupled with lifelong immune suppression.
  • the only source of autologous stem cells is bone marrow, the acquisition of which is invasive and painful.
  • 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).
  • ICAs pancreatic cell lineage resembling islet-like cell aggregates
  • the figure9B shows generation of islet like aggregates from dental pulp stem cells.
  • 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.
  • 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).
  • DPSCs into ICAs are 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 10 6 cells/cm 2 ].
  • 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 ⁇ 2-mercaptoethanol. The cells are cultured in this medium for 2 days.
  • 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.
  • IHMM Islet Hormone Maturation Medium
  • FIG. 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.
  • 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.
  • 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.
  • stem cells for generating insulin-producing ICAs in the prior art.
  • 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.
  • -Present method uses a combination of explants and enzymatic dissociation technique for dental pulp processing.
  • the Present invention provides a technique for processing and culturing of DPSC.
  • it also discloses cryopreservation of DPSCs at -196°C so as to make DPSC bank for preserving DPSCs for future use.

Abstract

La présente invention concerne un procédé largement reproductible et régulier pour la production rapide et à grande échelle de DPSC de grade clinique, haute qualité. Le présent procédé de production de DPSC est économique et produit des cellules en quantités commerciales pour une utilisation dans la thérapie de transplantation autologue et allogénique. La présente invention concerne en outre une composition de milieu de transport pour la conservation et la protection de la pulpe dentaire. La présente invention concerne également un procédé de production d'agrégats cellulaires de type îlots (ICA) à partir de DPSC en utilisant un protocole en 3 étapes et en utilisant une composition de différenciation spécifique. Les ICA obtenus sont utilisés sous la forme d'une composition pharmaceutique pour le traitement de conditions associées au diabète de type I et pour le criblage de nouveaux composés antidiabétiques.
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