Low-calcium serum-free defined medium selects for growth of normal prostatic epithelial stem cells

Abstract

Stage-specific differentiation markers were used to evaluate the cellular composition and the origin of nonimmortalized (PrEC) and immortalized (PZ-HPV7, CA-HPV10, RWPE-1, and 957E/hTERT) human prostate cell lines. These studies documented that immortalized and nonimmortalized prostate epithelial cells established and maintained in low (i.e., <300 micromol/L) Ca(2+) serum-free defined (SFD) medium were all derived from normal nonmalignant prostate tissues and contain CD133(+)/ABCG2(+)/alpha(2)beta(1)(Hi)/p63(-)/PSCA(-)/AR(-)/PSA(-) prostate stem cells. In these cultures, prostate stem cells are able to self-renew and generate two distinct cell lineages: the minor proliferatively quiescent neuroendocrine lineage and the major transit-amplifying cell lineage. Subsequently, CD133(-)/ABCG2(-)/alpha(2)beta(1)(Hi)/p63(+)/PSCA(-)/AR(-)/PSA(-) transit-amplifying cells proliferate frequently and eventually mature into proliferatively quiescent CD133(-)/ABCG2(-)/alpha(2)beta(1)(Lo)/p63(-)/PSCA(+)/AR(-)/PSA(-) intermediate cells. Such proliferatively quiescent intermediate cells, however, do not complete their full maturation into CD133(-)/ABCG2(-)/alpha(2)beta(1)(Lo)/p63(-)/PSCA(-)/AR(+)/PSA(+) luminal-secretory cells in low Ca(2+) SFD medium. Addition of universal type I IFN and synthetic androgen (R1881) to culture medium resulted in up-regulation of androgen receptor protein expression. However, it failed to induce full differentiation of intermediate cells into AR(+)/PSA(+) luminal-secretory cells. Our results indicate that such inability of prostate epithelial cells to complete their differentiation is due to continuous expression of Notch-1 receptor and its downstream effector, Hey-1 protein, which actively suppresses differentiation via its ability to transcriptionally repress a series of genes, including the GATA family of transcription factors.

Figure 1

Overview of phenotypic characteristics of the various cell subtypes within a prostate stem cell unit. NE, neuroendocrine cell.

Figure 2

A, ΔNp63 immunocytochemical staining (magnification, ×100) of CD133⁺ magnetic bead-selected stem cells (arrow). B, p63 gene can be alternatively spiced to generate a variety of splicing variants known as α, β, and γ isoforms of the TA or ΔN families of p63. C, expression of ΔNp63 and TAp63 proteins in human prostate normal and malignant cell lines. Actin was evaluated as a loading control. D, expression of AR, ΔNp63, PSCA, and actin (as a loading control) in the denoted lines.

Figure 3

Normal human prostate epithelial PrEC cultures are heterogeneous in cellular composition. A, phase contrast (magnification, ×20) of small, proliferating (arrow) transit-amplifying cells and corresponding ΔNp63 immunocytochemical staining (magnification, ×40). B, phase contrast (magnification, ×20) of large nonproliferating intermediate cells and corresponding immunocytochemical staining (magnification, ×60) for a subset of ΔNp63⁻ intermediate cells (arrow). C, phase contrast (magnification, ×20) of a neuroendocrine cell and immunocytochemical staining (magnification, ×20) of a ΔNp63⁻ neuroendocrine cell. D, flow cytometric analysis for PSCA expression of PrECcells after 4 and 10 days in culture. A nonspecific IgG was used as an isotype control. Cells were labeled with an anti-PSCA antibody followed by a FITC-conjugated secondary antibody and analyzed by flow cytometry. The percentage of PSCA⁺ cells in culture was 4.41% after 4 days and 10.40% after 10 days. Moreover, the size of the PSCA⁺ cells increased over time as indicated by an increase in forward scatter (FSC).

Figure 4

A, immunocytochemical ΔNp63 and PSCA staining (magnification, ×60) of 957E/hTERT cells. Time-lapse phase-contrast (magnification, ×20) monitoring of 957E/hTERT culture documented that large PSCA⁺ intermediate cells are proliferatively quiescent, whereas otherwise viable and mobile. For instance, two large intermediate cells (arrows) were monitored from day 1 (B) to day 4 (C) of the time-lapse experiment and were documented not to divide. In contrast, small cells in 957E/hTERT culture proliferated continuously. D, phase contrast (magnification, ×80) of neuroendocrine cells (arrows) in 957E/hTERT, CA-HPV10, and RWPE-1 cultures.

Figure 5

A, Western blot detection of AR protein expression in indicated cell lines in the presence or absence of 1 nmol/L of the synthetic androgen R1881. B, growth response to androgen (R1881) stimulation by the indicated cell lines. C, RWPE-1 cells were exposed for 24 hours to 1,000 units/mL universal type I IFN in the presence or absence of 1 nmol/L R1881 and AR protein levels were evaluated by Western blot. Immortalized WPMY-1 normal human prostate stromal cells in the presence or absence of R1881 were used as a control for weak AR expression. D, Western blot analysis of 110-kDa cleaved Notch-1 fragment, Hey-1, GATA-2, PSA, and actin (as a loading control) protein expression in the indicated normal and malignant prostate cell lines.