[Corneal differentiation following HSC70 and HSP72 expression in air-exposed limbal stem cells cultured on denuded amniotic membrane]

ABSTRACT

The aim of this study is to create an ex vivo model to examine the expression of two heat shock protein 70 [HSP70] family members, heat shock protein 72 [HSP72] and heat shock constitute protein 70 [HSC70], at the mRNA and protein levels in differentiating corneal cells from air exposed limbal stem cells. Limbal biopsies were cultured as explants on a cellular amniotic membrane for 14 days. The cells were then exposed to air for 16 extra additional days. The proposed expression of limbal stem cell markers [p63, ABCG2], corneal markers [K3/12, connexin 43], as well as HSP72 and HSC70 were analyzed by reverse transcription-polymerase chain reaction [RT-PCR] at the mRNA level, and by immunocytochemistry and flow cytometry at the protein level both pre and post air exposure. Fresh limbal and corneal tissues were used as control group. Air exposure decreased expression of p63 and increased expression of K3/ K12 indicating an increase in the number of corneal cells. Our data showed that HSP72 and HSC70 were expressed at the mRNA level before and after air exposure while their expression significantly increased post air exposure at the protein level. We assume HSC70 expression may be related to early and terminal stages of differentiation in cultured limbal stem cells. In addition, limbal stem cells were protected during normal development against oxidative stress thru increased HSP72 expression. These findings may have broader implications in development of therapeutic strategies for treating wound healing disorders by induction of HSPs