Project description:Tissue remodeling or regeneration is believed to initiate from organ-specific stem and/or progenitor cells. We report here the establishment of two spontaneously immortalized adult human benign prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1CD133(++)/CD44(++)/OCT4(++)/CK14(+++) was characterized as a putative progenitor cell, and BHPrE1CD133(+)/CD44(+)/OCT4(+)/CK14(++) as a putative intermediate cell. aCGH analysis demonstrated a largely intact diploid karyotype with DNA amplification of PTEN in NHPrE1 or Beta-Catenin in BHPrE1 cells, respectively. A tissue recombination-xenografting model was utilized to compare remodeling of human prostatic tissues in vivo by the NHPrE1 and BHPrE1 cell lines and their parental primary cells. A series of tissue recombinants, made by mixing different ratios of human prostatic epithelial cells and inductive rat urogenital sinus mesenchyme (UGM), were grafted to the renal capsule of SCID mice. Both cell lines were able to regenerate ductal/acinar structures in vivo containing basal and luminal epithelial populations confirmed by the expression of appropriate cytokeratin profiles. Androgen receptor (AR), prostate specific antigen (PSA), and 15-lipoxigenase-2 (15-LOX-2) expression were appropriately expressed in the regenerated epithelial structures. Regeneration of benign human prostatic ductal/acinar architecture could be achieved using as few as 10 NHPrE1 cells while 200,000 BHPrE1 cells were required to achieve prostatic structure. This suggests a greater proportion of progenitor cells in NHPrE1 facilitated regeneration of prostatic morphology and function. These cell lines provide important data on progenitor and intermediate cell phenotypes and represent significant new tools for the elucidation of molecular mechanisms of human prostatic regeneration, pathogenesis and carcinogenesis. Two channel CGH from Agilent platform was used to detect DNA amplification or deletion in genomic samples from NHPrE1 and BHPrE1 cell lines. A common pooled normal DNA refernce was used.

Project description:Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen, a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified key pathways and regulators of prostatic differentiation including Wnt5a, BPM7, PTEN, and NKx3.1 using E-PZ-iPS-lke cells. Our results suggest that iPS-like cells can be derived from prostatic epithelial cells. These cells are pluripotent and capable of prostatic differentiation, and therefore provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. Bisulphite converted DNA from the 10 samples were hybridised to the Illumina Infinium HumanMethylation450K Beadchip v1.2

Project description:Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen, a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified key pathways and regulators of prostatic differentiation including Wnt5a, BPM7, PTEN, and NKx3.1 using E-PZ-iPS-lke cells. Our results suggest that iPS-like cells can be derived from prostatic epithelial cells. These cells are pluripotent and capable of prostatic differentiation, and therefore provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.

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Functional remodeling of primary and immortalized progenitor and intermediate cells

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