In-vitro development of germ cell-like cells derived from human theca stem cells in reaggregated ovary
In-vitro development of germ cell-like cells derived from human theca stem cells in reaggregated ovary
Seyedeh Nasim Mirbahari,1Azam Dalman,2,*Fatemeh Hasani,3Mehdi Totonchi,4
1. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 2. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 3. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 4. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Introduction: In this study, human theca stem cells (hTSCs) were differentiated into human germ cell-like cells (hGCLCs) and the developmental potential of these cells was measured after being placed next to somatic cells in reaggregated ovary.
Methods: In this interventional experimental study, hTSCs were isolated from small antral follicles (3-5 mm in size). Isolated hTSCs were expanded and seeded in 6-well plates at a density of 5×104 cells/well in differentiation medium (DMEM-F12 containing 5% human follicular fluid and 5% FBS) for 40 days. The hGCLCs aggregated with somatic cells (cumulus cells and hTSCs) in ratio of 1 to 5 and cultured in growth medium (αMEM containing 3mg/ml BSA, 1% ITS, 0.005 IU/ml FSH and 0.11 mg/ml ascorbic acid) in suspension culture dish for 10 days. The size, morphology, viability and the expression of oogonial-specific marker (DAZL) and oocyte-specific markers (GDF9) in hTSCs, hGCLCs and reaggregated ovaries were evaluated by immunostaining to assess the development process.
Results: The morphology of hTSCs changed to round-shape cells (hGCLCs), similar to oocytes, after 12 days (size 20μm) and the diameters of these cells increased over time (50μm, P<0.05). The viability of hGCLCs cells after in vitro differentiation and development in reaggregated ovaries was 54%. The hTSCs did not express DAZL and GDF9, but the hGCLCs expressed DAZL marker, and did not express GDF9. The hGCLCs expressed GDF9 after being placed in reaggregated ovary.
Conclusion: To sum up, hTSCs have the ability to differentiate in to hGCLCs in-vitro. In addition, morphological similarities and protein presence imply that in-vitro hGCLCs development improved by co-culturing with ovarian somatic cells.