Functional characterization of human endometrial stem/progenitor cells in vitro and in vivo

Abstract

Human endometrium undergoes cyclic tissue breakdown and regeneration throughout a woman of reproductive life. It has been speculated that there is a subpopulation of stem/progenitor cells residing in the endometrium responsible to its remarkable capability. One key feature that distinguishes the adult stem cells is their quiescent state within the stem cell niche. Based on this unique stem cell property, slow-cycled cells from human endometrial tissues were examined and characterized. The first aim of this study was to examine label retaining cells (LRCs) in long-term engrafted human endometrial tissues using a mouse xenotransplantation model in vivo. After various initial approaches, the maximum initial labeling of LRCs was determined and, only stromal LRCs were detected after a 12-week chase. Mesenchymal stem cell (MSCs) and stem cell-like phenotypic appearances were detected in a small proportion of stromal LRCs. The second objective was to establish a novel method for identifying slow-cycled cells from cultivated endometrial epithelial and stromal cells in vitro. The method identified cells that retained fluorescence label after long-term culture. These cells were termed fluorescence retaining cells (FRCs). The variables and the different initial approaches leading to the establishment of the protocol for the identification of FRC were optimized. The third objective further characterized the endometrial stromal FRCs. Endometrial stromal FRCs were enriched with cells having higher colony forming and self-renewal abilities when compared with non-FRCs. At subsequent passage (P2) the adult stem cell characteristics of cells derived from FRCs was more prominent and differentiated into mesenchymal lineages. Clonally derived stromal FRCs expressed higher self-renewal and pluripotent genes (BMI-1, NANGOG, OCT4, and SOX2). They also expressed endometrial mesenchymal stem cell phenotypic surface markers: CD146/PDGFRβ and W5C5, suggesting these cells might be of MSC origin. The fourth objective was to examine the role of epithelial-stromal interactions on regulation of endometrial stem cells. Menstrual cycle day 2 epithelial conditioned medium significantly enhanced the colony forming ability of endometrial stromal cells. Protein expression analysis of the early menstrual phase conditioned medium showed two cytokines: angiogenin and interleukin 8, potentially involved in the regulation of endometrial stromal stem/progenitor cells. The fifth objective was to examine the reconstitution ability of the endometrial clonogenic cells in vivo using a mouse xenotransplantation model. Endometrial clonogenic stromal cells together with epithelial cells reconstituted muscle-like tissue. Although no endometrial-like tissue was detected, the findings provide the first in vivo evidence that stromal stem/progenitor cells are present within clonogenic cells. Overall, the thesis reports the existence of slow-cycled cells from human endometrium. By using various label retention techniques in vitro and in vivo a subpopulation of slow-cycled stromal cells which exhibit characteristics properties of adult stem cells was demonstrated for the first time.