Isolation of Human Prostate Epithelial Stem Cells and Demonstration of Their Stem Cell Character
Its current definition requires a tissue stem cell to be capable of giving rise to all cell types that are characteristic of the particular tissue or organ. Addressing this issue of multipotency requires isolation of the respective tissue stem cells and differentiation of the pure population in vitro or in vivo. Isolation of the stem cell subpopulation, however, has to be performed on the basis of a marker that is exclusive to this population. Hudson et al. undertook clonal analysis of human prostate epithelial cells in order to identify the stem cell subpopulation. Upon cloning of cells from biopsies of benign prostate hyperplasia, two types of colony could be distinguished. Type II colonies exhibited a smooth periphery and densely packed cells, whereas cells in type I colonies were less densely packed and colony borders were irregular; which is indicative of the presence of cell types with differing proliferative activity. Type II colonies were capable of generating three-dimensional duct like structures in Matrigel, with evidence of luminal-specific cytokeratin and androgen receptor expression in a subset of the cells. These results implied that type II colonies were founded by stem cells, whereas type I colonies were probably founded by transit-amplifying cells. Due to a lack of known prostate stem cell markers at this time, these experiments provided indirect evidence of the lineage of prostate epithelia. Collins et al took advantage of the observed association of stem cells with basement membranes in skin and testis to enrich for prostate epithelial stem cells from BPH tissue samples. About 1% of basal cells in the prostate exhibit a high affinity for the extracellular matrix component collagen I, which is attributed to a high-level expression of integrin o^Pi in these cells. Cells that adhere rapidly to collagen I are clonogenic in vitro and capable of regenerating prostatelike acini in vivo, with evidence of expression of the luminal cell markers PAP, AR, and PSA. Consistent with the findings reported by Hudson et al., however, differential adhesion to collagen I does not separate stem and transit-amplifying cells, as the retrieved cell population is capable of giving rise to both type I and type II colonies. Furthermore, rapidly adherent cells contain a subpopulation of approximately 5% of committed basal cells, as indicated by expression of the luminal-specific cytokeratin 18 in these cells. Cells adhering to collagen I after 20 minutes found small, terminal colonies of irregular shape (type III colonies), which indicates the high degree of differentiation of these cells.
Richardson et al. extended the cell surface markers for the isolation of prostate epithelial stem cells to include CD133, a five-transmembrane-domain cell surface glycoprotein which had originally been identified as a marker for hematopoietic stem cells and had previously been used to enrich for endothelial and neuronal stem cells. CD133 cells comprise a subset of the basal epithelial cells in prostate (0.75%) and are restricted to the a2(3ihl population of basal cells, which had previously been shown to be enriched for prostate epithelial stem cells.[25] Immunostaining patterns for CD133 and the proliferation antigen Ki-67 did not overlap, which suggests a replicative quiescence of the a2(3ihl/CD133+ cell fraction. This fraction, however, exhibits a higher proliferative potential and colony-forming ability than that of the CD133″ cells within the a2(3ihl cell fraction. Basal cells selected for a2(3ihl/CD133 expression are also capable of regenerating a fully differentiated prostatic epithelium in vivo with evidence of expression of the common differentiation markers, whereas a2Pihi/CD133- cells lack this ability.
In contrast to the subpopulation retrieved from basal prostate epithelial cells on the basis of high-level expression of o^Pi integrin alone, the a2(3ihl/CD133+ cell fraction consists solely of epithelial stem cells, whereas transit-amplifying cells are restricted to the CD133″ fraction among a2(3ihl cells. Integrin a2(3ilow cells among the basal population probably represent cells committed to luminal differentiation. A different method for the isolation of prostate stem cells from primary tissues was published by Bhatt et al. The isolation procedure was based on the SP (side population) phenotype, which is commonly exhibited by murine hematopoietic stem cells and is characterized by the ability of the respective cells to efflux Hoechst dye 33342. The dye-effluxing stem cells constitute an unstained side population upon flow cytometric analysis. Using this method for prostate epithelia, the authors estimated the stem cell population to comprise 1.4% of all prostate epithelial cells. However, gland regeneration activity has not been demonstrated in association with this putative stem cell fraction.
