Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme Barbara A. Foster 1 , Kalyan J. Gangavarapu 1 , Grinu Mathew 2 , Gissou Azabdaftari 3 , Carl D. Morrison 3 , Austin Miller 4 , Wendy J. Huss 1,5 * 1 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, United States of America, 2 Medical and Molecular Genetics, Indiana University School of Medicine, IUPUI, Indianapolis, Indiana, United States of America, 3 Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York, United States of America, 4 Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, New York, United States of America, 5 Department of Urologic Oncology, Roswell Park Cancer Institute, Buffalo, New York, United States of America Abstract Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population. Citation: Foster BA, Gangavarapu KJ, Mathew G, Azabdaftari G, Morrison CD, et al. (2013) Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme. PLoS ONE 8(1): e55062. doi:10.1371/journal.pone.0055062 Editor: Jean-Marc Vanacker, Institut de Ge ´nomique Fonctionnelle de Lyon, France Received August 15, 2012; Accepted December 22, 2012; Published January 31, 2013 Copyright: ß 2013 Foster 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 work was supported by NYSTEM (CO24292) and National Institutes of Health (NIH) (R01DK091240) to WJH; NIH (R01CA095367) to BAF; the NCI Cancer Center Support Grant (CA016056) to RPCI. 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 read the journal’s policy and have the following conflicts: WJH: RPCI patent holder titled ‘‘Methods for evaluating and implementing prostate disease treatments’’ Patent No. 8048640. * E-mail: wendy.huss@roswellpark.org Introduction Prostate epithelial stem cells are defined as possessing the capability to generate prostatic epithelium through the properties of self-renewal and multipotency. These essential features of prostate stem cells can be tested in vivo in the tissue recombination assay. Recombination of an epithelial stem cell with mesenchyme derived from embryonic urogenital sinus mesenchyme (UGM) and grafting the recombinant under the renal capsule of an immune compromised host animal re-establishes the stem cell niche and allows for the dynamic assaying of stem cell properties within an in vivo system. The classic application of urogenital tissue recombination technology was the demonstration that hetero- specific (between species) recombinations of UGM induced differentiation and branching morphogenesis in transplanted epithelium from different species [1]. The recombined mesenchy- mal/stromal environment has profound effects on the phenotype of the associated epithelium. Studies using adult human prostate epithelium in tissue recombination assays demonstrate that the stem cells in the prostate epithelial compartment can respond to the inductive effect of rodent UGM by committing to prolifera- tion, undergo branching morphogenesis and differentiation [2]. In addition, the human prostatic epithelium dictates smooth muscle differentiation in the rat UGM (rUGM), inducing the appearance of thick sheets of smooth muscle characteristic of human, not rat, prostate [2]. Tissue recombination has been used to demonstrate that the mouse prostate stem cell is located in the proximal region of the prostatic duct, and can be enriched by isolating Sca expressing cells [3,4]. Furthermore, single mouse prostate cells expressing Lin 2 Sca + CD133 + CD44 + CD117 + generated prostate tissue for one generation when recombined with rUGM at a low frequency [5]. Recent studies using lineage-tracing methods in prostate regeneration suggest that basal and luminal stem cells repopulate the respective compartments [6]. PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e55062