Characterization of endometrial mesenchymal stem-like cells obtained by endometrial biopsy during routine diagnostics

Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells. Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells. Pathologic conditions involving the endometrium such as endometriosis, adenomyosis, and endometrial carcinoma have been attributed to dysregulated stem cell (SC) function (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar, 2Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (255) Google Scholar, 3Gargett C.E. Uterine stem cells: what is the evidence?.Hum Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (268) Google Scholar, 4Du H. Taylor H.S. Stem cells and female reproduction.Reprod Sci. 2009; 16: 126-139Crossref PubMed Scopus (59) Google Scholar, 5Götte M. Endometrial cells get side-tracked: side population cells promote epithelial-mesenchymal transition in endometrial carcinoma.Am J Pathol. 2010; 176: 25-28Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Adult SCs have been isolated from menstrual blood (6Hida N. Nishiyama N. Miyoshi S. Kira S. Segawa K. Uyama T. et al.Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells.Stem Cells. 2008; 26: 1695-1704Crossref PubMed Scopus (258) Google Scholar, 7Patel A.N. Park E. Kuzman M. Benetti F. Silva F.J. Allickson J.G. Multipotent menstrual blood stromal stem cells: isolation, characterization, and differentiation.Cell Trans. 2008; 17: 303-311Crossref PubMed Scopus (276) Google Scholar), hysterectomy-derived endometrium (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar), and first-trimester decidua (9Dimitrov R. Kyurkchiev D. Timeva T. Yunakova M. Stamenova M. Shterev A. et al.First-trimester human decidua contains a population of mesenchymal stem cells.Fertil Steril. 2010; 93: 210-219Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), being characterized by SC marker gene expression (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar, 8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 10Matthai C. Horvat R. Noe M. Nagele F. Radjabi A. van Trotsenburg M. et al.Oct-4 expression in human endometrium.Mol Hum Reprod. 2006; 12: 7-10Crossref PubMed Scopus (92) Google Scholar, 11Wolf M. Kiesel L. Götte M. Endometrial stem cells. Potential relevance for the pathogenesis of endometriosis?.Gynakol Endokrinol. 2009; 7: 185-189Crossref Scopus (7) Google Scholar) and by their multilineage differentiation potential (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar, 13Wolff E.F. Wolff A.B. Hongling Du Taylor H.S. Demonstration of multipotent stem cells in the adult human endometrium by in vitro chondrogenesis.Reprod Sci. 2007; 14: 524-533Crossref PubMed Scopus (119) Google Scholar). Large-scale studies of endometrial SCs are hampered by the limited availability of hysterectomy tissue. Endometrial biopsies may represent a viable alternative, as they are less invasive, preserve the uterus, and its function and are routinely performed for diagnostic purposes. Endometrial SCs may preferably cluster in the deeper layer of the basalis, constituting a reservoir for monthly regeneration of the endometrium (3Gargett C.E. Uterine stem cells: what is the evidence?.Hum Reprod Update. 2007; 13: 87-101Crossref PubMed Scopus (268) Google Scholar). However, recent data indicate that putative endometrial SCs may also reside in the superficial layers accessible by endometrial biopsy (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar). In this study, we characterize adult SCs in human endometrium obtained by routine biopsy in an IVF setting by their functional properties, including serial clonal passaging, long-term culturing properties, SC marker expression, and multilineage differentiation potential. In a university-based reproductive medicine unit, 36 female patients undergoing pretreatment diagnostics including endometrial biopsy were included in the study, which was carried out according to the principles of the Helsinki Convention and approved by the local ethics commission (no. 2008-223-f-S). All subjects gave written informed consent. Transcervical endometrial biopsies were taken on cycle days 22–24 as part of the diagnostic routine with a Probet catheter (Gynemed, Lensahn, Germany) (14Revel A. Multitasking human endometrium: a review of endometrial biopsy as a diagnostic tool, therapeutic applications, and a source of adult stem cells.Obstet Gynecol Surv. 2009; 64: 249-257Crossref PubMed Scopus (18) Google Scholar). One part of the samples was analyzed by the pathologist applying established criteria (15Noyes R.W. Hertig A.T. Rock J. Dating the endometrial biopsy.Fertil Steril. 