Jiang, Strand, Fernandez et al. 355 Table 1. Tissue recombinants made by different ratios of epithelial cells with rat urogenital sinus mesenchyme NHPrE1 BHPrE1 Cell number per tissue recombinant 10 5,000 50,000 100,000 200,000 400,000 600,000 800,000 Tissue recombinants (recovered/total) 3/8 2/6 3/5 4/6 ND 2/5 6/6 5/7 Phenotypes Few small immature glands Glandular formation Immature glands Glandular formation Mature glandular formation Well differentiated functional ductal-acinar structure Well differentiated functional glands Tissue recombinants (recovered/total) ND ND ND ND 2/2 2/5 4/6 ND Phenotypes Few small immature glands Small immature glands Well differentiated functional glands Tissue recombinants made using different numbers of NHPrE1 or BHPrE1 cells with rat urogenital sinus mesenchyme were grafted under the renal capsule of male SCID mice supplemented with testosterone pellets and examined at 3 months after grafting. Histology of the regenerated human glandular prostatic tissues was evaluated microscopically. Abbreviations: ND, not done; N/A, not available. epithelial cells involved in the development process. It has long been supposed that a stem/progenitor cell hierarchy exists within the prostate [5, 43]. This concept enjoys some experimental support [14]. The precise nature of the cells con- cerned has not been clariﬁed; however, expression of a num- ber of markers, including CD44, SCA1, and OCT4, has been considered to be associated with ‘‘stemness’’ [44, 45]. In this study, we present two new cell lines, NHPrE1 and BHPrE1. The NHPrE1 cell line, which is shown by chromosomal anal- ysis to be a mixed population, was provisionally classiﬁed as including a self-renewing progenitor population (possibly the identiﬁed side population) on the basis of expression of a set of candidate stem and basal cell markers in vitro. The BHPrE1 cell line, which is also a genetically mixed popula- tion, behaves in bulk like an intermediate or transit-amplify- ing cell type due to its expression of previously characterized markers including p63, NKX3.1, b-catenin, and E-cadherin [46, 47]. 3D culture can be used to discriminate between normal and malignant cells [31]. NHPrE1 and BHPrE1 cells were both shown to be capable of forming polarized ductal or aci- nar structures in 3D Matrigel surface assays, and NHPrE1 cells were uniquely able to undergo branching in 3D Matrigel embedded assays, possibly suggesting a high ratio of stem/ progenitor-like cells. BHPrE1 cells were not able to make branching structures in 3D cultures, at least under these con- ditions, consistent with a high ratio of intermediate cell type. In tissue recombinants, made by mixing different ratios of epithelial cells with a ﬁxed quantity of rat UGM tissue, func- tional regeneration of mature human prostate glandular struc- ture was found to depend on the number and nature of implanted human prostate epithelial cells. These experiments support the standard model of an intermediate/transit-amplify- ing population that can recapitulate prostatic development. The BHPrE1 cells were able to differentiate into prostatic epi- thelial tissue containing both basal and luminal epithelial cells. However, it was noted that the more progenitor-like NHPrE1 population was able to perform this task more efﬁ- ciently and with a lower number of cells. As few as 10 NHPrE1 cells could give rise to glandular tissue, while the minimum number of BHPrE1 cells needed for the same task was 200,000. This may reﬂect characteristics of the bulk pop- ulation of cells or, alternatively, the relatively elevated num- ber of ‘‘stem-like’’ cells in the NHPrE1 population. The www.StemCells.com expression of stem cell–associated markers, notably CD133, OCT-4, and CD44 at elevated levels in NHPrE1 cells com- pared to BHPrE1, was consistent with the concept of these cells representing or containing a ‘‘progenitor’’ population. Conclusion Our data suggest that functional regeneration of benign pros- tate glandular architecture is more efﬁcient when initiated from a population representing or containing a major subpo- pulation of putative progenitor cells rather than of intermedi- ate/transit amplifying epithelial cells. We demonstrate that immortalized human prostatic epithelial cell lines are able to recapitulate prostatic development. One of the major strengths of these lines is their ability to form appropriate ductal-acinar architecture containing epithelial subpopulations with appro- priate expression of markers including androgen receptor, NKX3.1, PSA, and 15-LOX-2. While there are genomic alter- ations in the lines presented here associated with crisis and subsequent spontaneous immortalization, these cells are behaviorally benign as assessed with histopathological crite- ria. As such, these cell lines represent potentially useful mod- els in which to start to investigate mechanisms associated with both benign and malignant disease. ACKNOWLEDGMENTS The work was supported by National Institutes of Health (NIH) grants U54 CA126505 and R01 DK067049, and by DOD-PCRP, W81XWH-07-1-0479 (S.W.H.), by NIH 2T32 CA009592-21A1 and DOD-PCRP W81XWH-09-1-0222 (D.W.S.), and the Aus- trian Science fund FWF, project #P21919 (A.B.). The array- CGH analyses were performed by the Genomics Center of the Belfer Institute for Applied Cancer Science and supported by the NIH (grant U54 CA126505). We also thank the Joe C. Davis Foundation for support. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conﬂicts of interest.