Abstract: OBJECTIVE: To investigate whether Zichong granules(资冲颗粒, ZCKL), a very effective herbal formula for treating infertility, have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells(h ESCs) in vitro, and to explore the cellular mechanisms of its clinical effects.METHODS: Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from h ESCs for 6 d. Normal rat serum and a set of growth factors were used as negative and positive controls, respectively.RESULTS: ZCKL-medicated rat serum, but not normal rat serum, induced h ESCs-derived mesoderm cells to differentiate into functional ovarian granulosa-like cells(OGLCs) in a similar manner to defined growth factors. The induced OGLCs resembled the morphology of native human granulosa cells, expressed granulosa cell-specific markers at both the m RNA and protein levels, produced high levels of estradiol and strongly responded to follicle-stimulating hormone stimulation. Furthermore,m RNA levels of follistatin, mothers against decapentaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process.CONCLUSION: In the ZCKL treatment of infertility,one mechanism by which ZCKL may act is by influencing ovarian granulosa cell differentiation and development, possibly through the follistatin and BMP/SMAD signaling pathways.

Key words: human embryonic stem cells;;infertility;;serum;;mesoderm;;ovarian;;granulosa cells;;Zichong granules