WO2012152717A1 - Milieu de culture pour différencier des cellules souches en cellules de type β - Google Patents

Milieu de culture pour différencier des cellules souches en cellules de type β Download PDF

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WO2012152717A1
WO2012152717A1 PCT/EP2012/058292 EP2012058292W WO2012152717A1 WO 2012152717 A1 WO2012152717 A1 WO 2012152717A1 EP 2012058292 W EP2012058292 W EP 2012058292W WO 2012152717 A1 WO2012152717 A1 WO 2012152717A1
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cells
stem cells
free medium
serum free
medium
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Izzi LEO
Zanini CRISTINA
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Euroclone Spa
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/335—Glucagon; Glucagon-like peptide [GLP]; Exendin
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/115—Platelets, megakaryocytes
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1361—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from dental pulp or dental follicle stem cells

Definitions

  • the present invention relates to a serum-free culture medium for differentiating stem cells into ⁇ like cells.
  • the present invention originates in the field of serum-free culture media for stem cells.
  • the present invention concerns a formulation of a serum-free medium for culturing and differentiating mesenchymal stem cells into pancreatic ⁇ like cells.
  • Type I diabetes mellitus is an immunitary disease, having a genetic predisposition, characterized by an autoimmune response directed towards pancreatic ⁇ cells.
  • Type I diabetes mellitus is also referred to as juvenile or insulin-dependent diabetes, as it occurs in young subjects and requires insulin administration.
  • the treatment for this disease involves monitoring of blood glucose levels during the day, and subcutaneous administration of appropriate insulin doses divided up throughout the day as a function of the blood glucose levels detected.
  • pancreatic transplant or transplant of pancreatic islets An alternative treatment to pharmacotherapy, intended for the most severe cases, involves pancreatic transplant or transplant of pancreatic islets.
  • pancreatic transplant or transplant of pancreatic islets An alternative treatment to pharmacotherapy, intended for the most severe cases, involves pancreatic transplant or transplant of pancreatic islets.
  • such treatment is exposed to the organ rejection risks typical of transplant surgery, and requires chronic administration of cytotoxic immunosuppressants to the patient anyway.
  • stem cells have allowed stem cells to be selected as a possible valuable alternative to the treatment forms currently available for diabetes mellitus, in particular Type I diabetes mellitus, through neogenesys and regeneration of insulin-secreting ⁇ cells.
  • Stem cells are found in all multicellular organisms. They maintain their capability for regeneration through mitotic cell division and are able to differentiate into a wide range of possible types of specialized cells. Consequently, physicians working in the field of biotechnology view stem cells as a repair system suitable for integrating or providing cells of a specific organ or tissue of a human organism.
  • Stem cells plasticity allows for them to be subsequently manipulated ex-vivo with the aim to obtain specific insulin-secreting cells that are genetically identical to the patient's cells.
  • Such cell type can be used to regenerate pancreatic tissue through implant techniques that do not pose the known rejection risks, thus making the administration of immunosuppressants unnecessary.
  • a general object of the present invention consists in providing a medium formulation that is specific for the culture and differentiation of mesenchymal stem cells, these cells originating from several cellular sources such as, for example, pancreatic islets, bone marrow aspiration and dental pulp, into ⁇ like cells, for their possible use in treatments for diabetes mellitus.
  • a serum-free type of culture medium for mesenchymal stem cells, that is particularly suitable for the neogenesys of insulin-secreting cells, comprising a base medium for culturing stem cells comprising Platelet Lysate (PL), retinoic acid and derivatives and esters thereof, and GLPI-1 (Glucagon like peptide I).
  • PL Platelet Lysate
  • retinoic acid and derivatives and esters thereof retinoic acid and derivatives and esters thereof
  • GLPI-1 Glucagon like peptide I
  • PL Platelet Lysate
  • retinoic acid retinoic acid
  • GLPI-1 Glucagon like peptide I
  • the invention relates to the selection and incorporation of the three active components into a medium formulation suitable for culturing stem cells.
  • stem cells are cellule primitive, non-specialised cells endowed with the capability to develop into other body cell types.
  • stem cells include mesenchymal stem cells (MSCs), without being restricted thereto.
