Stem Cell-Like Properties of the Endometrial Side Population: Implication in Endometrial Regeneration Hirotaka Masuda 1,2 , Yumi Matsuzaki 1 *, Emi Hiratsu 1 , Masanori Ono 2 , Takashi Nagashima 2 , Takashi Kajitani 2 , Toru Arase 2 , Hideyuki Oda 2 , Hiroshi Uchida 2 , Hironori Asada 2 , Mamoru Ito 3 , Yasunori Yoshimura 2 , Tetsuo Maruyama 2 *, Hideyuki Okano 1 1 Department of Physiology, Keio University School of Medicine, Tokyo, Japan, 2 Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan, 3 Central Institute for Experimental Animals, Kanagawa, Japan Abstract Background: The human endometrium undergoes cyclical regeneration throughout a woman’s reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium. Methodology/Principal Findings: We found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo. Conclusions/Significance: These results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis. Citation: Masuda H, Matsuzaki Y, Hiratsu E, Ono M, Nagashima T, et al. (2010) Stem Cell-Like Properties of the Endometrial Side Population: Implication in Endometrial Regeneration. PLoS ONE 5(4): e10387. doi:10.1371/journal.pone.0010387 Editor: Manuel Tena-Sempere, University of Co ´ rdoba, Spain Received October 27, 2009; Accepted March 25, 2010; Published April 28, 2010 Copyright: ß 2010 Masuda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported, in part, by the Ministry of Education, Science, and Culture of Japan (to T.M., Y.Y., and H.M.), by a National Grant-in-Aid for the Establishment of a High-Tech Research Center in a Private University (to T.M.) and by a grant-in-aid from the Global Centers of Excellence program of the Ministry of Education, Science, and Culture of Japan to Keio University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: penguin@sc.itc.keio.ac.jp (YM); tetsuo@sc.itc.keio.ac.jp (TM) Introduction Human endometrium, which lines the uterine cavity, exhibits unique properties of cyclical regeneration and tissue breakdown under the influence of estrogen and progesterone throughout the course of a woman’s reproductive life. Retrograde shedding and ectopic implantation of menstrual endometrial cells and tissue fragments give rise to endometriotic lesions outside of the uterus. Endometrial cells prepared from the human endometrium are also capable of reconstituting functional endometrium in xenograft models of endometriosis [1]. When single cell suspensions of endometrial cells are transplanted under the kidney capsule of severely immunodeficient NOD/SCID/c c null (NOG) mice [1], the reconstructed ectopic endometrial tissues show menstrual cycle- related morphological and functional changes repeatedly in response to treatment with estrogen and progesterone [1]. These unique properties reflect the remarkable capacity of human endometrial cells for regeneration at eutopic and ectopic locations, and suggest the existence of stem/progenitor cells as well as an angiogenic system in the human endometrium. Indeed, it has been postulated that the endometrium contains a pool of multipotent stem cells within the deep basalis layer, capable of cyclically producing progenitor cells that further differentiate into each endometrial cell component [2,3]. Several groups have identified a number of endometrial cell subpopulations as candidate endometrial stem/progenitor cells. These include clonogenic endometrial cells [4], endometrial SP cells which possess a Hoechst 33342 low-fluorescence profile [5,6], CD146 + PDGFRb + stromal cells [7], and CD29 + CD73 + CD90 + stromal cells [8]. The phenotypic and functional stem cell-like PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10387