348 cells to rat UGM and on the postgrafting time course. Glandu- lar prostatic structures were found in tissue recombinants of NHPrE0 cells by 6 months after grafting [19] and with BHPrE0 cells by 4 months (Fig. 1A). A set of biomarkers and IHC staining showed protein expression patterns in the tissue recombinants made using BHPrE0 cells (Fig. 1B). These results were similar to NHPrE0-tissue recombinants [19]. Ku70-staining (data not shown), which distinguishes human cells by strong nuclear immunoreactivity, was strongly posi- tive in all epithelial cells. CK14 (Fig. 1B) and p63 (data not shown) were strongly expressed in the basal cells. CK18 (Fig. 1B) and CK8 (data not shown) were detected in the luminal cells. Androgen receptors (AR) were expressed in luminal and basal epithelial cells and in some stromal cells (Fig. 1B). PSA (Fig. 1B), which is a distinctive human prostate-speciﬁc marker, was expressed in the mature luminal epithelial cells appropriate to the observed differentiation status. These results demonstrate that benign, non-tumorigenic human pros- tate tissue architecture can be generated in mice from isolated primary cultures of epithelial cells using recombination with rat UGM. This is consistent with previous observations using primary digests of human prostatic epithelial organoids [9]. Generation and Characterization of Human Prostatic Epithelial Progenitor and Intermediate Cell Lines by Spontaneous Immortalization Two immortalized human prostatic epithelial cell lines desig- nated NHPrE1 and BHPrE1 were generated as described in Materials and Methods. These cells were derived from the pa- rental NHPrE0 and BHPrE0 strains, respectively. In culture, both NHPrE1 and BHPrE1 cells showed simi- lar prostate epithelial cell morphology, expressing predomi- nantly basal cell-associated markers (Fig. 2A). NHPrE1 cells proliferated somewhat more slowly under basal condi- tions than BHPrE1 cells (Fig. 2B), and the two lines showed similar cell cycle distribution proﬁles by FACS analysis compared to their parental NHPrE0 and BHPrE0 cells (sup- porting information). Using species-speciﬁc polymerase chain reaction primers to amplify human PSA (hPSA), rat Probasin, and mouse Probasin, genomic DNA of NHPrE1 and BHPrE1 cells was conﬁrmed as being of human origin (Fig. 2C). Ku70-IF staining (Fig. 2D) with strong nuclear immunoreactivity was strongly positive in all epithelial cells. A side population–FACS assay was used to detect a putative stem cell population in the NHPrE1 and BHPrE1 cell lines, and these data were compared to the parental pri- mary culture cells NHPrE0 and BHPrE0 [28]. The side pop- ulation numbers for NHPrE1 cells were approximately twice as great as those for BHPrE1. NHPrE1 demonstrated a 2.33% side population, which was reduced to 0.29% with verapamil treatment. BHPrE1 cells showed a 1.29% side population, which was reduced to 0.14% by verapamil treat- ment (Fig. 2E). The side population numbers for NHPrE0 cells were approximately 0.0%. BHPrE1 cells showed a 0.08% side population, which was reduced to 0.0% by vera- pamil treatment (supporting information), suggestive of rare stem cell population in primary cultured normal or benign human prostate epithelial cells. A soft agar colony formation assay was used to assess an- chorage-independent growth. No colony formation was detected in either NHPrE1 or BHPrE1 cells in contrast to C4- 2B positive control colony formation (supporting informa- tion). This ﬁnding is consistent with normal or benign cell types. With long-term continuous culture and more than 75 serial passages, both NHPrE1 and BHPrE1 cells maintained Remodeling of Prostatic Tissue by Progenitor Cell the basal epithelial cell type in vitro in the 5% FBS HPrE- conditional medium. Phenotypic Analysis of NHPrE1 and BHPrE1 Cells IF staining and Western blotting were used to characterize the NHPrE1 and BHPrE1 cell lines in vitro. Immunophenotyping conﬁrmed both cell lines as human (15-LOX-2), epithelial (WS-CK, CK14, p63, and b-catenin) (Fig. 3A), consistent with the observations described above. NHPrE1 cells expressed higher levels of the stem cell- associated CD133, CD44, and OCT-4, as well as lower levels of b-catenin and 15-LOX-2 proteins by IF staining compared with BHPrE1 cells (Fig. 3A). This is consistent with a puta- tive progenitor phenotype. In addition, the data generated were compared with those of the parental NHPrE0 and BHPrE0 cells. NHPrE1 cells expressed elevated levels of CD133, CD44, sonic hedgehog (SHH), PTEN (with high AKT and negative P-AKT expression (supporting informa- tion)), NKX3.1, p53, and RB by Western blotting, compared with NHPrE0 cells (Fig. 3B). BHPrE1 cells expressed higher levels of CD133, CD44, SHH, NKX3.1, p53, and RB pro- teins, and a lower level of PTEN protein, by Western blotting, compared with BHPrE0 cells (Fig. 3B). BHPrE1 cells showed similar levels of p53, NKX3.1, AKT, and Cyclin D1 (Fig. 3B and supporting information) as NHPrE1 cells. BHPrE1 cells expressed lower levels of CD133, CD44, PTEN, and OCT-4 (Fig. 3B and supporting information) proteins by Western blotting, compared with NHPrE1 cells. However, BHPrE1 showed higher levels of SHH, RB, C-MYC, p14 (ARF), p21 (WAF1), and the epithelial differentiation markers p63, b-cat- enin, and E-cadherin, than NHPrE1 cells did (Fig. 3B and supporting information). These data suggested characterization of the BHPrE1 line as a putative intermediate cell type. TERT protein expression was detected in both NHPrE0 and BHPrE0 cell lines, though at very low levels, along with a moderate level of p16 protein (Fig. 3B), suggesting that spontaneous immortalization may occur by progenitor/stem cell self-renewal and enrichment. Elevated levels of p16 pro- tein seen in BHPrE0 primary cells suggested the possibility of senescence in a subpopulation of cells. A few vimentin-posi- tive NHPrE1 cells were seen with IF staining (data not shown) and conﬁrmed with Western blotting; however, vimentin was not detected in BHPrE1 cells (supporting infor- mation). Synaptophysin, a biomarker characteristic of neuro- endocrine cells, was detected in a few NHPrE1 cells but was not seen in BHPrE1 cells (data not shown). A few PSA-posi- tive cells were detected with IF staining and a low level of PSA protein expression was seen with Western blotting in BHPrE1 cells, while PSA was not detected in NHPrE1 cells (data not shown). a-Smooth muscle-actin was not detected in either NHPrE1 or BHPrE1 cells (data not shown). No evi- dence of epithelial to mesenchymal transformation or stromal/ mesenchymal cell types was seen even after many passages of continuous culture in vitro. Karyotypic and Array-CGH and DIGMAP Analysis With long-term continuous culture in the serum-supplemented HPrE-conditional medium, NHPrE1 cells showed a near dip- loid karyotype with a 3n sideline. BHPrE1 cells were near 3n, 65–72 chromosomes with signiﬁcant chromosomal rearrange- ments. Array-CGH was used to detect DNA ampliﬁcation or deletion in genomic samples from NHPrE1 and BHPrE1 cell lines. Global views of the array-CGH results for NHPrE1 and BHPrE1 cell lines, their parental NHPrE0 and BHPrE0 cells were plotted (Fig. 4A). There was an obvious hemizygous de- letion of TP53, but not WAF1 (p21), Cyclin D1, or RB, in both NHPrE1 and BHPrE1 cells, compared to the array-CGH