  • MSCs mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • MSCs are pluripotent mesenchymal stromal cells and are a population of heterogenous cells from both a phenotypical and a functional point of view. MSCs normally divide to generate precursors or precursor cells that subsequently differentiate or develop into mature cells endowed with characteristic forms and specialised functions.
  • mesenchymal stem cells are normally generated by the bone marrow (BM), their generation is also possible starting from a variety of other tissues such as, for example, peripheral blood, adipose tissue, amniotic fluid, renal glomerulus, liver, and human stratified epithelia and, as proven by the applicant, also starting from pancreatic islets (Hl- MSCs) and dental pulp (PD-MSCs).
  • BM bone marrow
  • Hl- MSCs pancreatic islets
  • PD-MSCs dental pulp
  • culture medium for stem cell culture comprises media the formulations of which are suitable for culturing and growing stem cells, in particular mesenchymal-type stem cells.
  • a suitable medium is formulated based on amino acids, vitamins and inorganic salts, which allow for culturing mesenchymal stem cells.
  • a medium suitable for culturing the stem cells of the invention may comprise Dulbecco's Modified Eagle's Medium (DMEM) as base medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the Platelet Lysate (PL) component present in the formulation of the medium of the invention is a typical supplement component replacing foetal bovine serum (FBS) employed in media for cell culturing.
  • FBS foetal bovine serum
  • LP occurs as a straw-yellow liquid and is obtained from platelets isolated from whole blood after a freezing process at low temperatures followed by a thawing process, the latter being normally carried out the following day. The above procedure is usually repeated 3 or 4 times and the remaining surnatant is purified and stocked, typically at a temperature of -20°C.
  • PL features a high content in the growth factors required for cell expansion.
  • the retinoc acid component present in the formulation of the culture medium is vitamin A oxidized form.
  • Retinoic acid derivatives may also be used within the scope of the invention, including retinoic acid isoforms, for example the trans forms such as tretinoin, and the cis forms such as 9-cis-retinoic acid (Alitretinoin), 13-cis-retinoic acid (Isotretinoin), as well as retinoic acid esters such as, for example, the isobutyl ester, and in general derivatives and esters disclosed in the scientific literature such as those disclosed by Kutner et al in Proc. Natl. Acad. Sci USA, Vol. 83, pp. 6781 -6784, Sett. 1986 Biochemistry.
  • the GLPI-1 component is derived from the transcription product of the proglucagone gene.
  • the above is a potent antiglycemic hormone, typically produced in the bowel lumen by the L cells.
  • GLPI-1 also features the ability to increase beta cells mass and to increase the expression of the insulin gene.
  • the stem cell culture medium of the invention comprises one or more amino acid(s).
  • amino acid indicates natural occurring amino acids, non naturally occurring amino acids, amino acid analogs, in the forms of both D and L stereoisomers, if their structures allow for such stereoisomeric forms.
  • Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonin, triptofan, tyrosine, valine, and mixtures thereof.
  • Non naturally occurring amino acids include, without being limited thereto: azetidincarboxylic acid, 2-aminoadipic acid, 3-adipic acid, ⁇ -alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoeptanoic acid, 2- aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopymelic acid, 2,4 diaminoisobutyric acid, desmosine, isodesmosine, allo-isoleucine, norvaline, norleucine, ornithine, pipecolic acid and mixtures thereof.
  • Amino acid analogs include naturally occurring amino acids and those amino acids that are not chemically blocked, either reversibly or irreversibly, or modified on their N-terminal amino groups, or on their side chain groups as for example methionine, methionine sulfone, methionine sulfoxide, S-carboxymethyl-cysteine, S- carboxymethyl-cysteine sulfoxide.
  • the stem cells culture medium comprises the following naturally occurring amino acids: L-arginine, L-cysteine, L-glutamine, glycine, L-histidine, L- isoleucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-triptofan, L- tyrosine, L-valine.
  • the stem cells culture medium of the invention comprises glutamine as an amino acid.
  • the stem cell culture medium comprises one or more inorganic ion salts.
  • inorganic ion salt comprises any inorganic salt that is suitable for use in a typically solid elettrolytic medium.
  • the inorganic ion salt preferably contains at least one atom preferably selected from the group comprising Li, Na, K, Cs, Ag, Cu, and Mg.
