346 (Becton, Dickinson and Company) in HPrE-conditional medium were seeded on a 96-well plate coated with 10 ll GFR Matrigel. Each assay was performed for 9 days, after which structures were ﬁxed and stained with FITC-labeled phalloidin (Sigma-Aldrich) to detect F-actin. For the 3D-embedded assay, 1,500 NHPrE1 or BHPrE1 cells in 100 ll GFR Matrigel were seeded in a 96-well plate (Greiner tissue culture treated, M0687; Sigma-Aldrich) coated with 10 ll GFR Matrigel, and 100 ll HPrE-conditional medium was added once the gel solidiﬁed. Animals Adult male severe combined immunodeﬁcient (SCID) mice (C.B- 17/IcrHsd-SCID) were purchased (Harlan, Indianapolis, IN, http:// www.harlan.com). All work involving animals was performed under protocols reviewed and approved by the Vanderbilt Institu- tional Animal Care and Use Committee. Tissue Recombinants and Subrenal Capsule Xenografting Rat UGM, the inductive mesenchymal cells surrounding the epi- thelial core of the urogenital sinus, was prepared from E18.5 em- bryonic fetuses as previously described [23, 24]. To prepare tis- sue recombinants, rat UGM was mixed with human prostate epithelial cells at different ratios as discussed in the text. The cell mixture was pelleted and resuspended in 50 ll rat-tail collagen (pretitrated to pH 7.4). After polymerization, the collagen was overlaid with growth medium. After incubation overnight at 37 C, the tissue recombinants were grafted under the renal cap- sule of intact C.B-17/IcrHsd-SCID mouse. Hosts were euthanized at various time points after grafting as noted in Results. Antibodies and Reagents A set of primary antibodies were used in these experiments of immunohistochemical (IHC) and IF staining and Western blotting (supporting information). The secondary antibodies were FITC- conjugated anti-mouse (Sigma-Aldrich) or biotinylated anti-rabbit (DAKO, Glostrup, Denmark, http://www.dako.com). ABC kit was purchased from Vector Laboratories, Burlingame, CA (http:// www.vectorlabs.com). Hematoxylin and Eosin, IHC, and IF Staining Tissue recombinants were dissected and ﬁxed in 10% phosphate- buffered formalin overnight, transferred to 50% ethanol, and then processed to parafﬁn. Samples were sectioned for eight succes- sive layers at 5 lm intervals and stained with hematoxylin and eosin (H&E). IHC and IF were performed as previously described. After deparafﬁnization with Histoclear (National Diag- nostics, Atlanta, GA, http://nationaldiagnostics.com), antigens were unmasked by heating samples in unmasking solution (Vec- tor Laboratories). Endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide and blocked with Clean Vision (ImmunoVision Technologies, Burlingame, CA, http://www. immunovisiontech.com). Samples were then incubated with pri- mary antibodies. After washing in PBS, the slides were incubated in biotinylated secondary antibody for 1 hour. Then the slides were incubated in ABC solution to amplify the signals before vis- ualizing with 3,30-diaminobenzidine (DAB) and mounting. Human prostate epithelial cells were cultured on slides for 3 days and ﬁxed in 50% methanol/ethanol for 10 min at room tempera- ture and stored in PBS for IF staining. Western Blotting Cells were grown in T-75 ﬂasks for 3 days and lysed with TNN buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.5% NP40 (pH, 7.4)) containing Roche’s Complete Proteinase Inhibitor Cocktail Tablets (Roche Diagnostics, Basel, Switzerland, http:// www.roche-applied-science.com) after washing with cold PBS. The whole lysates were clariﬁed at 13,000 rpm for 20 min at 4 C. The total protein levels were quantiﬁed and samples were stored at 80 C for use. Approximately 50 lg protein/well was Remodeling of Prostatic Tissue by Progenitor Cell loaded and electrophoresed through 10% NuPAGE Bio-Tris gel (Invitrogen) and transferred to nitrocellulose membranes. Mem- branes were blocked for 1 hour in 1 TBS-T (TBS, 0.1% Tween 20) containing 5% Difco skim milk (Becton, Dickinson and Com- pany), incubated with the primary antibody TBS-T containing 5% skim milk overnight at 4 C, washed for 30 min in TBS-T, and followed with the horseradish peroxidase-conjugated second- ary antibody (1:2,000, Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) diluted in 1 TBS-T containing 5% skim milk for 1 hour at room temperature. Amersham ECL plus detection reagent (GE Healthcare, Pittsburgh, PA, http://www. gehealthcare.com) was used to visualize protein bands. The b- actin was used for the loading control. Array-CGH Analysis Genomic DNA from NHPrE1 and BHPrE1 cell lines and their pa- rental primary cells NHPrE0 and BHPrE0 were extracted with a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany, http:// www1.qiagen.com) according to the manufacturer’s instructions. Genomic DNA was fragmented and random-prime labeled as described [25, 26] and hybridized to human oligonucleotide microarrays. The oligonucleotide array contains unique 236,000 elements designed for CGH proﬁling (Human Genome CGH 244A; Agilent Technologies, Palo Alto, CA, http://www.agilent. com). The median interval between mapped elements was 8.9 kb. Fluorescence ratios of scanned images of the arrays were proc- essed to identify statistically signiﬁcant transitions in copy num- ber by using a circular binary segmentation algorithm [25, 26]. In this study, signiﬁcant copy-number changes were determined on the basis of segmented proﬁles only. To deﬁne ploidy level, array-CGH assay was supplemented with G-band karyotyping. At least 40 DAPI-stained metaphase spreads were evaluated per sample. DIGMAP Analysis Method Chromosome mapping of expression data (differential gene locus mapping (DIGMAP)), which aligns the known chromosomal location of a gene to its expression value deduced by array-CGH analysis, was performed as previously described [27]. The output ﬁles from these statistical analyses were processed by the DIG- MAP Viewer and differential ﬂag region mapping programs. The DIGMAP Viewer program reads the DIGMAP source ﬁle con- taining CGH data and partitions the CGH data into subsets by chromosome number and subchromosomal locations. A graphical presentation was generated using a heat map to represent each data point with a colored cell that quantitatively reﬂected the original differential copy number intensities. Genomic regions exhibiting differential gene expression were marked as differen- tial ﬂag regions by visual inspection of the graphical displays. Accession Number All array-CGH data have been deposited at the GEO database (accession number GSE18122). RESULTS Regeneration of Prostatic Glandular Tissues in Mice by Primary Human Benign Epithelial Cells To investigate whether cultures of human prostatic epithelial cells at early passages can regenerate functional glandular structure in vivo, we utilized two strains of cells, NHPrE0 (NHP8) and BHPrE0 cells, which were isolated from a benign prostate surgical sample (Fig. 1A). These cells were used to generate tissue recombinants with inductive rat UGM, which were xenografted into immunocompromised mouse hosts. NHPrE0 is a mixed population of epithelial cells. The major- ity of such cells were characterized as basal in vitro, with few