Generation of an in vitro model based on epidermal cells derived from murine foetal skin and investigation on p63 and CGI58 genes expression

Evidence from the newborns of p63 and CGI58 knockout mice have demonstrated that both genes are required for normal skin development, specifically in the maintenance of the epidermal stem cells (EpiSCs) and formation of a functional skin permeability layer, respectively. In the present study, the correlation of the p63 and the CGI58 genes was studied using 2-dimensional (2D) in vitro skin model established from mouse foetal skin EpiSC. The EpiSCs were first isolated using serum-free media supplemented with epidermal growth factor (EGF) and fibroblast growth factor (FGF). The cells of interest formed microspheres known as skin-derived precursor cells (SKPs). The isolated SKPs were found to demonstrate EpiSC characteristics, including the expression of basal cells marker (KRT5) and the ability to undergo multilineage differentiation (neuronal cells, astrocytes/Schwann cells and adipocytes). However, the EpiSC cultured in this methodology did not proliferate and showed loss of cells over passages. This was due to the continuous changes of the SKPs to express adherent characteristic and influence by the presence of adherent cells. Studies using the co-culture system showed continuous secretion of co-factor(s) by the adherent cells played major role in influencing the changes of SKPs characteristics, and not the direct contact between the adherent cells and SKPs. An improved methodology was established by removing the potential influence derived from the adherent cells during culture. This has yielded significantly higher numbers of EpiSC in comparison to the conventional approach without compromising the undifferentiated state of the cells and its differentiation abilities. The absence of p63 gene expression (TA and ΔN groups) in the isolated EpiSC, possibly due to the limitation of the isolation procedures hampered the investigation of the correlation between p63 and CGI58 in the established model. An alternative immunostaining of p63 and CGI58 approach using fluorescence techniques on the Formalin-fixed, Paraffin-embedded (FFPE) mouse foetal skin slides was adopted to determine the localisation and expression of these proteins and possibly establish its correlation. The data showed that p63 expression was confined in the basal layer while CGI58 was found to be localised in the granular layer of the epidermis without co-localisation of expression in any layer of the epidermis. These results suggested that both proteins are expressed by stages during skin development. Previous findings from p63 and CGI58 null mice have shown that both proteins are required for normal epidermal formation. However, we could not established the correlation between p63 and CGI58 using the currently suggested model. Full immunostaining profiles of these proteins in the p63 and CGI58 null mice are required in future work to further evaluate the correlation of the proteins in the skin epidermis. Furthermore, in situ RT PCR approach can be adopted in future study to determine the correlation between p63 and CGI58 at gene level.