Jiang, Strand, Fernandez et al. highly branched structures, which were never observed using BHPrE1 cells (Fig. 5B), possibly reﬂecting the signiﬁcant dif- ference in side population percentage (Fig. 2E). Functional Regeneration of Benign Human Prostatic Tissues in Mice Using NHPrE1 and BHPrE1 Cells with Inductive Rat UGM To investigate morphological and biological features of the NHPrE1 and BHPrE1 cell lines, a tissue recombination-xeno- grafting model was used to reconstruct human prostatic glan- dular architecture in an immunodeﬁcient SCID mouse model. Tissue recombinants made by mixing human prostate epithe- lial cell lines with inductive rat UGM in type I collagen were implanted under the renal capsule of testosterone-supple- mented male SCID mice (Fig. 6A). A series of different num- bers of NHPrE1 or BHPrE1 epithelial cells (10, 5,000, 50,000, 100,000, 200,000, 400,000, 600,000, and 800,000) to a single rat UGM (making four small pieces) were tested to empirically determine growth of tissue recombinants in vivo (Table 1). A minimum number of 10 NHPrE1 cells gave rise to small immature glands within 3 months (Fig. 6B). In contrast, a minimum of 200,000 BHPrE1 cells was required to generate similar structures. A ratio of 600,000 NHPrE1 or BHPrE1 cells to the mesenchymal cells derived from a single rat UGM gave rise to the best functional remodeling of benign human prostatic glandular tissues in mice (Fig. 6B). This ratio of epithelial cells/UGM was standardized for most experi- ments (Fig. 6C, 6D). Histologically, tissue recombinants of NHPrE1 cells gave rise to mature, well-differentiated glandular epithelium with organization closely resembling the human prostate after 3 months. Reconstituted glandular tissues showed the typical two layers of luminal and basal cells, and a glandular lumen ﬁlled with secretory ﬂuid (Fig. 6B). Epithelial cell origin was conﬁrmed using GFP-tagging, with subsequent IHC staining of NHPrE1-EGFP cells (Fig. 6C) and BHPrE1-EGFP (data not shown). These ﬁndings were also conﬁrmed by direct ob- servation of GFP by IF (data not shown). AR and NKX3.1 proteins were weakly expressed in the basal cells but strongly expressed in the 100% luminal cells (Fig. 6C). p63 protein was expressed in the basal cells (Fig. 6C). By double IF stain- ing, human prostate-speciﬁc biomarkers PSA and 15-LOX-2 proteins were expressed in the luminal cells of both NHPrE1- and BHPrE1-prostatic tissue recombinants, while p63 protein was found in the basal cells (Fig. 6D). b-Catenin (Fig. 6D) and E-cadherin (data not shown) were strongly expressed at the cellular membrane. Tissue recombinants made with BHPrE1 cells showed a phenotype consistent with mature prostatic acini by H&E and PAS (supporting information) by 3 months after grafting. His- tology and IF and IHC staining, which showed biomarker expression patterns in the reconstituted glandular tissues, resembled those seen both in the source of human prostate tis- sue (supporting information) and in recombinants made with BHPrE0 (Fig. 1A, 1B), NHPrE1 (Fig. 6B–6D), and NHPrE0 cells [19]. Consistent with many previous studies, control grafts of epithelial cells without rat UGM did not show any glandular organization and also did not form any appreciable masses in mice (data not shown), which conﬁrms the lack of malignant potential in NHPrE1 and BHPrE1 cell lines. These results, using human prostate-speciﬁc epithelial cell lines NHPrE1, a progenitor cell type with characteristics of CD133high/CD44high/OCT4high/PTENhigh, and BHPrE1, an in- termediate cell type with characteristics of p63high/p53high/ www.StemCells.com 353 p21(WAF1)high/RBhigh, mixed with inductive rat UGM dem- onstrate in an in vivo mouse model that mature human benign prostatic epithelial tissues are able to be functionally regener- ated from immortalized lines, with expression of a range of mature prostatic markers. DISCUSSION We established two adult, non-tumorigenic human prostatic epithelial cell lines: NHPrE1, which retains progenitor charac- teristics, and BHPrE1, which displays epithelial intermediate/ transit amplifying cell characteristics. These cell lines were generated without external DNA or viral modiﬁcation. Cells cultured in serum-supplemented HPrE-conditional medium in vitro were subsequently able to recapitulate key developmen- tal events in a tissue recombination-xenograft model, and to regenerate well-differentiated secretory prostatic tissues, expressing human prostatic biomarkers including PSA and 15- LOX-2 and the prostate-associated transcription factor NKX3.1. The NHPrE1 and BHPrE1 cell lines will be valuable tools to further develop our understanding of both cellular lin- eages and the molecular mechanisms underlying human pros- tatic development and pathogenesis. Possible Mechanisms of Spontaneous Immortalization Many methods have been used to immortalize cells. The use of viral oncogenes is efﬁcient but results both in signiﬁcant alterations to the cell cycle machinery and in the inactivation of key pathways such as p53 and RB, making the cells sus- ceptible to genomic instability and malignant transformation. Other approaches have included the overexpression of human TERT or knockdown of p16, which has some advantages in terms of reducing cellular changes [19, 32] but, like all model systems, also retains some problems (reviewed in [32, 33]). In this study, a spontaneous immortalization that allows cells to come through crisis and then derive immortalized lines has been used. The advantages of this approach include limiting genetic damage to key cell cycle checkpoints and allowing for the derivation of cells that are able to recapitulate key aspects of prostatic biology when appropriately stimulated by inductive mesenchyme. Negative results from the soft agar colony formation assay and the benign nature of both NHPrE1 and BHPrE1 cell lines in the tissue recombination assays conﬁrmed the lack of malignant transformation. Telo- merase expression was found to be elevated in both NHPrE1 and BHPrE1 cells compared with their parental primary cul- ture cells, while p16 protein expression was decreased in BHPrE1 cells, potentially representing routes contributing to their immortalization. High levels of p16 protein expression in BHPrE0 suggested possible senescence occurring in cell culture. In contrast, p16 protein was elevated in NHPrE1 cells compared to NHPrE0 cells, suggestive of progenitor/stem cel- lular self-renewal behaviors. Cell Lineage Commitments in Prostatic Organogenesis and Morphogenesis The development of the prostate results from androgen-driven mesenchymal-epithelial interactions [2]. Some key details of this process remain unclear. These include documenting the precise nature of the growth and transcription factors that mediate these interactions, an area that has been widely inves- tigated and in which much progress has been made [34–42]. A less well-understood aspect has been the nature of the