  • Suitable inorganic salts include CaCI 2 , MgS0 4 , CuCI 2, NaHC0 3 , NaCI, KCI, LiCI0 4 , LiCI, LiSCN, LiBF 4 , LiAsF 6 , LiCF 3 S0 3 , LiPF 6 , NaSCN, CsSCN, FeS0 4 , CuS0 4 , MgCI, AgN0 3 , MnS0 4 , (NH 4 ) 2 M0 4 , Na 2 HP0 4 , Na 2 Se03, NaSi0 3 , KH 2 P0 4 , SnCI 2 , ZnS0 4 , NiCI 2 , KCI, and Mg(CI0 4 ) 2 .
  • the salt may occur in its hydrate form such as, for example, Fe(N0 3 ) 3 .9H 2 0 and NaH 2 P0 4 .H 2 0.
  • the stem cell culture medium of the invention comprises one or more growth factor(s).
  • growth factor refers to one or more molecule(s) imparting an advantage or drawback to the host cell or replicator.
  • Typical growth factors include nutritive substances, enzymes required for the metabolism of nutritive substances, and binding as well as structural proteins, proteins involved in the replication, metabolism, formation and maintenance of the essential or cellular structural components, and in subcellular growth.
  • Growth factors include, without being limited thereto, epidermal growth factor, endothelial growth factor, transforming growth factor, platelet-derived growth factors, insulin, insulin-like growth factors, glial growth factor, basic fibroblast growth factor, growth hormone, bovine pituitary extract, recombinant epidermal growth factor, hydrocortisone, thymidine, triiodothyronine and mixtures thereof.
  • epidermal growth factor EGF
  • FGF fibroblast growth factor
  • the stem cell culture medium of the invention comprises one or more vitamin(s).
  • suitable vitamins include water-soluble vitamins such as, for example, B group vitamins, vitamin H, vitamin C in all its possible isomeric forms, L-ascorbate, L-ascorbic acid 2-phosphate, and/or non water-soluble vitamins such as vitamin A, vitamin E, and derivatives thereof.
  • the medium includes thiamine, riboflavin, pyridoxine, nicotinamide, D- pantothenic acid, myo-inositol, choline chloride, and folic acid.
  • the stem cell culture medium of the invention comprises one or more anti-oxidant(s).
  • anti-oxidants include vitamin C, vitamin E, N-acetyl-L- cysteine, and nicotinamide.
  • the stem cell culture medium of the invention comprises one or more components including one carbon source, typically a sugar such as, for example, glucose, galactose, and sugar mixtures.
  • a carbon source typically a sugar such as, for example, glucose, galactose, and sugar mixtures.
  • the stem cell culture medium of the invention comprises DMEM and optionally one or more of the following substances, sodium pyruvate, phenol red, sodium chloride, glutathione, bacto-peptone, and mixtures thereof.
  • the culture medium of the present invention further incorporates beta cellulin, a substance inducing the conversion of precursor cells of pancreatic islets into ⁇ like cells.
  • the culture medium of the present invention incorporates Activin A, which belongs to the "transforming growth factor-beta (TGF- ⁇ )" superfamily, and is essential for the formation of both mesoderm and exoderm during gastrulation.
  • the stem cell culture medium comprises DMEM, glucose preferably from 4.5 to 25 mM, Platelet Lysate (PL), preferably from 1 to 10% in volume, retinoic acid and/or its derivatives and/or esters, Activin, GLPI-1 , epidermal growth factor (EGF), fibroblast growth factor (FGF), beta cellulin, nicotinamide, and glutamine.
  • the culture medium of the present invention has a pH value of from 7 to 7.8, preferably of from 7.15 to 7.4.
  • the use of the medium selectively promotes the differentiation of the stem cells into ⁇ like insulin-secreting cells.
  • mesenchymal stem cells lose their fibroblastoid form after being stimulated for one week and matured for further 10-15 days, in the presence of the medium of the invention, to take on a less elongated conformation, and arrange themselves in aggregates while differentiating themselves into insulin-secreting ⁇ cells.
  • Figure 1 shows the multilineage differentiation of BM-MSCs and HI-MSCs.
  • Figure 2 shows the morphological changes and ultrastructural analysis of BM-MSCs and HI-MSCs during differentiation toward ⁇ like cells.
  • Figure 3 shows the analysis of phenotype modification by flow cytometry after differentiation of HI-MSCs toward ⁇ like cells.
