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

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Pietrus, Agata E.
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In previous years split-thickness skin grafts have been traditionally used to cover open wounds in the oral cavity. These grafts display disadvantages such as hair growth in intraoral use. An alternative, such as an oral mucosal graft is ideal, but is limited in supply. The development of Ex Vivo Produced Oral Mucosa Equivalent (EVPOME) has allowed for easier transplantation into the oral mucosa to reconstruct oral mucosa defects. To ensure the success of such grafts using EVPOME, genes must be efficiently delivered to the target cells and gene products must be expressed for long periods of time without having toxic effects on the host. To make EVPOME an efficient vehicle for gene delivery it is necessary to use stem cells to fabricate the graft. Stem cells are necessary in order to achieve long term gene expression and the release of growth factors. As of yet, there has been no research done to identify stem cells in the oral mucosa epithelium. Results from preliminary experiments in our lab suggested that the 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, indicating that small sized oral mucosa keratinocytes are the cells with the most stem cell-like characteristics. Using Fluorescent Activated Cell Sorter (FACS), oral mucosa keratinocytes were sorted into two groups, small sized cells and non-sorted, including all sized cells. Cells were transducted with lentivirus containing a reporter gene as well as green fluorescent protein (OFP) and were 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 greater care needs to be taken using sterile procedure if this experiment is to be repeated again. Recent experiments, not yet published, use different protocols and show that small sized oral mucosa keratinocytes are indeed the appropriate cells to use in fabricating EVPOME grafts for use in gene therapy.
vi, 30 p.
Kalamazoo College
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