Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

Abstract

Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways enrichment identified ribosome biogenesis and membrane estrogen-receptor signaling in stem cells with NF-κB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity

“…Our previous study demonstrated that AR À pluripotent CSCs isolated from patient biopsies differentiated into prostatic glandular structures containing all three epithelial cell types including AR þ luminal secretory cells as well as AR À basal and neuroendocrine cells when engrafted with embryonic mesenchyme under the renal capsule (2). Similarly, other studies report that AR À normal prostate stem-like cells differentiate into three prostatic epithelial cell lineages (3). This pattern of AR À expression mimics that observed in human and rodent prostate development where AR À epithelial anlagen grow into the urogenital mesenchyme until AR protein is induced by, as yet unknown mechanisms, to initiate glandular lumen formation, epithelial cell specification, and androgen-mediated secretory activity (4,5).…”

The role of WNT10B in normal prostate gland development and prostate cancer

“…A critical role for WNT10B in directing prostatic fate was directly supported by our previous work that showed WNT10B was essential for in vitro directed differentiation of hESCs into prostatic organoids, an effect that could not be replicated with canonical WNT3A. 22 More recently, human prostate stem cells isolated from disease-free primary specimens showed specific immunostaining for WNT10B while daughter progenitor cells were largely negative 36 which suggests a continued function for WNT10B in stem cell maintenance in the adult prostate. Implicated roles for WNT10B in regulating stemness have been reported in other organ systems including hematopoietic stem cells where WNT10B was shown to promote in vitro proliferation.…”

Concise Review: Prostate Cancer Stem Cells: Current Understanding

“…As a consequence of dampened citrate oxidation, normal prostate epithelial cells have a low level of TCA and glycolytic metabolism. Due to a relatively quiescent state, normal prostate SCs have low levels of TCA, oxidative phosphorylation (OXPHOS), and glycolysis [37] (Fig. 1).…”