  • Figure 4 shows the immunofluorescence analysis of HI-MSCs after differentiation toward ⁇ like cells.
  • Figure 5 shows the measurement of insulin secretion by ELISA.
  • Figure 6 shows the DE Western blot of BM-MSCs and HI-MSCs treated or untreated with differentiative medium for insulin.
  • Figure 7 shows the morphological changes (magnification 20X) during differentiation of HI-MSCs with different media toward ⁇ like cells.
  • Figures 8A-8F show the analysis of phenotype modification by flow cytometry of HI-MSCs after induction of differentiation with different media toward ⁇ like cells.
  • Figure 9 shows the morphological changes (magnification 4X) during differentiation of HI- MSCs with different media toward ⁇ like cells.
  • DMEM glucose 4.5 mM
  • Platelet Lysate (PL) 5% retinoic acid 10 ⁇
  • Activin 10 ng/ml Activin 10 ng/ml
  • GLPI-1 Glucagon like peptide I
  • EGF Epidermal growth factor
  • FGF Fibroblast growth factor
  • WHOLE medium is comprised of the BASE medium, DMEM-LG supplemented with the supplements of example 1 .
  • the supplements corresponding to 5 % of the final volume are calculated for a medium volume of 10 ml.
  • retinoic acid 100 mg are weighted and dissolved in DMSO up to a final volume of 33 ml, such that a 10 mM stock solution is obtained.
  • 500 ul aliquots are prepared and frozen.
  • the aliquotes should be stored away from the light; to this aim, they should be covered with aluminium paper.
  • DMEM-LG 0.122 mg is weighted and the powder is dissolved in a small volume of DMEM-LG. DMEM-LG is subsequently added up to a final volume of 1 ml, such that a stock solution of 1 M/L is obtained .
  • the thawed growth factor should be stored in the refrigerator for maximum one week.
  • Activin A For 10 ug of Activin A, 1 ml of DMEM medium is added to the vial and mixed thoroughly. 50 ul aliguots are prepared and frozen at -20°C.
  • DMEM medium For 10 ug of B cellulin, 1 ml of DMEM medium is added to the vial and mixed thoroughly. 50 ul aliguots are prepared and frozen at -20°C.
  • DMEM medium For 10 ug of FGF, 1 ml of DMEM medium is added to the vial and mixed thoroughly. 50 ul aliguots are prepared and frozen at -20°C.
  • the cryovial is thawed that contains mesenchymal stem cells from the pancreas or mesenchymal stem cells obtained from medullary fine-needle aspiration in a bath thermostated at 37 °C. Once thawed , the cryovial is removed from the bath and the vial is washed with 70% sterile ethanol. Cells should not be spun during thawing.
  • the cryovial is gently tilted for the cells to be mixed.
  • the cell suspension is sterile-transferred into a 50 ml conic tube containing 250-500 ul of 1 mg/ml DNase I (deoxyribonucleasi I) to prevent the formation of cell clusters.
  • DNase I deoxyribonucleasi I
  • cryovial is washed with 1 ml of the chosen medium and this medium is slowly added to 50 ml Falcon containing the cell suspension. Suspension is carried out by gentle rotation.
  • the cell suspension is centrifuged at 300 g at room temperature for 10'.
  • the surnatant is be carefully removed with a pipette while trying not to touch the pellet.
  • the cells should be resuspended by gently hitting the pellet, and the desired medium, preferably DMEM-LG with 2% FBS (1-2 ml), should be added to the cells for counting.
  • the desired medium preferably DMEM-LG with 2% FBS (1-2 ml)
  • DNase I should be added to the cell suspension final volume to avoid the formation of cell clusters.
  • the penicillin/streptomycin flask is thawed.
  • mesenchymal cells from medullary fine-needle aspiration 1 * 10 6
  • the cells are cultured for 5-10 days. Upon reaching about 80% confluence, the medium is replaced with whole medium containing the A+B supplement for inducing islet cell formation (5-7 ml).
  • thermostated bath is switched on at 37 °C.
  • PBS and medium are bathed upon reaching the preset temperature.
  • the medium is sucked.
  • a washing is performed inside the flask with PBS 9 ml.