1950; 1: 3-25Crossref Google Scholar), whereas the remaining part was transferred in HEPES-buffered Dulbecco’s modified essential medium (DMEM)/F-12 (Invitrogen, Carlsbad, CA). Endometrial stromal cells were isolated as described elsewhere (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Enzymatically dissociated stromal cells were negatively selected using Dynabeads (Dynal, Oslo, Norway) specific for epithelial cells (BerEP4) and leukocytes (CD45) (17Chan R.W. Schwab K.E. Gargett C.E. Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (477) Google Scholar). After erythrocyte lysis, cells were resuspended in DMEM/F-12/10% fetal calf serum (FCS) and cultured on fibronectin-coated 60-mm Petri dishes (BD Biosciences, Bedford, MA) at a density of 630 cells/3 mL in a humidified incubator (37°C/5% CO2). To determine cloning efficiency (CE) (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), cells were cultured for 15 days, changing culture media every 6–7 days. Ten percent formaldehyde-fixed cells were stained with Crystal violet. Cell clusters were considered as colonies when they contained >50 cells. CE was defined as number of colonies/number of cells seeded × 100%. For serial passaging, a part of the cultures was used to determine CE, whereas the remaining part was passaged every 7–10 days upon reaching 80%–100% confluence for additional analyses. Expression of CD146 (MCAM), NOTCH1, Musashi-1 (MSI1), and SOX2 relative to 18S rRNA was analyzed by quantitative polymerase chain reaction (qPCR) as described elsewhere (18Götte M. Kalkhake K. Ploeger S. Kiesel L. Stute P. Effect of testosterone on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro.J Steroid Biochem Mol Biol. 2009; 117: 168-175Crossref PubMed Scopus (5) Google Scholar, 19Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (116613) Google Scholar) using TaqMan gene expression systems hs00174838_m1, hs00413187_m1, hs0015929_m1, hs00415716_m1, and hs99999901 s1 (ABI, Darmstadt, Germany) on three or more replicates. For flow-cytometric analysis, cells were trypsinized, washed, and resuspended in phosphate-buffered saline/2% bovine serum albumin. Fifty microliters of cells (1 × 106/mL) were incubated with fluorochrome-labeled monoclonal antibodies CD73-PE, IgG1κ isotype control (BD-Pharmingen, Heidelberg, Germany), CD105-PE, CD146-PC5, CD90-PC5, IgG2a-PC5 isotype control, CD45-PC7, CD34-PC7, CD14-PC7, IgG1-PC7 isotype control (Beckman Coulter, Krefeld, Germany) for 30 minutes at 20°C. Samples were diluted with 1 mL ISOTON II (Beckman Coulter) and analyzed in a Beckman Coulter FC500 flow-cytometer. Osteogenic differentiation of clonal endometrial stromal cells (n = 6 cultures) and of the pluripotent embryonal carcinoma cell line NTera-2 (20Andrews P.W. Damjanov I. Simon D. Banting G.S. Carlin C. Dracopoli N.C. et al.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.Lab Invest. 1984; 50: 147-162PubMed Google Scholar) was performed as described (21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar). For 10 days before alkaline phosphatase activity staining using BCIP/NBT substrate (Sigma, Munich, Germany), 4.5 × 104 cells/1.5 mL were cultured on 35-mm cell culture dishes in NH-OsteoDiff Medium (Miltenyi, Bergisch-Gladbach, Germany). For adipogenic differentiation (21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar), 7.5 × 104 cells/1.5 mL (n = 3) were cultured on 35-mm cell culture dishes in NH-AdipoDiff Medium (Miltenyi) for 21 days before staining with 0.5% Oil Red-O (Sigma). Induction media were changed every 3 days. Reverse transcription (RT)-PCR for PPARγ2 was performed as described (12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar). For chondrogenic differentiation (n = 2) (21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar), 20,000 cells/200 μL were cultured in NH-ChondroDiff Medium (Miltenyi) for 24 days in 96-well plates before staining with 0.1 mg/mL Alcian Blue (Sigma). All negative control cells were cultured in DMEM/5% FCS. Conventional chromosome analysis was performed on long-term cultures analyzing 10 metaphase spreads at a 450-band level. Data are presented as mean ± SD. Unpaired t-tests were used to compare the significance between two groups. Data for cell yield were log transformed before t-test analysis. P<.05 was considered statistically significant. We obtained biopsies of 36 patients (mean age, 34.42 ± 5.10 years), of which 26 samples were secretory and 10 were proliferative. After plating immunopurified endometrial stroma cells at clonal density, clones emerged after 15 days (Fig. 1A ). Similar to hysterectomy-derived preparations (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17Chan R.W. Schwab K.E. Gargett C.E. Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (477) Google Scholar), large colonies containing a dense center of tightly packed cells with an overall swirly appearance (Fig. 1B) and small colonies of loosely packed cells (Fig. 1C) emerged. CE was 0.795% and increased to 4.329% after a second round of cloning (Fig. 1K), suggesting enrichment of SC activity. Among 16 independent clonal cultures that were passaged 12 times before cryoconservation, four cultures could be cultivated for >12 months and >30 serial passages without showing chromosomal aberrations (Fig. 1J). Endometrial cell clones were differentiated into multiple lineages (20Andrews P.W. Damjanov I. Simon D. Banting G.S. Carlin C. Dracopoli N.C. et al.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.Lab Invest. 1984; 50: 147-162PubMed Google Scholar, 21Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar). A subfraction of osteogenically induced clonal cells displayed alkaline phosphatase activity and a cuboidal morphology, demonstrating successful differentiation (Fig. 1D–1F). Successful formation of lipid vacuoles as a readout of adipocyte formation (22Okano H. Kawahara H. Toriya M. Nakao K. Shibata S. Imai T. Function of RNA-binding protein Musashi-1 in stem cells.Exp Cell Res. 2005; 306: 349-356Crossref PubMed Scopus (307) Google Scholar) was demonstrated by Oil Red-O staining and independently confirmed by PCR analysis for PPARγ2 (Fig. 1G–1I). Chondrogenic induction (20Andrews P.W. Damjanov I. Simon D. Banting G.S. Carlin C. Dracopoli N.C. et al.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.Lab Invest. 1984; 50: 147-162PubMed Google Scholar) resulted in the formation of comparably small (25–35 μm) Alcian Blue–positive cartilage-like nodular aggregates (not shown). We can only speculate whether these structures represent early stages of chondrogenic differentiation, as the formation of larger aggregates would be expected upon successful differentiation of mesenchymal SCs (MSCs) into chondrocytes. Expression of four adult SC markers and one pluripotency-associated SC marker was confirmed by qPCR (Fig. 1L–1N). Conforming with immunohistochemical data (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar), MSI1 and NOTCH1, which maintain SCs in an undifferentiated state (22Okano H. Kawahara H. Toriya M. Nakao K. Shibata S. Imai T. Function of RNA-binding protein Musashi-1 in stem cells.Exp Cell Res. 2005; 306: 349-356Crossref PubMed Scopus (307) Google Scholar), were expressed in clonal endometrial cells (Fig. 1L). Similar to hysterectomy-derived endometrium (12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar), CD146 was expressed in endometrial biopsy–derived cells. Expression of SOX2 (23Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14451) Google Scholar) was considerably lower compared with adult SC markers (Fig. 1M). A second round of culturing at clonal density resulted in enrichment of CD146, whereas MSI1 expression decreased (Fig. 1N). Flow-cytometric analysis of long-term clonal cultures of passages 18–34 revealed high expression of the MSC markers CD90 (98.45%, SD = 0.6, n = 2), CD73 (96.2%, SD = 2.3, n = 4), CD105 (96.8%, SD = 1.8, n = 2), and CD146 (75.3%, SD = 20.3, n = 4; Fig. 1O). Expression of CD34 (2.45%, SD = 0.2, n = 2) and CD14 (2.6%, SD = 1.4, n = 2) was at the limit of detection. Of two samples investigated for CD45 expression, one sample showed 12.3% CD45-positive cells, whereas a second sample contained 71.6% CD45-positive cells. Our study demonstrates the existence of MSC-like cells in human endometrium obtained by transcervical endometrial biopsy. Possibly owing to underrepresentation of the basalis layer, the CE in biopsy-derived endometrial stromal cell clones was slightly lower compared with hysterectomy-derived clones (0.8% vs. 1.1%–1.6%) (17Chan R.W. Schwab K.E. Gargett C.E. Clonogenicity of human endometrial epithelial and stromal cells.Biol Reprod. 2004; 70: 1738-1750Crossref PubMed Scopus (477) Google Scholar). In accordance with Schwab et al., no difference was observed between secretory and proliferative samples (16Schwab K.E. Chan R.W. Gargett C.E. Putative stem cell activity of human endometrial epithelial and stromal cells during the menstrual cycle.Fertil Steril. 