br Introduction The adult prostate gland contains a simple c

Introduction The adult prostate gland contains a simple columnar epithelium composed of luminal secretory and underlying basal PCP inhibitor with a scarce neuroendocrine cell component. These epithelial cells are derived from a rare, relatively quiescent stem cell population that maintains glandular homeostasis throughout life (Leong et al., 2008). While prostate epithelial stem cells and their progeny have been investigated in rodent models and humans, their unique characteristics and lineage hierarchy remain a topic of debate. Credible evidence suggests that there may be a common precursor stem cell for all lineages while other findings support distinct basal and luminal stem cell populations within the adult prostate. These two scenarios are not mutually exclusive as emerging data indicates inherent plasticity and stage/context-specific utilization of stem and progenitor cell populations. In rodent models, prostate homeostasis appears to be maintained by both luminal and basal unipotent progenitor cells as well as bipotent stem/progenitor cells that exist in both compartments (Ousset et al., 2012; Toivanen et al., 2016; Wang et al., 2015; Wang et al., 2013; Xin et al., 2007). Although stem cell traits and lineage hierarchy for the human prostate epithelium are less studied, lineage tracing techniques using mitochondrial mutations have clearly demonstrated that basal, luminal and neuroendocrine cell lineages in the adult prostate are derived from a common precursor stem cell (Blackwood et al., 2011; Gaisa et al., 2011). Most current evidence from human prostate tissues suggests that normal stem cells primarily reside within the basal cell compartment (Goldstein et al., 2008; Zhang et al., 2016). Advances in prostate cancer research have identified resident cancer stem-like cells that are intrinsically resistant to standard treatments and reseed tumor growth following ablative therapies (Chen et al., 2016; Collins et al., 2005; Yun et al., 2016). Furthermore, gene profiling analysis has shown that prostate cancer increases in a stem-like state as it progresses from organ-confined to metastatic disease (Smith et al., 2015). Consequently, it is imperative to develop therapeutic modalities that target the prostate cancer stem-like population for effective disease management. Although prostate cancer stem cells will be distinct from normal prostate stem cells (Chen et al., 2016), similarities could be capitalized on for therapeutic advantage. Thus a fundamental understanding of normal human prostate stem cell properties and the factors that modulate their self-renewal and lineage commitment may provide new insights into the origin and treatment of prostate cancer. Approaches for isolating prostate stem cells have primarily utilized flow cytometry and 3D spheroid culture (Hu et al., 2011; Leong et al., 2008; Xin et al., 2007). However, a detailed characterization has been hindered by their lack of specificity and selectivity. Utilization of FACS with different antibodies against multiple surface antigens have yielded variable results (Collins et al., 2005; Vander Griend et al., 2008; Williamson et al., 2013), raising questions on the identity of the isolated cells. While resident stem cells are typically growth quiescent in vivo, when placed in 3D matrix culture without niche restraints, they undergo asymmetric division, generating progenitor cells that rapidly proliferate and lineage commit. Whereas the prostasphere (PS) culture system has been useful to enrich stem and progenitor cell populations, the resulting spheroids are a heterogeneous mixture of these cells types (Fig. 1C), making the identification of unique stem cell properties inconclusive. Clearly, improved assays to recognize and separate prostate stem cells are essential to move the field forward. Towards that end, the overall goal of the present study was to develop a system that permits clear identification and isolation of purified stem cells from human prostate specimens and conduct robust downstream analysis of their functional properties.