  • the cells are detached with trypsin and pelleted at 1200 rpm for 5 min. One washing is performed in PBS to block the trypsin effect; seed T25 ultra-low- attachment in the plate with base medium + B supplement (5- 7ml). From this point in time onwards, the cells will grow in suspension
  • the cells differentiate and mature into islet cells that appear as spheroidal aggregates.
  • the cells are pelleted by centrifuging at 1200 rpm for 5 min. The surnatant is removed and a washing is performed in PBS.
  • the cells normally mature over 14-20 days.
  • HI-MSCs human islet-mesenchymal stem cells
  • BM-MSCs medullary fine-needle aspiration
  • PD-MSCs dental pulp
  • HI-MSCs Mesenchymal stem cells from pancreatic islets
  • pancreatic islets were digested with Liver Digestion Medium (Gibco, Grandlsland, NY) for 10 minutes at 37°C and set to adhere in the presence of fibronectin (0.5 microgram/ml) in a medium comprised of: alpha-Minimum essential medium/Endothelial cell basal medium-1 (aMEM/EBM) (3: 1 ) (Gibco/Cambrex) with glutamine (5 mM), Hepes (12 mM, pH 7.4), penicillin (50 lU/ml), streptomycin (50 mg/ml) (Sigma, St. Louis, MO), and 10% FCS.
  • Liver Digestion Medium Gibco, Grandlsland, NY
  • fibronectin 0.5 microgram/ml
  • aMEM/EBM alpha-Minimum essential medium/Endothelial cell basal medium-1
  • glutamine 5 mM
  • Hepes (12 mM, pH 7.4
  • penicillin 50 lU/
  • the isolated cells were then analyzed by cytofluorimetry and immunofluorescence to determine their phenotypes.
  • the precursors isolated from the islets were exposed to high glucose concentration to stimulate insulin release.
  • the surnatants of the cultured cells were then withdrawn and subjected to ELISA test for insulin assessment.
  • the inventors could therefore isolate a population of resident stem cells displaying mesenchymal markers capable of differentiating into insulin-secreting cells, adipocytes, chondrocytes, epatocytes, and osteocytes.
  • Such cell express markers that are typically expressed by such mesenchymal medullary stem cells as: CD105, CD29, CD73, CD166, CD146, CD44, CD90, vimentin and nestin (mesenchymal medullary stem cells and liver-residing cells). Moreover, such cells do not express hematopoietic and endothelial markers such as: CD34, CD45 and CD133 (hematopoietic stem cells), KDR and CD31 (endothelial cells).
  • embryonic markers as nanog, oct4 and musashi (embryonic stem cells) and a fetoprotein (expressed during embryonic development by the exocrine pancreas and by common pancreas and liver precursors). Furthermore, they express glucose receptor Glut2.
  • BM-MSCs medullary fine-needle aspiration
  • PD-MSCs dental pulp
  • mesenchymal stem cells from the pancreas, fine-needle aspiration and pulp were induced to differentiate into adipocytes with appropriate culture media by using the EUROMED Human Adipogenic differentiation kit medium, into chondrocytes by using EUROMED Chondrogenic differentiation kit and into osteoblasts by using EUROMED Human Osteogenic differentiation kit.
  • MSCs should be induced to adipogenic differentiation after having been expanded with Euromed Mesenchymal medium MSC e having been taken to confluence. The cells at confluence are then placed in a medium with EUROMED Human Adipogenic differentiation kit. The medium is able to promote MSC differentiation into adipocytes in about 2-3 weeks. MSCs should be induced to chondrogenic differentiation in pellet.
  • 2.5x10 5 MSCs are placed in a 15 ml Falcon in 1 ml of Euromed Mesenchymal MSC medium and subsequently centrifuged at 150 x g for five minutes at room temperature, while sucking and removing the surnatant (this operation is to be repeated twice).
  • Five ml of Chondrogenic differentiation kit whole medium are added to the thus formed pellet. Every two-three days, the medium is sucked via a sterile pipette and replaced with fresh medium. For complete differentiation, 14-28 days are required.
  • the pellet is fixed in formalin or alcohol and embedded in paraffin for the preparation of histological sections.
  • the sections are then stained with safranin O to detect glicosylaminoglycans.
  • safranin O By placing 1X10 5 MSCs in a T25, MSCs differentiate into osteoblasts with EUROMED Human Osteogenic over about 14-21 days. The cells are then stained with Alizarin Red to identify the calcium depositions.
