We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications. IntroductionSomatic cells have a limited lifespan, gradually slow in growth, and stop dividing, a process known as cellular senescence. This process is thought to limit the vulnerability of aging cells to disease. Human keratinocytes are invaluable for the study of skin biology and the pathogenesis of skin-related diseases, but their short lifespan in culture is a limitation. Different conditions have been developed to optimize the culture of keratinocytes; for example, the presence of fibroblast feeder cells increases the proliferative capacity of primary keratinocytes from approximately 20 to 40-60 population doublings (1, 2). Spontaneously immortalized keratinocyte lines, for example HaCaT (3) and NIKS (4), have been used for skin-related research. However, these cell lines have genetic abnormalities, such as mutations in p53 (5) or isochromosomes (4).Continuous replication of primary human cells is blocked by 2 separate events: mortality stage 1 (M1; replicative senescence) and mortality stage 2 (M2; crisis). At M1, signaling by shortened telomeres results in activation of the p53 and pRB pathways. M2 represents a critical period of genomic instability, with extremely eroded telomeres, resulting in chromosomal fusions and translocations. In retinal pigment epithelial cells and foreskin fibroblasts (which have decreased expression of components of the p16INK4A/pRB pathway), telomerase expression is sufficient to bypass both M1 and M2 and stabilize and elongate chromosome ends (6). However, telomerase expression is not sufficient for immortalization of keratinocytes, and p16INK4A function must also be disrupted (7,8).Keratinocytes expressing the E6 and E7 proteins encoded by high-risk human papillomavirus (HPV) types bypass both M1 and M2 blocks and become immortal (reviewed in ref. 9). E7 inactivates and degrades the pRB retinoblastoma tumor suppressor protein to induce G 1 /S phase progression of th...

IntroductionWe previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research.MethodsIn this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression, proliferation, behaviour and lifespan in the presence and absence of Y-27632.ResultsWe demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely, keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification.ConclusionsROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research, as well as for regenerative and personalized medicine.

Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication.