Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Abstract

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.

Figure 1. Y-27632 stabilizes the growth rate of primary keratinocytes.

Growth rate of human keratinocytes from foreskin (HFK strain c; black), ectocervix (HCK; red), and vaginal tissue (HVK; blue) cultured in the presence (filled squares) or absence (open diamonds) of 10 μM Y-27634 (Y). The arrows indicate cells lines that continued to divide indefinitely. The growth rate is measured as population doubling per day.

Figure 2. The morphology of Y-27632–immortalized cells resembles that of early pass primary keratinocytes.

Images of human foreskin, ectocervical, and vaginal keratinocytes at P1 are shown in the left column. Images of keratinocytes near senescence (HFK P15, HCK P9, and HVK P5) are shown in the center column. Images of keratinocytes immortalized by 10 μM Y-27632 (HFK P100, HCK P29, and HVK P26) are shown in the right column. Scale bar: 10 μm.

Figure 3. Telomerase expression increases over time, and the length of telomere ends stabilizes after culture with Y-27632.

(A) Relative levels of TERT mRNA from HFK strain a, cultured in the absence or presence of 10 μM Y-27632 at the pass indicated, as quantitated by real-time PCR. Each bar represents the mean of replicated samples ± SD. (B) Relative length of telomeres in HFK strain a, cultured in the absence or presence of 10 μM Y-27632 at the pass indicated, as quantitated by real-time PCR. Each bar represents the mean of replicated samples ± SD.

Figure 4. Expression of p16INK4, p53, p21CIP1, and MYC proteins in cells cultured with Y-27632.

(A) Immunoblot analysis of MYC and p16INK4 proteins in HFK strain c, HVK, and HCK cells, cultured in the absence or presence of 10 μM Y-27632 and collected at the pass indicated. Cells containing oncogenic HPV31 and HPV18 viruses are included as controls. α-Tubulin was detected as a loading control. (B) DNA damage was induced by treatment of cells with actinomycin D (ActD). The response was measured by immunoblot analysis of p53 protein levels and those of its downstream target p21CIP1. HFKs grown without Y-27632 were assayed at P4, and those cultured in 10 μM Y-27632 were assayed at P122.

Figure 5. Keratinocytes can differentiate in organotypic raft culture after long-term culture with Y-27632.

H&E-stained histological sections of primary keratinocytes at (A) P1 or (B) after 18 passes in 10 μM Y-27632, cultured in organotypic raft culture for 17 days, are shown. Scale bar: 20 μM.

Figure 6. HFKs express appropriate differentiation markers in organotypic rafts after long-term culture with Y-27632.

After 17 days in organotypic raft culture, tissue sections from normal primary keratinocytes at P1 (HFK) or after 18 passes in 10 μM Y-27632 (HFK + Y) were stained for DNA (DAPI; blue), keratin K14 (green; basal layer), involucrin (red; spinous layer), and filaggrin (cyan; granular and cornified layers). A magnified image from the merge is shown on the bottom row. Scale bar: 50 μM.