  • BM-MSCs and HI-MSCs differentiate towards those histotypes.
  • Panel (A) shows cultured BM-MSCs and Panel (B) shows HI-MSCs before differentiation.
  • Panels (C-D) show representative micrographs of osteogenic differentiation: calcium depositions were detected by Alizarin Red after culturing BM-MSCs (C) and HI-MSCs (D) for 21 days in specific osteogenic medium.
  • Panels (E-F) show representative micrographs of adipogenic differentiation showing the presence of lipid droplets after 21 days in adipogenic differentiating medium in of BM- MSCs (E) and HI-MSCs (F) respectively, (magnification x 200).
  • Panels (G and H) show representative micrographs of chondrogenic differentiation shown by formation of a pellet positive to safranin O (G) and alcian blue (H) after culturing Hl- MSCs in chondrogenic medium for 28 days (magnification x 100).
  • MSCs obtained from all of the above mentioned sources show remarkable plasticity. This accounts for these cells' capability to differentiate towards different phenotypes such as ⁇ like cells.
  • MSCs Mesenchymal stem cells from pancreas, fine needle aspiration, and pulp, were cultured by plating 1x10 5 cells on a T75 in EuroMed Human Mesenchymal Stem Cell Kit (Codice: ECM0888K - Euroclone). Upon reaching 60% confluence, the medium was replaced with the formulation of example 1 to assess the differentiation capabilities and neogenesys of the ⁇ like cells. Microscopy morphology of MSCs at day six of the plate culture is illustrated in Fig. 2, Panel A. After seven days, the whole medium is replaced with fresh medium. The culture can be performed either in adhesion or in suspension.
  • the differentiation into ⁇ like cells was conducted by culturing MSCs originating from the above-mentioned sources with the serum-free formulation of example 1 in chambers slides treated with fibronectin (SIGMA) and in T25. After being stimulated for 1 week in the presence of whole medium, the cells lose their fibroblastoid form to take on a less elonged conformation, and they arrange in clusters.
  • Panel B shows the microscope morphology of such cells by displaying the aggregation leading to differentiation into ⁇ like cells.
  • the cells were cultured on low attachment plastic surfaces to facilitate the formation of cell clusters (viable spherical aggregates) using the formulation reported above. Subsequently, they were placed in chamber slides with fibronectin to assess differentiation by immunofluorescence.
  • Panel C shows ⁇ cell phenotype at the maturation stage, after 18 days of plate cu Iture.
  • Panels D, E, and F show the ultrastructural analysis of MSCs prior to and after differentiation induced by whole medium.
  • the HI-MSCs and BM-MSCs were fixed in 2.5% sodium cacodylate-buffered glutaraldehyde immediately after in vitro differentiation.
  • Pelleted cells were post-fixed in osmium tetroxide in the same buffer, dehydrated in ethanol and embedded in Araldite. Thin sections stained with uranyl acetate and lead citrate were studied in a Philips 400T transmission electron microscope.
  • Cytofluorimetric analyses were performed using the following monoclonal antibodies, all of them conjugated to Alexa Fluor: CD44 (Cell Signaling), CD29, CD33,CD151 (BD), Insulin, C Peptide, PDX1 , GLUT-2. All incubation runs were performed for 30 minutes at 4°C in 100 ul phosphate buffer (PBS) and 0.1 % TRITON 100X. For each sample, 10,000 cells were analyzed using FACSCALIBUR cytometer (BD). Using Cell Quest software (BD), absolute values as well as population percentages were obtained for the populations selected for each experiment.
  • the cells do not express hematopoietic markers such as CD34, CD45, and CD33.
  • cytofluorimetric analysis shows about 24% of positive cases for the PDX-1 (Pancreas Duodenum Homeobox-1 ) which plays a major role in inducing differentiation of ⁇ like pancreatic cells.
  • C-Peptide is a protein fraction secreted from the pancreas into the circulation in amounts comparable to those of insulin. By assaying this substance, an accurate assessment can be obtained of the amount of insulin produced in the pancreas.
