Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model  An-Pei Kao, M.S., Kai-Hung Wang, M.S., Chia-Cheng Chang, Ph.D., Jau-Nan Lee, M.D., Ph.D., Cheng-Yu Long, M.D., Ph.D., Hung- Sheng Chen, M.D., Cheng-Fang Tsai, B.S., Tsung-Hua Hsieh, M.S., Eing-Mei Tsai, M.D., Ph.D.  Fertility and Sterility  Volume 95, Issue 4, Pages 1308-1315.e1 (March 2011) DOI: 10.1016/j.fertnstert.2010.09.064 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) A diagram showing the stepwise isolation procedure for endometrial stromal cells, epithelial cells, and MSCs by serial filtration of collagenase-digested endometrial tissues through different pore sizes of meshes and selection of large colonies after limiting dilution and colony formation. Bars = 200 μm. (B) Endometrial epithelial and stromal cells showed the expression of CK7 and vimentin, respectively, by immunostaining. (C) At passage 3, 97.5% ± 2.2% of stromal cells expressed vimentin, whereas only 3.8% ± 3.3% of stromal cells expressed CK7 by flow cytometric analysis. (D) Endometrial stromal cells were competent of gap junctional intercellular communication as indicated by Lucifer Yellow dye transfer; in contrast, both eutopic and ectopic endometrial MSCs were deficient in gap junctional intercellular communication. Bars = 200 μm. EN-MSCs = endometrial mesenchymal stem cells. Fertility and Sterility 2011 95, 1308-1315.e1DOI: (10.1016/j.fertnstert.2010.09.064) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Both eutopic and ectopic endometrial MSCs (EN-MSCs) were capable of adipogenesis (shown by Oil Red O staining of lipid droplets, red color), osteogenesis (shown by von Kossa staining of calcified extracellular matrix, black staining), and chondrogenesis (shown by Alcian blue staining of sulfated proteoglycans, blue color). (B) These eutopic and ectopic endometrial MSCs also were able to differentiate into cardiomyocytes as shown by the expression of troponin-I (cTnI) and α-myosin heavy chain (α-MHC) after 1-week induction. At 2 weeks, some aggregated cells formed ball-like structures (indicated by triangles). Bars = 100 μm. (C) Both eutopic and ectopic endometrial MSCs were also capable of transdifferentiation into neural cells (ectodermal origin), as indicated by the expression of microtubule-associated proteins (MAP) and neural-specific enolase (NSE), neural cell markers. The majority of these cells showed a stellate cell body with many dendritelike protrusions. Bars = 200 μm. Fertility and Sterility 2011 95, 1308-1315.e1DOI: (10.1016/j.fertnstert.2010.09.064) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) At lower cell density, the symmetric divisions of serpiginous-shaped ectopic endometrial MSCs can be observed in culture (indicated by triangles). Both eutopic and ectopic endometrial MSCs had high proliferation potential. The cumulative population doublings were higher for ectopic endometrial MSCs (cumulative population doubling level= 33.9, 31.5, and 32.9 respectively, for EN12, 17, and 19) compared with eutopic endometrial MSCs (cumulative population doubling level= 25.9 for EN18). Triangles indicate symmetric divisions of serpiginous cells. (B) The growth rates of ectopic endometrial MSCs (EN-MSCs) were significantly higher than those of eutopic endometrial MSCs (P<.05). The eutopic and ectopic stromal cells had significantly slower growth rate compared with endometrial MSCs. (C) Cells were plated at a density of 200 viable cells per 100 mm dish in triplicate for colony development for 21 days. The colony-forming efficiency was determined from the number of colonies developed as the percentage of the total number of cells inoculated. The colony-forming efficiency of ectopic endometrial MSCs was not significantly different from that of eutopic endometrial MSCs; however, the average colony size of eutopic endometrial MSCs was significantly larger than that of ectopic endometrial MSCs (14.6 ± 3.4 mm and 10.1 ± 2.2 mm). (D) Ectopic endometrial MSCs showed higher ability of migration and invasion than other endometrial cells (P<.05). Fertility and Sterility 2011 95, 1308-1315.e1DOI: (10.1016/j.fertnstert.2010.09.064) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Eutopic and ectopic endometrial MSCs (EN-MSCs) were seeded in scaffolds and implanted into the SCID mice to test the invasion ability in vivo. The tissues formed by the scaffolds were harvested 1 month after surgery. The surfaces of eutopic endometrial MSC–seeded scaffolds appeared slightly rugged and showed new tiny blood vessels around the scaffold (indicated by arrow). The ectopic endometrial MSC–seeded scaffolds appeared as irregular spheres covered with many blood vessels on the surface (indicated by arrow). Two months after implantation, the tissues formed by scaffolds containing ectopic endometrial MSCs appear to be wrapped by capsulelike membrane and did not decrease significantly in size. (B) New stromal tissue was found inside the tissue formed by ectopic endometrial MSC–containing scaffold, interlaced with the few remaining scaffold materials that were not absorbed completely (arrow); new blood vessels were found in the newly developed tissue as determined by histologic study with H&E staining (triangle). (C) Human cells expressing HLA marker were clearly identifiable by green fluorescence in immunostaining of transplanted ectopic endometrial MSCs. Mouse skin did not show the expression of this marker. The invasiveness of ectopic endometrial MSCs was observed in the surrounding mouse tissue (yellow dotted lines). Bars = 100 μm. Fertility and Sterility 2011 95, 1308-1315.e1DOI: (10.1016/j.fertnstert.2010.09.064) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

By flow cytometry analysis, eutopic and ectopic endometrial MSCs (EN-MSCs) expressed mesenchymal stem cell markers. These cells did not show specific markers for hematopoietic stem cells (CD34), leukocytes (CD45) and embryonic stem cells (SSEA4). Fertility and Sterility 2011 95, 1308-1315.e1DOI: (10.1016/j.fertnstert.2010.09.064) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions