Method for obtaining human brown/beige adipocytes
The present invention concerns a method of producing brown/beige adipocytes from white adipose tissue cells and/or mesenchymal stem cells, in particular from subcutaneous white adipose tissue cells, and the use of said brown/beige adipocytes in a cell based therapy of a subject or in screening platforms.
1. An in vitro method of producing brown/beige adipocytes comprising:
contacting cells selected from white adipose tissue cells and mesenchymal stem cells, with a differentiation medium comprising serum, a glucocorticoid and a mix of growth factors comprising a growth factor of the Vascular Endothelial Growth Factor (VEGF) family, a growth factor of the Epidermal Growth Factor (EGF) family, an insulin-like growth factor and a growth factor of the Fibroblast Growth Factor (FGF) family, until obtaining adipogenic progenitor cells,
contacting said adipogenic progenitor cells with an adipogenic agent until obtaining brown/beige adipocytes, and
optionally recovering said brown/beige adipocytes.
2. The method according to claim 1 , wherein said cells are white adipose tissue cells or subcutaneous white adipose tissue cells.
3. The method according to claim 1 , wherein the differentiation medium comprises:
from about 0.1 ng/mL to about 20 ng/mL of a growth factor of the EGF family,
from about 0.1 ng/mL to about 20 ng/mL a growth factor of the FGF family,
from about 5 ng/mL to about 40 ng/mL of an insulin-like growth factor, and/or
from about 0.05 ng/mL to about 5 ng/mL of a growth factor of the VEGF family.
4. The method according to claim 3 , wherein the growth factor of the EGF family is EGF, the growth factor of the FGF family is FGF2, the insulin-like growth factor is IGF-1 or an analog thereof, and the growth factor of the VEGF family is VEGF.
5. The method according to claim 1 , wherein the growth factor of the EGF family is EGF, the growth factor of the FGF family is FGF2, the insulin-like growth factor is IGF-1 or an analog thereof, and/or the growth factor of the VEGF family is VEGF.
6. The method according to claim 1 , wherein said differentiation medium comprises from about 0.5% to about 10% serum, and/or from about 0.05 μg/mL to about 5 μg/mL of a glucocorticoid.
7. The method according to claim 1 , wherein the serum is fetal bovine serum and the glucocorticoid is hydrocortisone.
8. The method according to claim 1 , wherein said differentiation medium further comprises a free-radical scavenger or anti-oxidant.
9. The method according to claim 1 , wherein said adipogenic agent is selected from the group consisting of insulin or analogs thereof, non-selective phosphodiesterase (PDE) inhibitors, beta-adrenergic agonists, thiazolidinediones, glucocorticoids, Bone Morphogenetic Proteins (BMPs), derivatives and mixtures thereof.
10. The method according to claim 9 , wherein said adipogenic agent comprises one or more adipogenic agents selected from the group consisting of insulin, dexamethasone, indomethacin, IBMX, rosiglitazone, BMP4 and BMP7.
11. The method according to claim 1 , wherein said adipogenic agent comprises a Bone Morphogenetic Protein and, optionally, insulin.
12. The method according to claim 1 , wherein white adipose tissue cells or mesenchymal stem cells are contacted with the differentiation medium in a culture system enabling 3D spheroid formation, thereby producing spheroids of adipogenic progenitor cells.
13. The method according to claim 12 , wherein spheroids of adipogenic progenitor cells are transferred into a 3D culture matrix mimicking the extracellular matrix and allowing three-dimensional growth, before to be contacted with the adipogenic agent.
14. The method according to claim 1 , wherein said adipogenic agent comprises a Bone Morphogenetic Protein and, optionally, insulin and the growth factor of the EGF family is EGF, the growth factor of the FGF family is FGF2, the insulin-like growth factor is IGF-1 or an analog thereof, and the growth factor of the VEGF family is VEGF.
15. The method according to claim 1 , wherein the glucocorticoid is hydrocortisone, the serum is fetal bovine serum and the mix of growth factors comprises VEGF-A, EGF, IGF-1 or an analog thereof, and FGF2.
16. The method according to claim 1 , wherein the glucocorticoid is hydrocortisone, the serum is fetal bovine serum and the mix of growth factors comprises VEGF-A, EGF, IGF-1 or an analog thereof, and FGF2 and the adipogenic agent comprises a mixture of adipogenic agents and, optionally, insulin.
17. The method according to claim 16 , wherein the mixture of adipogenic agents comprises one or several adipogenic agents selected from the group consisting of insulin, dexamethasone, indomethacin, IBMX, rosiglitazone, BMP4 and BMP7.
18. The method according to claim 1 , wherein the differentiation medium consists essentially of fetal bovine serum, hydrocortisone, VEGF, EGF, IGF-1, insulin and FGF2.
19. The method according to claim 1 , said method comprising:
contacting cells selected from white adipose tissue cells and mesenchymal stem cells, with a differentiation medium consisting essentially of fetal bovine serum, hydrocortisone and a mix of growth factors, said mix of growth factors consisting essentially of VEGF, EGF, FGF2, and IGF-1, until adipogenic progenitor cells are obtained;
replacing the differentiation medium with a replacement medium consisting essentially of one or more adipogenic agent and contacting said adipogenic progenitor cells with said replacement medium agent until brown/beige adipocytes are obtained, wherein the at least one adipogenic agent is BMP7, insulin, or a combination thereof; and
recovering said brown/beige adipocytes.
20. The method according to claim 19 , wherein said replacement medium consists essentially of BMP7 and insulin.