  • Figure 3 shows cytometric analysis in HI-MSCs, the phenotype is compatible with ⁇ like cells. Positivity for Insulin (A) and Glut-2 (E) is higher than 50%, while it ranges between 20 and 40% for PDX-1 (B) and C-peptide (D) IMMUNOFLUORESCENCE
  • Indirect immunofluorescence was performed on differentiated cells after 2% paraformaldehyde fixation. Cells were permeabilized with methanol (MetOH) for some antigens.
  • Primary antibodies were diluted according to the manufacturer's instructions. The following antisera were utilized: pancreatic duodenal homeobox gene-1 (PDX1 ), Insulin, C Peptide. Secondary antibodies were Anti-Rabbit IgG Alexa Fluor 488 conjugate, Anti- Mouse IgG Alexa Fluor 488 conjugate. Non-immune rabbit or mouse sera were used as negative controls at a 1 :50 dilution.
  • the cells turned out positive at fluorescence microscopy for PDX1 , C Peptide, Insulin.
  • PDX1 PDX1
  • C Peptide C Peptide
  • Insulin Insulin
  • Insulin release was checked on the surnatants by ELISA test using the Insulin Kit from Dako (Code: K6219). Test principle.
  • the small format gel was transferred to a nitrocellulose membrane.
  • Membranes were blocked with 3% w/v BSA in PBS/Tween 20-0.1 % v/v for 1 hour; membranes were then washed three times with PBS and probed with a 1 :200 dilution of Insulin O.N. at 4°C.
  • Membranes were washed six times with PBS, incubated for 1 hour with a 1 : 1000 dilution of anti-rabbit IgG peroxidase-labeled antibody and immunoreactivity was detected with an enhanced chemiluminescence kit.
  • 2-DE Western blotting for Insulin detected the hormone in both cell populations, thus confirming the immunological data.
  • Figure 6 shows that insulin expression was higher in HI-MSC differentiated cells compared to the BM counterpart.
  • FBS foetal bovine serum
  • the other relevant piece of data refers to insulin; the insulin content, as measured by cytofluorimeter ( Figure 8) is very low, being about 6% in contrast with the clearly positive value (higher than 50%) in the formulation object of the present invention. Moreover, the contents of both C-peptide and PDX- 1 are also very low, as can be observed from Figure 8 medium 1 phenotype.
  • GLP-1 was not included in the whole formulation prepared according to example 1.
  • the formulation contained: DMEM, glucose 4.5 mM, Platelet Lysate (PL) 4%, retinoic acid 10 ⁇ , Activin 10 ng/ml, EGF (Epidermal growth factor) 20 ng/ml, FGF (Fibroblast growth factor) 10 ng/ml, Beta-cellulin 10 ng/ml, nicotinamide 10 mM/L, glutamine 2 mM.
  • Retinoic acid was not included in the whole formulation prepared according to example 1.
  • the formulation contained: DMEM, glucose 4.5 mM, Platelet Lysate (PL) 4%, Activin 10 ng/ml, GLPI-1 (Glucagon like peptide I) 200 ng/ml, EGF (Epidermal growth factor) 20 ng/ml, FGF (Fibroblast growth factor) 10 ng/ml, Beta-cellulin 10 ng/ml, nicotinamide 10 mM/L, glutamine 2 mM.
  • the spheres appear morphologically smaller as is shown in figure 7 Panel C, however the most relevant piece of data refers to the phenotype, with the % of positive cases for the markers insulin and PDX- -1 being lower than that found for the whole medium (less than 20%) as is observed in Figure 8 phenotype medium 4.
  • GLP-1 nor retinoic acid were included in the whole formulation prepared according to example 1.
  • the formulation contained: DMEM, glucose 4.5 mM, Platelet Lysate (PL) 4%, Activin 10 ng/ml, EGF (Epidermal growth factor) 20 ng/ml, FGF (Fibroblast growth factor)10 ng/ml, Beta-cellulin 10 ng/ml, nicotinamide 10 mM/L, glutamine 2 mM.
  • GLP-1 was replaced with exendin-4.
  • the formulation contained: DMEM, glucose 4.5 mM, Platelet Lysate (PL) 5%, retinoic acid 10 ⁇ , Activin 10 ng/ml, exendin ⁇ l 200 ng/ml, EGF (Epidermal growth factor) 20 ng/ml, FGF (Fibroblast growth factor) 10 ng/ml, Beta-cellulin 10 ng/ml, nicotinamide 10 mM/L, glutamine 2 mM.
