Methods of differentiating preadipocytes and uses thereof
View Patent ↗Described herein are methods of transdifferentiating preadipocytes, populations of transdifferentiated preadipocytes, and methods of using the transdifferentiated preadipocytes.
1. A method of transdifferentiating mammalian preadipocytes, the method comprising:
(a) culturing mammalian preadipocytes;
(b) infecting the mammalian preadipocytes with a viral vector encoding Oct4 such that the mammalian preadipocytes overexpress Oct4;
(c) culturing the infected cells until about 100% confluent;
(d) subculturing the cells of step (c), wherein subculturing comprises seeding at least a portion of the cells onto a mouse embryonic fibroblast (MEF) feeder cell layer;
(e) expanding the cells seeded onto the feeder layer of step (d) for a period of time sufficient to produce a population of cells which produce glucagon and which express a pancreatic alpha cell marker gene selected from the group consisting of CGC, CNTN1, PCSK1, PDK4, RGS4, IRX2, LPPR4, LOXL2, KCTD12, KL, and any combination thereof.
2. The method of claim 1 , further comprising overexpressing exogneous Klf4 in at least one of the mammalian preadipocytes.
3. The method of claim 2 , wherein the exogenous Klf4 is simultaneously overexpressed with exogenous Oct4 in the same mammalian preadipocyte.
4. The method of claim 2 , wherein overexpression of exogenous Klf4 occurs via transfection or transduction of the at least one mammalian preadipocyte with an exogenous Klf4.
5. The method of claim 1 , further comprising overexpressing exogenous Sox2 in at least one of the mammalian preadipocytes.
6. The method of claim 5 , wherein overexpression of exogenous Sox2 occurs via transfection or transduction of the at least one mammalian preadipocyte with an exogenous Sox2.
7. The method of claim 1 , further comprising overexpressing exogenous cMyc at least one of the mammalian preadipocytes.
8. The method of claim 7 , wherein exogenous cMyc is simultaneously overexpressed with exogenous Oct4 in the same mammalian preadipocyte.
9. The method of claim 1 , wherein the mammalian preadipocytes have been passaged 3 times or less prior to overexpression of exogenous Oct4.
10. The method of claim 1 , wherein the cells seeded onto the feeder layer are expanded on the feeder layer for about 3, 4, 5, 6, 7, 8, 9, or about 10 days.
11. The method of claim 1 , wherein culturing the population of cells is performed without passaging for about 1,2,3, 4, or 5 days prior to performing subculturing of cells.
12. The method of claim 1 , further comprising the step of contacting the population of cells of producing glucagon with a compound.
13. The method of claim 12 , further comprising the step of evaluating a characteristic of the cells producing glucagon, wherein the characteristic is selected from the group consisting of cell growth, cell development, cell differentiation, glucagon production, apoptosis, cytotoxicity, and any combination thereof.
14. The method of claim 1 , further comprising overexpressing exogenous Kif 4, exogenous cMyc, and exogenous Sox2 in the at least one mammalian preadipocyte.
15. The method of claim 1 , wherein expanding the cells is performed using a serum free media.
16. The method of claim 1 , wherein the cells producing glucagon express NeuroD.