The approach for stem cell identification utilized herein is

The approach for stem cell identification utilized herein is functional, based on the relative quiescence and thus label retention property of stem buy BMS-907351 within a mixed epithelial population. Long-term 5-bromo-2′-deoxyuridine (BrdU) retention has been previously used to label stem cells in vivo and in vitro based on their prolonged doubling time (Cicalese et al., 2009; Klein and Simons, 2011). In addition, the immortal strand DNA hypothesis suggests that as stem cells undergo asymmetric division, the older parental DNA segregates into one daughter stem cell while the other daughter cell receives newly synthesized DNA and becomes a committed progenitor cell (Cairns, 1975). This unique situation allows the opportunity to BrdU-label DNA in parental stem cells within primary cultures and monitors their properties following BrdU-washout upon transfer to 3D spheroid culture. In the present studies, this pulse-chase approach was applied to primary prostate epithelial cultures derived from healthy organ donors, as opposed to benign regions from patient specimens, to ensure lack of a modifying disease field effect. While primary prostate epithelial cells adapt a basal and transit amplifying phenotype in 2D culture, they also contain the rare multipotent stem cells as evidenced by formation of fully differentiated organoids or differentiated spheroids upon transfer to 3D systems (Hu et al., 2011; Karthaus et al., 2014). By using PS-based BrdU/CFSE/Far red retention assays followed by FACS sorting, we herein identify label-retaining spheroid cells at a single cell resolution. Importantly, they exhibit stem cell characteristics including asymmetric cell division with segregation of parental DNA in daughter stem cells, in vitro serial passage and in vivo prostate regenerative capacity, augmented autophagy flux, increased ribosome biogenesis and reduced metabolic activity relative to the lineage committed progenitor cells within early-stage spheroids. RNA-seq revealed differentially expressed genes in the stem-like cells including cytokeratin 13 (KRT13) and prostate cancer susceptibility candidate 1 (PRAC1) that may serve as novel biomarkers for human prostate stem cells. Application of this approach to cancer specimens and cell lines identified a small number of label-retaining cancer stem-like cells which may provide translational opportunities to target this therapeutic resistant population.
Materials and methods
Results
Discussion The present study utilized a long-term label-retaining approach to identify, isolate and characterize adult human prostate stem cells from young disease-free organ donors. Detailed analyses confirm the bona fide stem cell nature of these isolated cells using in vitro and in vivo assays of regenerative capability, unique gene expression profiles and the documentation of symmetric/asymmetric cell division, classic indicators of stemness. By use of this biologic-based approach, we identify several novel properties of prostate stem cells hitherto unknown or underappreciated. These include heightened autophagy flux and asymmetric DNA inheritance to maintain stem cell integrity, decreased mitochondrial metabolism, heightened ribosome biogenesis and elevated expression of specific genes that may be useful as prostate stem cell markers in tissue and cultured cell populations. That this approach can similarly be utilized for identifying cancer stem-like cells from primary specimens and cell lines provides an avenue for detailed interrogation of this elusive cancer cell population. Several novel markers for normal prostate stem cells were identified in the present studies. E-cadherin, a cell adhesion-junction protein, is an epithelial differentiation marker that is down-regulated in stem-like cells and cancer cells undergoing EMT (Graff et al., 1995; Toivanen et al., 2016). Herein, we find E-cadherin is absent in normal prostate stem cells but robust within daughter progenitors. Since RNA-seq data found comparable E-cadherin mRNA in the cell types, the limited protein in stem cells may reflect increased endocytosis or protein degradation (Bryant and Stow, 2004). Wnt10b is an early and specific marker of prostate epithelial buds during murine embryogenesis (Allgeier et al., 2010) and is essential for directed differentiation of hESC into prostate organoids (Calderon-Gierszal and Prins, 2015). The present data is the first to identify the selective expression of canonical WNT10B in prostate stem cells. As Wnt signaling is known to play a crucial role in stem cell homeostasis (Clevers et al., 2014), we propose that WNT10B may provide this necessary function in the prostate stem cell niche. Selective expression of KRT13 and PRAC1 was also documented in prostate stem cells relative to progenitors and knockdown experiments confirmed their critical role in maintaining stemness properties. A recent study similarly identified KRT13 as enriched in human fetal prostate epithelial cells and confirmed it as a marker for the stem/progenitor population in adult prostate tissue (Liu et al., 2016). Of note, KRT13 plays a directive role in prostate cancer metastasis and its expression in primary prostate tumors is predictive of metastasis and lower survival (Li et al., 2016). Little is known about PRAC1, which encodes a small nuclear protein in human prostate and bladder cancer with prognostic capability (Kim et al., 2015; Lenka et al., 2013). That KRT13 and PRAC1 are high in normal prostate stem cells and re-expressed in prostate cancers supports the emerging concept that during cancer progression, epithelia transition to a stem-like state that permits their robust growth potential. Together, these stem cell proteins as well as other identified differentially expressed genes may be useful markers in future studies to distinguish stem cells within human prostate tissues.