Microenvironmental regulation of human endometrial mesenchymal stem-like cells.

Abstract

Title: Microenvironmental regulation of human endometrial mesenchymal stem-like cells. Authors: RWS Chan, MZ Cao, EHY Ng, WSB Yeung. Institution: The University of Hong Kong, Obstetrics & Gynecology, Hong Kong, Hong Kong; Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong-Shenzhen Hospital. Study question: (25 words) Do myometrial cells function as niche cells regulating the biological activities of endometrial stem cells? Study answer: (25 words) Soluble factors derived from myometrial cells increased self-renewal and proliferation activities of endometrial mesenchymal stem-like cells (eMSCs) by activating WNT/β-catenin signaling. What is known already: (100 words) Adult stem cells contribute to regeneration of endometrium. Data from human and mouse endometrium localized eMSCs preferentially to regions adjacent to the myometrium. In mice, active β-catenin was highly expressed in proliferating endometrial label-retaining cells after parturition. We hypothesized that the myometrial cells provided niche signals regulating the biological activities of eMSCs. Study design, size, duration: (75 words) Sequential beading with magnetic beads coated with anti-CD140b and anti-CD146 antibodies was used to isolate eMSCs from endometrial tissues from proliferative (n = 24) and secretory (n = 16) phase. The eMSCs were seeded at low density, and indirectly co-cultured with myometrial cells at ratio of 1:90 for 15 days. Functional stem cell assays were performed and the proportion of cell expressing the eMSCs markers was evaluated. Participants/materials, setting, methods: (75 words) Proliferative and secretory phase samples obtained from women aged 35- to 50-years undergoing total abdominal hysterectomy. Endometrial cells were isolated enzymatically and the percentage of eMSCs after co-culture with myometrial cells was analyzed by flow cytometry. Western blot analysis for active β-catenin validated the activation of Wnt/β-catenin signaling. The role of WNT/β-catenin signaling in eMSCs self-renewal was confirmed by gain-of (activator: Wnt3a conditioned medium and recombinant WNT3A) and loss-of (inhibitors: XAV939 and IWP-2) function approaches. Main results and role of chance: (200 words) In vitro co-culture of myometrial cells enhanced the colony forming and self-renewal ability of eMSCs. The expanded eMSCs after coculture retained multipotent characteristic and exhibited a greater total cell output when compared to medium alone culture. The level of active β-catenin in eMSCs increased significantly after co-culture with myometrial cells suggesting activation of the WNT/β-catenin signaling. Secretory factors from myometrial cells produced the same stimulatory effect on eMSCs. The functional WNT/β-catenin signaling in eMSCs self-renewal was determined with WNT activator (Wnt3a conditioned medium) and WNT inhibitors (XAV939 and IWP-2). Addition of Wnt3a CM or recombinant WNT3A increased the clonogenic activity and number of eMSCs. While, the stimulatory effect of myometrial cells on eMSCs was suppressed by the Wnt inhibitors. Limitations, reasons for caution: (50 words) The behavior of the cells may be altered during culture. The functionality of myometrial derived factors on eMSCs needs to be determined with in vivo studies. Wider implication of the findings: (50 words) Myometrial cells provide paracrine factors to eMSCs via the Wnt/β-catenin signaling. Secretory factors derived from the myometrial cells offer a specialized microenvironment modulating eMSC activities. By gaining a better understanding of the niche composition, it will be possible to recreate a milieu for stem cell expansion and control their biological activities. Study funding: This project was supported by Research Grant Council in Hong Kong.