Jiang, Strand, Fernandez et al. notably PSA and 15-lipoxygenase-2 (15-LOX-2), in human but not rodent prostate [10, 11]. The concept of primitive stem and progenitor cells with the capacity for self-renewal and multilineage differentiation has been important in developing a model to understand the molec- ular mechanisms of normal prostatic development, maturation, and functional homeostasis [5, 12]. This idea is also important to comprehend how tissues are remodeled during inﬂammatory repair or in carcinogenesis resulting from genomic insult, meta- bolic oxidative stress, or inﬂammation [13, 14]. However, at a practical level there have been few human cell lines available that accurately recapitulate prostatic development and that can be used to examine these concepts. To pursue studies relevant to normal human prostate biology and as a starting point for studies on human disease, there is an urgent need for human prostate epithelial cell lines that show phenotypes that match human tissue samples. Tissue recombination is a valuable tool for the analysis of the functional remodeling of benign human prostatic tissues in an immunodeﬁcient mouse model [9]. While a number of non-tumorigenic immortalized human prostate epi- thelial (HPrE) cell lines have been established using viral SV- 40Tag or E6/E7 infection including BPH-1 [15], and RWPE-1 [16], none of these accurately recapitulate normal human pros- tatic growth and function. Tissue recombinants generated using BPH-1 cells exhibit solid cords and squamous hyperplastic dif- ferentiation [17]. RWPE-1-tissue recombinants show a more differentiated acinus-like structure [18]. These cells can be used to answer speciﬁc questions. However, the viral onco- genes seriously impair their relevance to many aspects of human prostatic biology. None of the immortalized lines avail- able accurately recapitulates the well-differentiated mature duc- tal-acinar system characteristic of human prostate. In this report, we describe the establishment and characteri- zation of adult, non-tumorigenic human prostate-speciﬁc epi- thelial progenitor and intermediate cell lines. NHPrE1, a spon- taneously immortalized ‘‘normal’’ human prostate epithelial cell line, that was established from parent NHPrE0 cells, and BHPrE1, a spontaneously immortalized benign human prostate epithelial cell line from BHPrE0. Cell lines were characterized using immunoﬂuorescence (IF) staining, Western blotting, and array-comparative genomic hybridization (array-CGH) analysis as organ-speciﬁc epithelial progenitor and intermediate cell types, respectively. We further demonstrated that these cells differentiate into mature, functional human prostatic ductal-aci- nar structures in vivo using a tissue recombination-xenografting assay with inductive rat urogenital sinus mesenchyme (UGM). The in vitro characteristics of the cells in relation to putative progenitor and intermediate/transit amplifying cell marker expression are shown to be reﬂective of their behavior in vivo. MATERIALS AND METHODS Primary Culture and Spontaneous Immortalization HPrE cells (NHP8, designated NHPrE0 here), primary cultured ‘‘normal’’ human prostate epithelial cells isolated from a 41-year- old healthy male prostate, were purchased from Cambrex (Charles City, IA, http://www.cambrex.com) and cultured in the supplied se- rum-free conditional medium [19]. These cells went into crisis af- ter four serial passages showing classic signs of senescence and ap- optosis. We tested different cell culture media and optimized a formulation, here designated ‘‘HPrE-conditional medium,’’ using 50/50 Dulbecco’s modiﬁed Eagles medium (DMEM)/F12 contain- ing 5% fetal bovine serum (FBS; Atlanta Biologicals, Lawrence- ville, GA, http://www.atlantabio.com), 1% insulin-transferrin-sele- nium-X, ITS (Gibco, Grand Island, NY, http://www.invitrogen. www.StemCells.com 345 com), 0.4% bovine pituitary extract (BPE; Hammond Cell Tech, Windsor, CA, http://www.hammondcelltech. com), and 1:1,000 10 ng/ml epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) with 1% antibiotic-antimycotic mix (Ab/Am; Life Technologies, Rockville, MD, http://www. lifetech.com), similar to a previously described formulation [20]. This formulation was used for culture, growth, and passaging of NHPrE0 cells. The NHPrE0 cells adapted to the HPrE-conditional culture medium for 3 days and were subsequently passaged ﬁve times at the dilution ratio (1:2) per 3 days. At this point a number of colonies composed of small cells grew out and were passaged in excess of 25 times at a suitable dilution ratio (1:2) per 3 days. Two cell lines were established by this method, NHP8-No.1.1 and NHP8-No.1.2. The NHP8-No.1.2 cells, re-designated as NHPrE1, were used for the following experiments, and, after more than 75 subsequent passages, they are considered to be an immortalized line. These cells were routinely passaged in HPrE-conditional me- dium containing 5% FBS and 1% Ab/Am. A benign human prostate surgical sample was processed by col- lagenase hyaluronidase digestion to release epithelial organoids as previously described [20, 21]. Organoids were stored in liquid nitro- gen. Organoids were recovered and plated on the bottom of a T-25 tissue culture ﬂask and cultured in keratinocyte serum-free medium supplemented with EGF and BPE. These gave rise to primary cul- tures containing small epithelial-like cells that grew out from the organoids in 3–7 days. Primary cultures of BHPrE0 cells resulted in small cells that were passaged when they reached >75% conﬂuence. After four or ﬁve passages, the cells stopped growing, remaining alive but quiescent. The culture medium was changed to the HPrE- conditional medium, and the cells were passaged 10 times at a 1:2 dilution. Small progenitor-like colonies in the primary cultures were identiﬁed and trypsinized for collection and enrichment. Cells were passaged in excess of 25 times (1:2) per 3 days and designated BHPrE1. Both NHPrE1 and BHPrE1 have now been maintained in excess of 75 passages with no detected phenotypic changes. Both NHPrE1 and BHPrE1 cells were retrovirally infected by a pBird–cytomegalovirus–enhanced green ﬂuorescent protein (EGFP) vector [22] and selected by FACS–green ﬂuorescent pro- tein (GFP) sorting. When cultured, these cells were uniformly green on ﬂuorescence microscopy. Cellular Viability and Proliferation NHPrE1 and BHPrE1 cells were seeded at 2,000 cells per well in 24-well plates in the HPrE-conditional medium. Cell proliferation was determined using the Cell Titer 96 assay (Promega, Madison, WI, http://www.promega.com) at indicated time points according to the manufacturer’s protocol. After 2 hours of incubation at 37 C, absorbance at 490 nm wavelength was read using a micro- plate reader. Side Population Analysis NHPrE1 and BHPrE1 cell lines and their parental primary cells NHPrE0 and BHPrE0 were suspended at 1 106 cells/ml in pre- warmed DMEM plus 5% FBS. Then 5 lg/ml Hoechst 33342 (bis-benzimide, from a 1 mM stock; Sigma-Aldrich) was added, as well as 50 lM verapamil (from a 5 mM stock in ethanol; Sigma-Aldrich) in the þ verapamil samples. The cell suspensions were incubated for 120 minutes at 37 C with frequent intermit- tent mixing. The tubes containing the cell suspensions were placed on ice, and cells were pelleted at 4 C and resuspended in cold PBS with 2% FBS. Propidium iodide (2 lg/ml) was added to discriminate dead cells. Side population was analyzed using 325 nm ultraviolet laser excitation and emission bandpass ﬁlters 400/40 nm and 510/20 nm, separated by a dichroic 470-nm long- pass ﬁlter (LSR I; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Three-Dimensional Culture For a three-dimensional (3D)-on-top assay, 1,500 NHPrE1 or BHPrE1 cells in 2% growth factor–reduced (GFR) Matrigel