Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells  Aymara Mas, Ph.D., Irene Cervelló, Ph.D., Claudia Gil-Sanchis, Ph.D., Amparo Faus, B.Sc., Jaime Ferro, M.D., Antonio Pellicer, M.D, Carlos Simón, M.D.  Fertility and Sterility  Volume 98, Issue 3, Pages 741-751.e6 (September 2012) DOI: 10.1016/j.fertnstert.2012.04.044 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Identification, isolation, and characterization of the human leiomyoma SP cells. (A) Distribution of the SP and NSP living cells isolated from human leiomyoma cell fraction (left panel). SP cells represent 0.63% of total living cells stained with Hoechst 33342. Coaddition of 50 μM reserpine (inhibitor of ABC transporters) resulted in the disappearance of the SP fraction, negative control (right panel). (B) Molecular characterization showing the mRNA expression of ABCG2, hormonal receptors (ESR1, ESR2, FSHr, LHr), and muscular markers (smoothelin and calponin) in leiomyoma SP, leiomyoma NSP, and myometrial tissue. The hESC line VAL-9, obtained from the Spanish Stem Cell Bank (http://www.isciii.es/htdocs/terapia/terapia_lineas.jsp), was used as a positive control (C+) for ABCG2. Endometrium and myometrium were used as positive controls (C+) for hormone receptors and smooth muscle markers, respectively. Negative control (C−): water (no RNA samples). GAPDH gene expression was used as a housekeeping gene for all the samples analyzed (last row). (C) Highlighted macroscopic morphology showing the colonies formed by sorted SP (left) and NSP (right) from the human leiomyoma cells isolated and cultured at clonal densities for 15 days under hypoxic conditions. The lower graph shows the cloning efficiency (%) of sorted SP (1.185%) versus NSP (0.775%) cultured under hypoxic conditions (data are mean ± SEM). Fertility and Sterility 2012 98, 741-751.e6DOI: (10.1016/j.fertnstert.2012.04.044) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Karyotype, molecular characterization, phenotype, and in vitro differentiation of the human LeioSP1-2 cell lines. (A) Cytogenetic analysis of LeioSP1-2 cell lines showing normal karyotypes corresponding to 46,XX in both cases. (B) PCR assays demonstrating the presence and/or absence of undifferentiated, mesoderm (MESO), endoderm (ENDO), and ectoderm (ECTO) genes in both cell lines. The hESC line VAL-9 was used as a positive control (C+) for undifferentiated markers, and several tissues like heart, kidney, liver, and brain were used to test mesodermal, endodermal, and ectodermal origin. (C) A positive expression (highlighted in red) was observed for the mesenchymal SC markers like CD73, CD90, and CD105 in both LeioSP cell lines. A negative expression in LeioSP1 and LeioSP2 was observed for hematopoietic SC markers (CD45 and CD34), endothelial marker (CD31), and endothelial progenitor marker (CD133). Antibodies isotypes (black) were used as negative controls: FITC-labeled IgG1 (Millipore), FITC-labeled IgG2b (Chemicon), APC-labeled IgG1 (Milteny Biotec), Alexa647-labeled IgM (Biolegend), and PE-labeled IgG1 (Abcam). (D) (Upper panel) Adipogenic differentiation of the induced LeioSP1 and LeioSP2 cell lines was determined by the presence of oil red-O staining. Adipocytes from the culture explant were included as a positive control with typical oil red-O staining (control cells). LeioSP1 and LeioSP2 cell lines, not treated with differentiation media, were included as negative controls (see small picture) and were negative for specific adipogenic staining. (Lower panel) Induction of osteogenic differentiation was detected by the reactivity against bone sialoprotein in LeioSP1 and LeioSP2. Osteocytes from the culture explant were included as a positive control for this protein (control cells), and nontreated cells with differentiation media were included as a negative control (see small picture). All histological images were examined under a ×20 objective lens. Fertility and Sterility 2012 98, 741-751.e6DOI: (10.1016/j.fertnstert.2012.04.044) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Reconstruction of human leiomyoma-like tissue from the LeioSP cell lines. (A–D) Gross appearance (indicated by an arrow) of LeioSP cell lines xenografts on the injected kidney and SC tissue. Histology of LeioSP cell lines xenografts by H&E staining on the SC tissue (E–G) and injected kidney (F–H), both treated with E2 or E2 + P4, 8 weeks after grafting. The histological analysis of both injection sites treated with E2 + P4 demonstrated high reconstruction appearence (right panels) in comparison with non-P treatment (left panels). Fertility and Sterility 2012 98, 741-751.e6DOI: (10.1016/j.fertnstert.2012.04.044) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 In vivo differentiation of human leiomyoma-like tissue from the LeioSP cell lines. (A) Characteristic expression of typical ECM (Alcian Blue) and cells with hormone component (PR) was demonstrated in an animal injected with LeioSP cells in both hormone treatment groups (E2 or E2 + P4). Positive controls corresponding to small intestine (Alcian Blue) and endometrium (PR) are shown (right panels). (B) Gross appearance and histology of host kidney injected with LeioSP mixed with myometrial cells, treated with E2 + P4. Positive expression for Alcian Blue and PR was observed as a result of combination cells. All histological images were examined under a ×10–20 objective lens. Fertility and Sterility 2012 98, 741-751.e6DOI: (10.1016/j.fertnstert.2012.04.044) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions