Ex Vivo Development of Transfected Oral Mucosal Grafts

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DESCRIPTION: Our long-term goal is to produce a "smart" transduced oral mucosal graft that will be used for reconstruction of major oral defects. Currently, an unfractionated unsorted human adult single keratinocyte suspension is used to manufacture a "naive" non-transduced Ex Vivo Produced Oral Mucosa Equivalent (EVPOME) that is suitable to perform FDA-approved human clinical trials. Our success with fractionated unsorted cultured adult human oral mucosal cells provides compelling empiric evidence that a subpopulation exists within our cell population that represents the "stem" cell compartment. In order for our technology/gene therapy to become a clinically viable option we need to isolate a progenitor/stem cell population. Our hypothesis states: "A subpopulation of fractionated sorted oral mucosa keratinocytes enriched for progenitor/stem cells can be: 1) isolated and 2) manipulated to alter their replication and differentiation, to enhance their ability to fabricate a superior EVPOME graft for intraoral grafting and for use as a device for gene therapy. Specific Aims: 1. Isolate and characterize an "enriched population" of oral mucosa progenitor/stem cells from fractionated sorted cultured primary human oral keratinocytes. 2. Demonstrate the safety and fate of a transduced enriched population of oral mucosa progenitor/stem cells in a "smart" EVPOME by grafting into both SCID mouse and canine models. 3. Alter expression of nuclear transcription factor PPARy to enhance replication and decrease differentiation of an enriched population of oral mucosa progenitor/stem cells. This research project is a logical and necessary iteration of the "bench to bedside" paradigm as it applies to the EVPOME. The results to establish expanded cultures of an enriched population of oral mucosa progenitor/stem cells, under chemically defined conditions and FDA guidelines, can lead to greater advances in cell replacement therapy. These types of translational studies are needed to generate new cell based tissue-engineered devices to improve patient care.