Differentiation Of Human Embryonic Stem Cells Into Endometrium-like Cells

Endometrium of women in the child-bearing age is affected by estrogen and progesterone. Its proliferation stag, production stage appear alternately during menstrual cycle. However, Congenital, pathological, artificial factors damage the balance of endometrium, resulting in thin endometrium which decreases embryo implantation rate. This situation is common in assisted reproductive technology.At present, there are several ways of improving thin endometrium:drug therapy, surgery therapy, transplantation of stem cells, etc. Estrogen and progesterone are widely used and have positive effect on improving endometrium thickness; surgery treatment also can help to some extent; however,transplantation of stem cell has not been applied in clinic. Studies showed that adult stem cells can differentiate into endometrial cells in vitro. At the same time, researchers reported that transplantation of mesenchymal stem cells could improve the thin endometrium in mice model. Because it is convenient to aquire embryonic stem cells in our center, so the aim of experiment is to investigate the condition of hESCs differentiating into endometrial-like cells. We aim to lay a experimental basis for the treatment of thin endometrium. ObjectiveAim to get endometriun-like cells from human embryonic stem cells by different differentiation Protocol and compare the efficiency.Methods1. human endometrial cells were isolated,cultured, characterizated and preservated. Meanwhile, if the surgical options of gaining endometrium influence the obtaining of endometrial stromal cells.2.human embryonic stem cells having been successfully built and identified in our laboratory, could be amplified by passage culture. Then they were used for differentiation.3.According to the different induction protocol, there were3groups in the experiment:Spontaneous differentiation group, simple differentiation group, co-culture differentiation group. Differentiation process is divided into two stages.4. Analysis expression of keratin and vimentin in terminal differentiation stage cells with immunofluorescence. Also, Verified the related gene expression by RT-PCR (Reverse Transcriptase Polymerase Chain Reaction).Results1.Origin of endometrial tissue affects the acquisition of endometrial stromal cells.2.With the extension of differentiation? the shapes of hESCs have been changed.3.In terminal inducing differentiation stage, According to the results of immunofluorescence, expression of keratin was positive in3groups. Expression of vimentin was positive in spontaneous differentiation group, but it was negative in simple differentiation group and co-culture differentiation group. Mean immunofluorescence intensity was compared and the results showed that:the mean immunofluorescence intensity difference among3groups was statistically significant (P<0.05). Then, we compared the mean immunofluorescence intensity between any two groups, there was statistically significant difference between co-culture differentiation group and simple differentiation group, co-culture differentiation group and spontaneous differentiation group (P<0.05),however, there was no statistically significant difference between simple differentiation group and spontaneous differentiation group (P>0.05). Results of RT-PCR revealed that expression of Keratin gene was positive, and no gene of Hoxa10and ER stripe was detected in3groups.Conclusion1.Different surgical method (curettage vs. endometrial biopsies) of obtaining endometrial tissue affects the acquisition of endometrial stromal cells.2.hESCs can differentiate into endometrial-like cells in the condition of inducing, while differentiate into mesenchymal cells and epithelial cell in the spontaneous condition.3.Chemical reagents and cell factors(17-β estradiol, TGF-α, EGF, PDGF-BB) can induce hESCs into endometrial-like cells. Differentiation from hESCs to endometrial-like cells is more efficient in co-culture condition than in simple differentiation condition.