Propagation and immortalization of human adult epithelial cells. A: Prostate, breast, tracheal, and liver (hepatocellular carcinoma) tissues were harvested and digested with trypsin-collagenase, as described in Materials and Methods. Cells isolated from the tissues were plated on a feeder layer of irradiated (3000 rad) Swiss 3T3 cells (J2 subclone) and grown in F medium containing 10 μmol/L ROCK inhibitor (Y-27632). Small colonies could be observed after 1 day. At day 5 (shown), there were large islands of epithelial (epi) cells that compressed the surrounding feeder cells (white arrows). B: The prostate and breast epithelial cells were passaged repetitively using trypsinization techniques described for keratinocytes. 21 The cell number was recorded at each passage, and a plot of population doublings versus time (days) was constructed. The cells were grown under four different conditions: with both feeder cells and Y-27632 in F medium, with feeders only in F medium, with Y-27632 in mammary epithelial growth medium or PrEGM or mammary epithelial growth medium or PrEGM alone. Only cells grown in F medium containing feeders and Y-27632 continued to proliferate with a constant growth rate.

Characterization of late-passage breast and prostate cells. A: The differentiation potential of prostate and breast cells was evaluated by culture in Matrigel, without feeders or Y-27632, as described in Materials and Methods. Both prostate and breast cells formed normal prostaspheres (A, top row, left panel) and mammospheres (A, top row, second panel from left) with glandular lumens. The mammospheres showed normal polarized laminin deposition around the periphery of the sphere (A, bottom row, left panel). In addition, the mammospheres also showed the characteristic cytoplasmic/plasma membrane localization of β-catenin observed in normal epithelial cells (A, bottom row, second panel from left, green). In contrast, mammary cells immortalized with the Myc T58A mutant showed abnormal differentiation and formed abnormally shaped solid spheres with extensions into the Matrigel (A, top row, second panel from right). Scale bars: 25 μm (white); 20 μm (yellow). These Myc-immortalized cells also exhibited abnormal deposition of laminin within the sphere and without polarized localization to the colony periphery (A, bottom row, second panel from right). Mammary cells immortalized by hTERT also formed abnormal solid spheres (A, top row, right panel) with aberrant, dispersed distribution of laminin (A, bottom row, right panel). B: The cause of the high hTERT expression in the epithelial cells is the result of the in vitro culture conditions. When prostate cells are grown in commercial medium (PrEGM), the level of hTERT mRNA is low. However, when those cells are transferred to F medium with feeders (with or without Y-27632), there is an approximate 20-fold increase in hTERT expression. The greatest impact is the presence of feeder cells. C: Induction of hTERT mRNA in breast and prostate cells is similar and occurs early during passaging. Quantitative real-time PCR for hTERT was performed at the indicated passage numbers. Both cell types show an early induction of telomerase expression. In prostate, for example, the induction is maximal already at passage 2, indicating that selection during in vitro culture is not the cause of this induction. D: Chromosomal analysis of late-passage prostate cells revealed a normal 46,XY karyotype. E: DNA fingerprinting of early- and late-passage prostate cells demonstrated that they have nine identical STR loci and the Y-specific Amelogenin locus, thereby verifying their genetic identity. Data are presented as mean ± SEM.

Establishment of normal prostate and prostate adenocarcinoma cell cultures from the same patient. Biopsy specimens of normal and malignant prostate tissue from the same patient were processed and propagated in vitro, as described in Materials and Methods. For two-dimensional cultures, cells were photographed at days 2 and 3, demonstrating the rapid and equivalent outgrowth of cells from the biopsy specimens. For in vitro three-dimensional cultures, matched cells were also grown in the three-dimensional Matrigel system, as described in Materials and Methods. The normal prostate cells formed prostaspheres, whereas the tumor cells formed large undifferentiated aggregates. For an in vivo xenograft, 1 million normal and tumor cells were injected s.c. into the flanks of five mice with severe combined immunodeficiency at two different sites (a total of 10 sites for each cell type). The prostate cancer cells induced tumors at 7 of 10 sites within 8 weeks; a representative tumor is shown (arrow). The normal prostate cells did not induce tumors (0 of 10 sites). epi, epithelial cells.

Colony-forming efficiency of primary human cells. Human keratinocytes and cervical cancer cells (Caski) were plated at the indicated cell numbers onto the feeder cell cultures. After 7 to 10 days, the feeder cells were removed and the epithelial colonies were fixed for 5 minutes with 3.7% paraformaldehyde, and then stained for 30 minutes with 0.05% crystal violet. The plates were photographed and the colonies were counted with ChemiDoc and QuantityOne software (Bio-Rad Laboratories, Inc, Hercules, CA). A: The number of cells plated was plotted versus the number of colonies observed after 7 to 10 days. B: The efficiency of colony formation for the cervical cancer cells is depicted for the indicated number of Caski cells plated. The plating efficiency for low-density seeding ranged from 40% to 70%. Data are presented as mean ± SEM.

Potential model for the cooperative effects of F medium plus feeders and the ROCK inhibitor in cell immortalization. Class 1 genes induce telomerase activity, and class 2 genes alter both the p16/Rb pathway and the cell cytoskeleton. The connecting lines indicate documented interactions between class 1 and class 2 genes that result in cell immortalization.