A key element of the culture

A key element of the culture conditions is the balanced combination of growth factors and signaling molecules (e.g., EGF, bFGF, IGF, insulin, transferrin, and Rock inhibitor), which apparently generates an artificial androgen-independent PESC microenvironment that promotes the self-renewal and maintenance of 2prostate stem cell fate. Although these factors have been known for a long time in the cell-culture field, it was critical to discover the exact composition of the media in combination with a hydrophobic surface that would allow significant expansion of undifferentiated murine basal PESCs as compared with the widely used standard method (PrEGM). The necessary adaptations to enable human basal PESC amplification included the switch to surface-treated flasks and the addition of sodium selenite and progesterone. Progesterone has also been reported to induce mammary epithelial progenitor cell expansion, indicating that it may promote hormone-controlled epithelial stem corticotropin-releasing factor in general (Joshi et al., 2010). Expanded basal PESCs not only show expression signatures similar to those of pluripotent ESCs and other somatic stem cells but also harbor functional stem cell potential, as demonstrated by their capacity to generate prostatic tubules in vivo. These results are comparable to those obtained in transplantation experiments performed with PESCs isolated from primary prostate biopsies (Goldstein et al., 2010, 2011; Lukacs et al., 2010). The method described here now allows the robust expansion of such primary cells and thus facilitates an in-depth analysis of the molecular programs employed. One can induce expanded PESCs to differentiate at any desired time point by transferring the cells from adherent conditions into previously described prostasphere culture conditions (Xin et al., 2007). However, the described method cannot be used to study the role of luminal PESCs, which have also been reported to be a self-sustaining lineage (Karthaus et al., 2014). In addition to basal PESCs, luminal PESCs have also been suggested to be the putative cell of origin for prostate cancer (Choi et al., 2012; Goldstein et al., 2010; Wang et al., 2009). Furthermore, in vitro differentiation into spheres can only a serve as a model system and does not resemble the full luminal differentiation program of prostate gland development in vivo. This limitation and the putative presence of transit-amplifying (intermediate) cells have to be considered when using these methods (Ousset et al., 2012; Pastrana et al., 2011). Additionally, the methods we have described for murine cells cannot be used to replace lineage-tracing mouse models—they can only complement the findings from such models. In particular, work by Wang et al. (2013) clearly shows that prostate basal cells develop a substantial plasticity ex vivo when they are removed from their normal environment. In line with this, our experiments confirm the finding that a significant discrepancy exists between the high in vitro sphere-forming capacity of basal PESCs and their capacity to form glands in vivo. In vivo, only a small proportion of basal cells were shown to have a graft-regenerating capacity (Wang et al., 2013). Nevertheless, our methods additionally facilitate the analysis of primary human cells, allowing such cells to be amplified, manipulated, and studied in detail. Clearly, the direct analysis of human cells holds the potential to provide data that are of more relevance to the biology of human development and disease. The culture method described here creates a novel platform for studying prostate disease etiology and progression. PESCs grown as adherent feeder-free cultures are easy to manipulate (e.g., for transfection and infection) and can be induced to differentiate or transplanted to form prostate tubules in vivo. Thus, this method will provide the basis for various in-depth analyses of epithelial prostate stem cells. First, it provides the basis to selectively expand and study murine basal PESCs isolated from different genetically engineered mice, such as in the PTEN prostate cancer model (Di Cristofano et al., 2001). This may help to identify molecular mechanisms during differentiation and the progression from normal prostate basal stem cells to hyperplastic and possibly even neoplastic epithelium (Carver et al., 2011). However, one has to keep in mind that prostate cancers that arise from basal stem cells may have a different phenotype and clinical outcome compared with those derived from luminal prostate stem cells (Choi et al., 2012; Lu et al., 2013). Second, using co-culture techniques that combine basal PESCs with cellular prostate stromal components (e.g., associated fibroblasts and smooth muscle cells), one can dissect and study important cross regulations between primary PESCs and their corresponding microenvironmental niche to better understand prostate-gland regulation at a more global level. Third, human basal PESCs isolated from patients with BPH can be isolated and studied at the molecular and genomic levels, and subsequently linked to their biologic behavior in vitro and in vivo. An estimated 50% of men show histologic evidence of BPH by the age of 50 years, and 40%–50% of these men become clinically significant, demonstrating the clinical relevance of this novel method. Finally, mouse- or patient-derived and expanded PESCs can be used for high-throughput screens using knockdown or chemical compound libraries. This novel mouse and human method to expand functional PESCs may boost research on normal prostate gland biology and may open up new possibilities for studying the etiology of prostatic diseases.