Role of niche cells in the regulation of endometrial mesenchymal-like stem cells during the menstrual cycle

Abstract

Human endometrium is highly regenerative in a woman’s reproductive life. A population of endometrial mesenchymal-like stem cells (eMSCs) contributes to this remarkable process. Adult stem cells remain quiescent during normal homeostasis but divide and differentiate efficiently in response to appropriate stimuli primarily from the microenvironment in which the cells reside (niche). Endometrial regeneration starts at menstruation. There is limited understanding of the microenvironmental regulation of eMSCs during endometrial repair. The hypothesis of this thesis is that endometrial stem/progenitor cells are activated for tissue repair in each menstruation. The first objective of this study was to evaluate the percentage and the stem cell properties of eMSCs in different menstrual phases and endometrial layers. The co-expression of CD140b and CD146 surface markers was used to enrich eMSCs. Flow cytometry analysis showed the percentage of the eMSCs across the menstrual cycle was stable. However, the basal layer of the endometrium contained more number of eMSCs than the functional layer. The eMSCs from menstruation phase exhibited better self-renewal and proliferative activity than those from secretory phase. These findings indicate a reservoir of the stem/progenitor cells residing in the basalis are activated at menstruation. Therefore, the second objective was to demonstrate at menses, specific regulatory factors contribute to maintenance of eMSCs. Indirect co-culture of the endometrial cells (epithelial or stromal) from menstruation phase enhanced the colony forming and self-renewal activities of the eMSCs. Such phenomenon was not observed from proliferative phase niche cells. Secretory factors secreted by menstruation niche cells also enhanced the stem cell phenotype of eMSCs. These findings indicate that at menstruation the endometrial cell derived secretory factor(s) are responsible for maintaining the stem cell activities of eMSCs in vitro. The third objective was to search for potential cytokine regulators at menstruation. Four candidates were chosen based on the cytokine array, namely C-X-C motif chemokine 1 (CXCL1), C-X-C motif chemokine 5 (CXCL5), interleukin 6 (IL6) and granulocyte macrophage colony stimulating factor (GM-CSF). Functional assay of IL6 on eMSCs suggest it may be involved in stem cell maintenance. The fourth objective was to evaluate and identify the molecules involved in the observed actions mediated by soluble secretory factors of the Wnt/β-catenin signaling pathway. Clonally derived stromal cells after co-cultured with endometrial cells from menstruation phase confirmed an increased in expression active β-catenin. Therefore, the effect of Wnt related molecules (Wnt,3a, Wnt5a and RSPO1) were analysed using functional assays. Our preliminary data revealed that Wnt5a stimulated the proliferation and differentiation of the eMSCs while RSPO1 is involved in maintaining the stem cell population. In summary, the proliferative and self-renewal activities of eMSCs changed across the menstrual cycle. Mature endometrial cells provide secretory factors to stimulate the self-renewal of the eMSCs at menstruation, possibly through the activation of the Wnt/β-catenin signaling.