Original articleIdentification and characterization of the human leiomyoma side population as putative tumor-initiating cells
Section snippets
Human Tissue Collection and Sample Preparation
Samples of human leiomyomas were obtained from Caucasian patients (n = 16) undergoing laparoscopic myomectomy for symptomatic uterine fibroids. Use of human tissue specimens was approved by the Institutional Review Board and Ethics Committee of Instituto Universitario, Instituto Valenciano de Infertilidad (registered under number 1006-C-072-CS-F). All patients signed a written informed consent.
Leiomyoma fragments were carefully dissected out and minced manually into small pieces (1–2 mm3). Then
Isolation, Molecular Characterization, and Clonogenicity Assays of Human Leiomyoma SP
Cell suspensions obtained from intramural leiomyomas (n = 16) were stained with PI and Hoechst 33342 dye to evaluate cell viability and to isolate SP cells by flow cytometry. Cell viability after our procedure ranged from 60% to 75%, and the leiomyoma SP cells represented 0.63% ± 0.21% of the total living cells analyzed. In all cases, the ABCG2 inhibitor, reserpine, was used as a negative control (Fig. 1A).
At the mRNA level, leiomyoma SP cells expressed ABCG2 transporter in accordance with
Discussion
The existence of myometrial SCs has been validated by the identification of label-retaining cells in an animal model (49) and in human myometrium by the SP method (31). In our study, we went one step forward, demonstrating the existence of SP cells in uterine leiomyoma with SC features of tumor-initiating cells. We demonstrated that the proportion of SP in the whole leiomyoma fraction was 0.63% ± 0.21%. These percentages of leiomyoma SP cells are in accordance with recent publications in this
Acknowledgments
The authors thank Verónica Ruiz and Amparo Galán from the Valencia Stem Cell Bank for their assistance in molecular biology; Enrique O'Connor and Alicia Martínez-Romero for their valuable experimental support in the flow-cytometric assays; Viviana Bisbal from the Prince Felipe Research Centre for expert advice in animal procedures; and Sonia Herráiz for her experience in the immunohistochemistry assays. Finally, we thank Jose Antonio Martinez Conejero from IVIOMICS for his expert guidance in
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A.M. has nothing to disclose. I.C. has nothing to disclose. C.G.-S. has nothing to disclose. A.F. has nothing to disclose. J.F. has nothing to disclose. A.P. has nothing to disclose. C.S. has nothing to disclose.
A.M. and I.C. contributed equally to this article.
This work has been funded by grant no. SAF 2008-02048 from the Spanish Ministry of Science and Innovation (principal investigator: C.S.), by Fundació Gent per Gent 08/09 (principal investigator: C.S.), and by PROMETEO/2008/163 (principal investigator: C.S.), which is supported by the Regional Valencian Ministry of Education.