Induced pluripotency of human prostatic epithelial cells

Abstract

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 (PSA), 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 epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.

Figure 1. Generation of iPS cells from E-PZ cells by lentiviral transduction.

(A) A diagram of the experimental design. The three compounds added to the medium at day 7 were 2 µM SB431542, 0.5 µM PD0325901, and 0.5 µM Thiazovivin. (B) Representative images of colonies derived from two E-PZ cultures at different time points.

Figure 2. Optimization of culture conditions to maintain ESC-like morphology of E-PZ-iPS-like cells.

Colonies lost ESC-like morphology in mTeSR-1 medium on feeder layers (A); however, they maintained ESC-like morphology in DMEM/F12 (1∶1) supplemented with 5 ng/ml bFGF, 10 ng/ml LIF and 5% knock out FBS on feeder layers (B) or Matrigel-coated plates (C). E-PZ-iPS-like cells formed spheres when cultured on ultra-low attachment plates in DMEM/F12 (1∶1) supplemented with 5 ng/ml bFGF, 10 ng/ml LIF and 5% knock out FBS (D).

Figure 3. Immunofluorescence detection of marker expression in E-PZ-1-iPS-like-4 cells.

E-PZ-1-iPS-like-4 cells showed strong cell membrane staining of TRA-1–81 (A) and SSEA-3 (D), and nuclear staining of Nanog (G), Sox2 (J), Oct4 (M), and c-Myc (P). These cells displayed strong nuclear staining of Ki67 (S) and the human nuclear antigen Ku70 (V). They did not express basal cell marker CK5 (Y) except in a few cells mostly located along the edge of the colonies. (B), (E), (H), (K), (N), (Q), (T), (W), and (Z) are DAPI staining of the nuclei. (C), (F), (I), (L), (O), (R), (U), (X), and (AA) are merged images of staining of DAPI and antibodies against specific markers.

Figure 4. Determination of expression levels of pluripotent genes and methylation levels of their promoters in E-PZ-1 and E-PZ-1-iPS-like cells.

mRNA levels of Nanog (A), Rex1 (B), Oct4 (C), Klf4 (D), c-Myc (E), Sox2 (F), and CD133 (G) were measured by qRT-PCR and normalized against TBP. Methylation of Nanog (H) and Oct4 (I) promoters were determined by bisulfite pyrosequencing. The Y-axis is the fold-level of gene expression or promoter methylation in E-PZ-1-iPS-like cells compared to those in E-PZ-1 cells, which were set as 1. (J) and (K) were histograms of the number of chromosomes in 100 E-PZ-1-iPS-4 and -7 cells, respectively, determined by metaphase chromosome counting.

Figure 5. In vitro differentiation of E-PZ-1-iPS-like cells.

E-PZ-1-iPS-like-4 and -7 cells were subjected to conditions that induced differentiation into neural cells (ectoderm), adipocytes (mesoderm), osteoblasts (mesoderm), or hepatocytes (endoderm). Osteoblast induction produced cells positive for osteocalcin (A and B). Neural induction generated cells positive for the neural cell marker MAP-2 (D and E). Adipocyte induction produced cells with lipid droplets that stained with oil red O (G and H). Hepatocyte induction generated cells positive for human α-fetoprotein (α-FP) (J and K). (C), (F) and (L) are negative controls that were stained with secondary antibodies only. (I) is a negative control without oil red staining. Some spheres derived from E-PZ-iPS-like cells and cultured in E-PZ medium expressed basal prostatic epithelial cell markers including CD44 (M), MAO-A (N), and p63 (O). In addition, some spheres expressed CK18 (P) and PCNA (Q). The spheres also expressed AR (R) in the presence of R1881. When co-cultured with rat UGS, a subset of the spheres expressed PSA (S, inserts showing UGS negative for PSA). In the absence of UGS, no PSA expression was detected (T).

Figure 6. In vivo differentiation of E-PZ-1-iPS-like cells.

E-PZ-1-iPS-like cells injected under the renal capsule of immunodeficient mice expressed human-specific nuclear antigen Ku70 (A) and basal prostatic epithelial markers including p63 (B) and CD44 (C). A subset of cells was positive for the transit amplifying epithelial cell marker CK18 (D) but not the secretory cell markers AR (E) or PSA (F). When combined with UGM, E-PZ-1-iPS-like cells gave rise to tissue expressing Ku70 (G) and CK18 (J). In addition, p63 was expressed only by cells at the edge of the tissue (H) and CD44 expression was reduced (I). Although the cells were negative for PSA (L), they expressed an intermediate level of AR in the nuclei (K). White dotted lines mark the boundary of grafts derived from E-PZ-1-iPS-like cells and mouse kidney. All magnifications are 40×.

Figure 7. In vivo differentiation of F-iPS cells.

F-iPS cells injected under the renal capsule of immunodeficient mice showed histological characteristics typical of teratoma including cartilage (A), gut-like epithelium (B), muscle (C), adipose tissue (D), pigmented cells (E), and neuroepithelial rosettes (F). Many glands were positive for CK18 (G), but not p63 (H) or PSA (I). When combined with UGM, F-iPS cells gave rise to cell clusters expressing both CK18 (J) and p63 (K), but not PSA (L). All magnifications are 20×.

Figure 8. Identification of DNA methylation changes in prostatic differentiation using E-PZ-1-iPS-4 cells.

(A) Genes that showed significantly higher (in red) or lower (in green) levels of methylation in cells cultured under AR- or PSA- inducing conditions compared to control across different time points of the induction process. (B) Canonical pathways identified by IPA that are enriched with genes hypermethylated in AR and PSA induced cells at late time points compared to early time point. Red arrows point out key pathways known to be involved in prostatic cell differentiation.