2005; 84: 1124-1130Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Furthermore, when serum P as a continuous marker of secretory transition was evaluated, no correlation was observed with PCR markers (not shown). Similar to hysterectomy-derived samples (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar), CE increased significantly after the second round of cloning. Similarly (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar), we demonstrated high expression of CD146 and a coexpression of the MSC markers CD73, CD105, and CD90 (24Dominici M. Le Blanc K. Mueller I. Slaper-Cortenbach I. Marini F. Krause D. et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (11882) Google Scholar) in long-term cultures of serially passaged clones. Our cells were negative for markers of endothelial and primitive hematopoietic cells, monocytes, and macrophages. Our study therefore adds to the evidence for the presence of MSC-like cells in the human endometrium: Gargett and coworkers (8Gargett C.E. Schwab K.E. Zillwood R.M. Nguyen H.P. Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.Biol Reprod. 2009; 80: 1136-1145Crossref PubMed Scopus (369) Google Scholar, 12Schwab K.E. Gargett C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium.Hum Reprod. 2007; 22: 2903-2911Crossref PubMed Scopus (410) Google Scholar) could previously demonstrate that CD146+PDGFRß+MSC-like cells are located perivascularly in the functional and basal layers, and Hida et al. (6Hida N. Nishiyama N. Miyoshi S. Kira S. Segawa K. Uyama T. et al.Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells.Stem Cells. 2008; 26: 1695-1704Crossref PubMed Scopus (258) Google Scholar) could detect CD29+/CD105+/CD34−/CD45−MSC-like cells in menstrual blood. Since we immunodepleted CD45-positive cells during the isolation procedure, it is not fully clear whether flow-cytometric detection of CD45 in a subpopulation of our cultures indicates inefficient immunodepletion or a secondary reexpression induced by long-term culturing, possibly indicating a bone marrow–derived origin of our cells (2Sasson I.E. Taylor H.S. Stem cells and the pathogenesis of endometriosis.Ann N Y Acad Sci. 2008; 1127: 106-115Crossref PubMed Scopus (255) Google Scholar, 4Du H. Taylor H.S. Stem cells and female reproduction.Reprod Sci. 2009; 16: 126-139Crossref PubMed Scopus (59) Google Scholar). Inclusion of analytical steps monitoring CD45 expression in early culture passages may be a reasonable addition to future isolation protocols. Quantitative PCR revealed similar CD146, MSI1, and NOTCH1 expression levels (1Götte M. Wolf M. Staebler A. Buchweitz O. Kelsch R. Schüring A.N. et al.Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma.J Pathol. 2008; 215: 317-329Crossref PubMed Scopus (160) Google Scholar). The finding that CD146 expression increased upon serial passaging, while MSI1 expression dropped, may indicate that these markers are expressed in different subpopulations. Specific growth factor supplementation or extracellular matrices may be required for propagation of MSI1-expressing cells. Detection of SOX2 is in accordance with endometrial expression of pluripotency-associated transcription factors such as OCT4 (10Matthai C. Horvat R. Noe M. Nagele F. Radjabi A. van Trotsenburg M. et al.Oct-4 expression in human endometrium.Mol Hum Reprod. 2006; 12: 7-10Crossref PubMed Scopus (92) Google Scholar) and NANOG (11Wolf M. Kiesel L. Götte M. Endometrial stem cells. Potential relevance for the pathogenesis of endometriosis?.Gynakol Endokrinol. 2009; 7: 185-189Crossref Scopus (7) Google Scholar), possibly suggesting the existence of different endometrial SC pools characterized by adult SC markers (CD146, MSI1, NOTCH1) or by an embryonic SC-like signature (SOX2, OCT4, NANOG), respectively. Our study provides novel evidence that routine endometrial biopsy is suitable to obtain MSC-like cells. Because the procedure is easy and performed routinely, a higher number of tissue samples can be obtained compared with hysterectomy. Since integrity and function of the organ is preserved, promising research strategies elucidating endometrial SC function during the female reproductive period will be possible. In the future, SCs derived by endometrial biopsy could provide an easily available source for the generation of induced pluripotent SCs. We thank Birgit Pers for expert technical assistance and Dr. Tobias Cantz, Dr. Andree Zibert, and Ramsi Siaj for helpful discussions.