Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme

Barbara A Foster,Gissou Azabdaftari,Austin Miller,Kalyan J Gangavarapu,Wendy J Huss,Grinu Mathew,Carl D Morrison,Jean-Marc Vanacker

doi:10.1371/journal.pone.0055062

Barbara A Foster, Gissou Azabdaftari + Show 6 more

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https://doi.org/10.1371/journal.pone.0055062

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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.

Highlights

Prostate epithelial stem cells are defined as possessing the capability to generate prostatic epithelium through the properties of self-renewal and multipotency
ABC transporter G2 (ABCG2) enriched side population cells were determined by gating the population of cells that were restricted from DyeCycle Violet (DCV) efflux due to the inhibition of transporter activity in the presence of ABCG2 inhibitor fumitremorgin C (FTC) (Figure 2 and Figure S2)
While fresh undigested human tissue that contains stem cells has been used in tissue recombination as a classic example of this assay [2], most human prostate stem cell studies have focused on using transformed/immortalized cell lines or cells maintained in culture to generate spheres or organoids [9,28,29,30]

Summary

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. The classic application of urogenital tissue recombination technology was the demonstration that heterospecific (between species) recombinations of UGM induced differentiation and branching morphogenesis in transplanted epithelium from different species [1]. 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 proliferation, undergo branching morphogenesis and differentiation [2]. Recent studies using lineage-tracing methods in prostate regeneration suggest that basal and luminal stem cells repopulate the respective compartments [6]

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