Use of a Rock Inhibitor to Sustain Primary Human Keratinocytes in a Proliferative State

Abstract

Disclosed herein is the finding that treatment with a ROCK inhibitor increases proliferation and induces immortalization of primary keratinocytes. Accordingly, provided is a method of immortalizing primary keratinocytes by exposure to a ROCK inhibitor. Also provided are immortalized primary keratinocytes produced by the described method, as well as organotypic tissue equivalents and cell cultures comprising the immortalized primary keratinocytes. Furthermore, ROCK inhibitor-treated cells show a greatly increased ability to support viral DNA replication of both “low risk” and “high risk” HPV genomes, indicating that ROCK inhibitors will be useful for studying the life cycles of a wide range of HPVs.

1 - 26 . (canceled) 
     
     
         27 . A method of immortalizing primary keratinocytes, comprising (i) culturing the primary keratinocytes in the presence of fibroblast feeder cells and in media containing an effective amount of a ROCK inhibitor for a period of time sufficient to allow immortalization of the primary keratinocytes; and (ii) continuing to culture the immortalized keratinocytes in media lacking the ROCK inhibitor, wherein the immortalized keratinocytes retain the capacity to differentiate when cultured in media lacking the ROCK inhibitor. 
     
     
         28 . The method of  claim 27 , wherein continuing to culture the immortalized keratinocytes comprises culturing the immortalized keratinocytes until they form an organotypic tissue equivalent. 
     
     
         29 . The method of  claim 27 , wherein the primary keratinocytes are foreskin keratinocytes, vaginal keratinocytes or cervical keratinocytes. 
     
     
         30 . The method of  claim 27 , wherein the ROCK inhibitor is Y-27632. 
     
     
         31 . The method of  claim 30 , wherein the effective amount of Y-27632 is about 1 to about 100 μM. 
     
     
         32 . The method of  claim 30 , wherein the effective amount of Y-27632 is about 5 to 25 μM. 
     
     
         33 . The method of  claim 27 , wherein the primary keratinocytes are cultured in media containing the ROCK inhibitor for at least 15 days. 
     
     
         34 . The method of  claim 30 , wherein the effective amount of Y-27632 is about 10 μM. 
     
     
         35 . The method of  claim 27 , wherein the ROCK inhibitor is a small molecule inhibitor. 
     
     
         36 . The method of  claim 35 , wherein the effective amount of the ROCK inhibitor is about 1 to about 100 μM. 
     
     
         37 . The method of  claim 35 , wherein the effective amount of the ROCK inhibitor is about 5 to about 25 μM. 
     
     
         38 . The method of  claim 35 , wherein the effective amount of the ROCK inhibitor is about 10 μM. 
     
     
         39 . The method of  claim 27 , wherein the ROCK inhibitor is fasudil. 
     
     
         40 . The method of  claim 27 , wherein the primary keratinocytes are cultured in media containing the ROCK inhibitor for at least 20 days. 
     
     
         41 . The method of  claim 27 , wherein the primary keratinocytes are cultures in media containing the ROCK inhibitor for at least 40 days. 
     
     
         42 . The method of  claim 27 , wherein the primary keratinocytes are cultured in media containing the ROCK inhibitor for at least 60 days. 
     
     
         43 . The method of  claim 27 , wherein the primary keratinocytes are cultured in media containing the ROCK inhibitor for at least 100 days.