Abstract
BackgroundThin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro.MethodsWe prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells.ResultsCo-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model.ConclusionsWe herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.
Highlights
Thin endometrium adversely affects reproductive success rates with fertility treatment
We further explored protein expression and hormone responsiveness (Fig. 1h–l)
The decidualization markers PRL and IGFBP1 were significantly upregulated in the decidualized cells (P = 0.0017 and 0.0033, respectively) (Fig. 1k, l), suggesting that hormone responsiveness is preserved in endometrial stromal cells even after the freeze-thaw procedure and subsequent passages
Summary
Thin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Several treatment modalities are presented to patients with thin endometrium to improve endometrial thickness [9]. Regenerative medicine including application of platelet-rich plasma (PRP) and cell therapy utilizing menstruation-derived stem cells is expected to be a promising therapeutic strategy [10, 11]. These strategies are not broadly available and there is no reliable clinical evidence supporting their use. Considering these limitations, further study is needed to determine how to maintain endometrial epithelial cells efficiently under xeno-free conditions
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