  • GLP-1 is more effective than exendin ⁇ l in the formulation object of the present invention.

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Abstract

Selon un premier aspect, l'invention concerne un milieu de culture sans sérum pour différencier des cellules souches mésenchymateuses en cellules de type β sécrétant de l'insuline, ledit milieu comprenant un milieu de base pour la culture de cellules souches et une combinaison de lysat plaquettaire (PL), d'acide rétinoïque et de GLPI-1 (peptide I de type glucagon).
PCT/EP2012/058292 2011-05-06 2012-05-04 Milieu de culture pour différencier des cellules souches en cellules de type β WO2012152717A1 (fr)

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NL2010222C2 (en) * 2013-02-01 2014-08-04 Conradus Ghosal Gho Composition and method for generating a desired cell type and/or tissue type from hair follicular stem cells.
NL2010225C2 (en) * 2013-02-01 2014-08-04 Conradus Ghosal Gho Composition and method for preserving, transporting and storing living biological materials.
CN105039239A (zh) * 2015-07-08 2015-11-11 深圳爱生再生医学科技有限公司 细胞转化诱导液及其应用
CN107937335A (zh) * 2017-11-15 2018-04-20 广州润虹医药科技股份有限公司 用间充质干细胞制备成纤维细胞的培养基及方法
CN109112101A (zh) * 2017-06-26 2019-01-01 拜西欧斯（北京）生物技术有限公司 一种成纤维细胞培养基及其应用
CN109182262A (zh) * 2018-09-25 2019-01-11 深圳市五零生命科技有限公司 一种间充质干细胞无血清培养基
CN111944752A (zh) * 2020-08-28 2020-11-17 广州同康生物科技有限公司 骨骼肌干细胞无血清培养基及其制备方法
CN117398525A (zh) * 2023-12-05 2024-01-16 北京大学人民医院 表达Exendin-4蛋白的间充质干细胞及其用途
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WO2014087435A1 (fr) * 2012-12-06 2014-06-12 Euroclone S.P.A. Méthode de diagnostic de maladies auto-immunes
NL2010222C2 (en) * 2013-02-01 2014-08-04 Conradus Ghosal Gho Composition and method for generating a desired cell type and/or tissue type from hair follicular stem cells.
NL2010225C2 (en) * 2013-02-01 2014-08-04 Conradus Ghosal Gho Composition and method for preserving, transporting and storing living biological materials.
WO2014120014A1 (fr) * 2013-02-01 2014-08-07 Conradus Ghosal Gho Composition et procédé pour conserver, transporter et stocker des matières biologiques vivantes
US11840706B2 (en) 2013-02-01 2023-12-12 Conradus Ghosal Gho Composition and method for generating a desired cell type and/or tissue type from hair follicular stem cells
CN105039239A (zh) * 2015-07-08 2015-11-11 深圳爱生再生医学科技有限公司 细胞转化诱导液及其应用
CN109112101A (zh) * 2017-06-26 2019-01-01 拜西欧斯（北京）生物技术有限公司 一种成纤维细胞培养基及其应用
CN107937335A (zh) * 2017-11-15 2018-04-20 广州润虹医药科技股份有限公司 用间充质干细胞制备成纤维细胞的培养基及方法
CN109182262A (zh) * 2018-09-25 2019-01-11 深圳市五零生命科技有限公司 一种间充质干细胞无血清培养基
EP4092106A4 (fr) * 2020-01-13 2024-03-06 Qingdao Restore Biotechnology Co., Ltd. Inducteur pour induire des cellules souches mésenchymateuses à se différencier en cellules d'îlots
CN111944752A (zh) * 2020-08-28 2020-11-17 广州同康生物科技有限公司 骨骼肌干细胞无血清培养基及其制备方法
CN111944752B (zh) * 2020-08-28 2021-09-03 广州同康生物科技有限公司 骨骼肌干细胞无血清培养基及其制备方法
CN117398525A (zh) * 2023-12-05 2024-01-16 北京大学人民医院 表达Exendin-4蛋白的间充质干细胞及其用途
CN117398525B (zh) * 2023-12-05 2024-03-12 北京大学人民医院 表达Exendin-4蛋白的间充质干细胞及其用途

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