Context: Granulosa cells are important for the development and maturation of oocytes. The dysfunction of granulosa cells may contribute to abnormal folliculogenesis and steroidogenesis. Objective: Our objective was to establish an effective culture system to differentiate human embryonic stem cells (hESCs) into granulosa cells. Design: For differentiation of hESCs to granulosa cells, we used multistep approaches comprising in vitro treatments with cocktails of growth factors. Expression of mesendoderm/intermediate plate mesoderm markers, and granulosa cell markers were analyzed by real time-PCR, Western blotting, immunofluorescence, and flow cytometry. The production of estradiol and anti-Mullerian hormone (AMH) was analyzed by ELISA. Results: Gene expression analyses showed the progress of hESCs to primitive streak-mesendoderm, intermediate plate mesoderm, and finally to functional granulosa-like cells that expressed the granulosa cell-specific forkhead transcription factor FOXL2, estrogen synthetase cytochrome P450 19A1 (CYP19A1),AMH, the type 2AMHreceptor (AMHR2), and the FSH receptor (FSHR). However, they did not express the LH receptor (LHR). Western blot showed that AMHR2 and CYP19A1 levels in differentiated hESCs were higher than in undifferentiated cells. Flow cytometry showed that the percentage of AMHR2-, FSHR-, and CYP19A1-positive cells increased to 36%, 12%, and 34%, respectively, after differentiation for 12 days. These granulosa-like cells were also capable of producing AMH and aromatizing testosterone to estradiol, suggesting that they were biologically functional. Conclusions: We successfully established an effective protocol to generate functional ovarian granulosa-like cells from hESCs. The derivation of these cells opens new avenues for the further study and potential application of these cells in human folliculogenesis and steroidogenesis.
|Number of pages||11|
|Journal||Journal of Clinical Endocrinology and Metabolism|
|Publication status||Published - Sept 1 2013|
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry
- Biochemistry, medical