Ex Vivo Development of Transfected Oral Mucosal Grafts

描述：我们的长期目标是生产一种“智能”的转导口腔粘膜移植物，用于口腔主要缺陷的重建。目前，一种未分离、未分选的成人单一角质细胞悬浊液被用于制造“初始”非转导的体外产生的口腔黏膜等效物（EVPOME），适合进行fda批准的人体临床试验。我们在未分选培养成人口腔粘膜细胞分离方面的成功提供了令人信服的经验证据，证明在我们的细胞群中存在一个亚群，代表“干细胞”区室。为了使我们的技术/基因治疗成为临床可行的选择，我们需要分离祖细胞/干细胞群。

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.