The Experimental Study On Derivation Of Human Umbilical Cord Mesenchymal Stem Cells Into Functional Granulosa-like Cells

Background: Premature ovarian failure(POF),referring to the women who have ovarian failure before the age of 40,and is characterized by anovulation,amenorrhea,E2 deficiency and infertility.The etiology of POF is complicated and involves many factors such as environment,heredity,autoimmunity,infection and iatrogenicity.There is no safe and effective treatment and most of them use hormone replacement therapy(Hormone Replacement Therapy,HRT)at present.However,this therapy can only improve the clinical symptoms of patients due to hormonal decline,and can not fundamentally restore ovarian function and fertility in patients with POF,and increase the risk of thrombotic diseases,ovarian tumors and breast cancer.Follicles,as a basic units of ovary,mainly composed of granulosa cells(GCs)and theca cells,play an important role on development and maturation of oocytes.The dysfunction of GCs may lead to abnormal steroidogenesis and folliculogenesis,occurrence of POF and infertility.With the study and application of stem cell therapy technology in variety of tissual lesion diseases,some scholars try to reconstruct ovarian function with Mesenchymal stem cells(MSCs)and get some exciting results.In this study,a co-culture system of human umbilical cord mesenchymal stem cells(HUMSCs)with human follicular fluid and granulosa cells(GCs)were established in order to induced HUMSCs differentiating into functional GCs,and seek for a new effective therapeutic approach for POF.Objective: To establish a induction and differentiation system of HUMSCs through co-culture with human follicular fluid and GCs for obtaining functional GCs.Methods: HUMSCs were pretreatmented with BMP4,then co-cultured with human follicular fluid and cumulus GCs.The morphology and expression of FSHR?AMH and CYP19A1 as well as OCT4 of cultured cells were observed and detected through immunofluorescence and fluorescent real time-PCR(RT-PCR).Results: 12 days after co-cultured of HUMSCs with human follicular fluid and GCs,the morphology of cultured cells was more like those human GCs with FSHR positive,and more intensive fluorescence signal of FSHR in human cumulus GCs(positive control)were observed,while HUMSCs before co-culture(negative control)showed no green fluorescence of FSHR.Moreover,the expression of pluripotency-related genes OCT4 in co-cultured cells down-regulated significantly(P<0.05)and were close to that in human GCs(positive control),while the expression of GCs specific makers such as FSHR?AMH and CYP19A1 in co-cultured cells up-regulated significantly(P<0.05),suggesting that the pluripotency of stem cell in co-cultured cells were losing and differentiating into functional GCs.Conclusion: HUMSCs were successfully induced and differentiated into functional granulosa-like cells under co-culture system of human follicular fluid and GCs.