Methods for growth and maturation of ovarian follicles
The present technology provides for methods for generation and isolation of granulosa cells and/or granulosa cell precursors from multi-potent cells, wherein the granulosa cells and/or granulosa cell precursors are useful in methods for growth and maturation of follicles or follicle-like structures. Additionally, the present technology also provides for methods of increasing steroidal hormones in a subject in need thereof.
1. A method to promote the growth and maturation of a follicle or an immature oocyte in a subject, the method comprising:
introducing into a plurality of mammalian multi-potent cells a recombinant DNA construct comprising a reporter coding sequence under the control of a promoter of an ovarian granulosa cell-specific gene, wherein the ovarian granulosa cell-specific gene is forkhead box L2 (Foxl2), and said multi-potent cells comprising an expressed reporter have differentiated into granulosa cells or granulosa cell precursors;
culturing the plurality of mammalian multi-potent cells comprising the recombinant DNA construct in vitro, under conditions suitable for differentiation of the multi-potent cells into the granulosa cells or the granulosa cell precursors;
isolating an enriched population of the granulosa cells or the granulosa cell precursors, wherein the enrichment is based on expression of the reporter; and
contacting the enriched population of cells comprising the granulosa cells or the granulosa cell precursors with mammalian ovarian tissue in vivo, wherein the mammalian ovarian tissue comprises at least one of the follicle and the immature oocyte, and wherein the granulosa cells or the granulosa cell precursors promote the growth and maturation of at least one of the follicle and the immature oocyte.
2. The method of claim 1 , wherein the population comprising the granulosa cells or the granulosa cell precursors and the mammalian ovarian tissue are autologous.
3. The method of claim 1 , wherein the mammalian ovarian tissue is human.
4. The method of claim 1 , wherein the reporter is a fluorescent reporter.
5. The method of claim 4 , wherein the fluorescent reporter is selected from the group consisting of: Discosoma sp. red (DsRed), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and orange fluorescent protein (OFP).
6. The method of claim 1 , wherein the mammalian multi-potent cells are selected from the group consisting of: embryonic stem cells (ESCs), induce pluripotent stem cells (iPSCs), reprogrammed somatic cells, bone marrow derived cells, and peripheral blood-derived cells.