Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy

In human prostate epithelium, morphologically basal and luminal cells can b e discriminated. The basal cell layer that putatively contains progenitor c ells of the secretory epithelium is characterized by the expression of kera tins (K) 5 and 14. Luminal cells represent the secretory compartment of the epithelium and express K8 and 18. We developed a technique for the simulta neous analysis of K5, 14, and 18 to identify intermediate cell stages in th e prostate epithelium and to study the dynamic aspects of its differentiati on in vitro. Nonmalignant prostate tissue and primary epithelial cultures w ere immunohistochemically characterized using triple staining with antibodi es for K5, K14, and K18. Antibodies for K18 and K5 were conjugated directly with fluorochromes Alexa 488 and 546. K14 was Visualized indirectly with s treptavidin-Cy5. Keratin expression was analyzed by confocal scanning micro scopy. The occurrence of exocrine and neuroendocrine differentiation in cul ture was determined via antibodies to prostate-specific antigen (PSA), chro mogranin A, and serotonin. We found that basal cells expressed either K5(+)/14(++)/18(+) or K5(++)/18(+). The majority of luminal cells expressed K18 (++), but colocalization of K5(+)/18(++) were recognized. Epithelial monola yer cultures predominantly revealed the basal cell phenotype K5(++)/14(++)/ 18(+); whereas intermediate subpopulations expressing K5(+)/14(+)/18(++) an d K5(+)/18(++) were also identified. On confluence, differentiation was ind uced as multicellular gland-like buds, and extensions became evident on top of the monolayer. These structures were composed of K18(++)- and K5(+)/18( +)-positive cell clusters surrounded by phenotypically basal cells. Few mul ticellular structures and cells in the monolayer showed exocrine differenti ation (PSA(+)), but expression of chromogranin A and serotonin was absent. We conclude that simultaneous evaluation of keratin expression is useful fo r analyzing epithelial differentiation in the prostate. During this process , putative stem cells phenotypically resembling K5(++)/14(++)/18(+) differe ntiate toward luminal cells (K18(++)) via intermediate cell stages, as iden tified by up-regulation of K18 and down-regulation of K5 and 14.