Gene transduction with lentiviral vectors has already been used for basic and clinical studies of human keratinocytes. In this study, we demonstrated the efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector by using polybrene and Y-27632. This work has three major findings concerning lentiviral transduction into keratinocytes. First, polybrene markedly enhances the efficiency of lentiviral gene transduction, but reduces keratinocyte proliferation and negatively impacts the maintenance of keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Second, ROCK inhibitor Y-27632 significantly interferes with the lentiviral transduction into cultured human keratinocytes. Third, a suitable combination of polybrene and Y-27632 can effectively expand keratinocytes carrying a transgene.
Polybrene is widely used in viral gene transduction. Polybrene has no cytotoxic activity at low concentrations, but negatively affects the proliferation of corneal keratinocytes at concentrations greater than 10 μg/ml . It also negatively impacts the proliferation of human mesenchymal stem cells, even if used at 1 μg/ml . It has been reported that treatment with 2 to 6 μg/ml of polybrene results in the highest efficiency of gene transduction with adenoviruses in human epidermal keratinocytes . In our experiments, 20 μg/ml of polybrene was required to induce the highest expression of transgene in lentiviral transduction. However, polybrene significantly reduced the proliferation of keratinocytes, even if used at low concentrations. Furthermore, we demonstrated that polybrene negatively impacted the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. In serial culture, keratinocyte stem cells progressively lose their proliferative capacity to become transient amplifying cells with limited growth, a phenomenon termed clonal conversion . Clonal conversion is accelerated by stress, suboptimal culture conditions, serial cultivation and age of donor . It is also governed by the balance of Rac1 and Akt signaling activity and actin filament organization . As polybrene modulates the charge of the cell surface, it might affect these intracellular signaling pathways and actin network, and then increase the rate of clonal conversion. Retroviral gene transfer into keratinocytes is also enhanced by another polycationic polymer, protamine sulfate  and by their cultivation on a substrate of fibronectin . The effects of these reagents on transduction efficiency and clonal conversion should be further investigated.
The process of retroviral infection involves the absorption of viral particles on the host plasma membrane, the entry of the viral protein-RNA complexes into the cytoplasm, the reverse transcription of viral RNA into DNA and the intracellular trafficking and entry of viral protein-DNA complexes into the nucleus, which is followed by the integration of viral DNA into the host genome . We have clearly demonstrated here that the ROCK inhibitor Y-27632 interfered with the lentiviral transduction in human epidermal keratinocytes. ROCK regulates both actin polymerization and phosphorylation of myosin regulatory light chain . Recently, it has also been shown that ROCK regulates microtubule dynamics through phosphorylation of the tubulin polymerization promoting protein 1 (TPPPA/p25) . The cytoskeleton, including actin filaments and microtubules in the host cells, contributes to intracellular transport of retroviral genomes from the cytoplasm into the nucleus [36–38]. As further investigations should be required, ROCK activity could be involved in intracellular trafficking of the retroviral genome.
Interestingly, van den Bogaard et al. have recently reported that Y-27632 enhances lentiviral transduction into human keratinocytes . This report seems to be contrary to our results. However, van den Bogaard et al. have used high-passage adult keratinocytes for lentivirus transduction and the generation of human skin equivalents (HSEs), and then assessed the efficiency of lentiviral transduction into keratinocytes in HSEs, but not in cultured keratinocytes. Human adult keratinocytes significantly decrease their proliferative capacity by serial cultivation and stress in culture, including gene transduction . Therefore, the enhanced expansion of EGFP-expressing adult keratinocytes in HSEs by Y-27632 might be due to the fact that the expansion of Y-27632-treated adult keratinocytes expressing EGFP is more efficient than that of untreated EGFP-expressing adult keratinocytes during the formation of HSEs.
van den Bogaard et al. has also reported high levels of GFP expression in the differentiated layers of HSEs . We also observed that EGFP was strongly expressed in stratified and differentiating cells in the center of colonies. These results suggest that CMV promoter activity is enhanced in the stratified and differentiating keratinocytes. Y-27632 inhibits keratinocyte differentiation [28–30]. These observations alone suggest that Y-27632 inhibits EGFP expression under the control of the CMV promoter, but not lentiviral transduction itself. However, our quantitative PCR analysis has revealed that the integration of the exogenous EGFP gene into keratinocytes significantly interfered with the treatment of Y-27632. Consequently, reduced expression of EGFP after lentiviral transduction with Y-27632 might be, at least partially, due to the inhibition of keratinocyte differentiation and, therefore, CMV promoter activity by Y-27632, as well as lower transduction efficiency by the treatment with Y-27632.
We have demonstrated in this study that keratinocytes gave rise to colonies derived from their stem/progenitor cells carrying a transgene after lentiviral transduction with lower concentration of polybrene. We have also shown that the keratinocyte stem/progenitor cells carrying EGFP transgene could maintain both self-renewing and terminal differentiation abilities that are indispensable for making a functional epidermal sheet ex vivo, and permanent engraftment after transplantation [33, 39]. These results indicate that a homologous clone of a keratinocyte stem cell carrying a transgene can be isolated and expanded massively for cell therapy for genetic disorders of the skin [7, 40].