Investigation of Small Sized Oral Keratinocytes and their Role in Fabricating Longer Lived EVPOME Grafts for Use as Devices in Gene Therapy

Abstract

In previous years split-thickness skin grafts have been used to cover open wounds in the oral cavity. These grafts display disadvantages such as hair growth in intraoral use. Oral mucosa is an ideal graft, but is limited supply. The development of EVPOME, an ex vivo produced oral mucosa equivalent, has allowed for easier transplantation into the oral mucosa to reconstruct oral mucosa defects. To make EVPOME an efficient vehicle for gene delivery it is necessary to use longer lived (stem) cells to fabricate graft to achieve long term gene expression and release growth factors. To this point, there has been no research trying to identify stem cells in the oral mucosa epithelium. Preliminary experiments from our lab allowed us to hypothesize that use of small sized oral mucosa keratinocytes would allow for the fabrication of the longer lived EVPOME grafts for use as devices in gene therapy. Oral mucosa keratinocytes were sorted into two groups, small sized cells and non-sorted, including all sized cells, using Fluorescent Activated Cell Sorter (FACS). Cells were transducted with lentivirus with a reporter gene, green fluorescent prolein (GFP) and allowed to grow in the equivalents (EVPOME). Bacterial contamination did not allow us to continue keratinocyte equivalent culture and thus a firm conclusion could not be made, It appears that keratinocytes were contaminated during cell sorting. These results show that great care needs to be taken using sterile procedure if this experiment is to be repeated again. Recent experiments using different protocols (Results not yet published) show that small sized oral mucosa keratinocytes are indeed the appropriate cells [0 use in fabricating EVPOME grafts for use in gene therapy.