Abstract
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.
Highlights
We 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
Primary keratinocytes have a finite lifespan in culture, but we previously demonstrated that culture in the presence of a Rho kinase (ROCK) inhibitor greatly increased the proliferation and resulted in apparent immortalization of human keratinocytes derived from several anatomical sites [1]
In these experiments we removed Y-27632 at different times from cells that had been continually grown in its presence, and added it to cells that were only a few passages from senescence. The controls for these experiments are shown in Figure 1A: human foreskin keratinocytes (HFKs) from three different donors were cultured in parallel in the presence or absence of Y-27632
Summary
We 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. We demonstrated that the resulting keratinocytes were very similar to primary keratinocytes in that they had a normal karyotype, an intact DNA damage response and could differentiate into a stratified epithelium [1] These cells have proven to be very useful for basic research studies and for clinical research. We show that the ROCK inhibitor can be added at late stages of the replicative life span, when cells are close to senescence, and it will still efficiently promote indefinite proliferation of the cells We extend these studies to show that animal keratinocytes can be induced to proliferate indefinitely using this technique. This effect is immediate and increased proliferation can be observed within days of addition of the ROCK inhibitor
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