Ex Vivo Development of Transfected Oral Mucosal Grafts

描述：我们的长期目标是生产一种“智能”的转移式口腔黏膜移植物，用于重建主要的口腔缺损。目前，未分选的成人单个角质形成细胞悬浮液被用于制造适合进行FDA批准的人体临床试验的“天然”非转导的Ex Vivo Produced口腔黏膜等效物(EVPOME)。我们在分离培养的成人口腔粘膜细胞上的成功提供了令人信服的经验证据，证明在我们的细胞群体中存在代表“干细胞”隔间的亚群。为了使我们的技术/基因疗法成为临床上可行的选择，我们需要分离出祖细胞/干细胞群体。 我们的假说是：“分离分离的口腔黏膜角质形成细胞亚群富含祖细胞/干细胞：1)分离，2)操纵以改变它们的复制和分化，增强它们制造用于口腔内移植和用作基因治疗装置的优质EVPOME移植物的能力。 1.从分离培养的原代人口腔角质形成细胞中分离并鉴定口腔黏膜祖细胞/干细胞的“富集群”。 2.通过移植到SCID小鼠和犬模型中，证明了在“智能”EVPOME中转导的丰富的口腔黏膜祖细胞/干细胞群体的安全性和命运。 3.改变核转录因子PPARy的表达以促进口腔黏膜祖细胞/干细胞丰富群体的复制和减少分化。 这项研究项目是适用于EVPOME的“长凳到床边”范例的合乎逻辑和必要的迭代。在化学定义的条件和FDA指南下，建立丰富的口腔黏膜祖细胞/干细胞群体的扩大培养的结果可以导致细胞替代治疗的更大进展。需要这些类型的转译研究来产生新的基于细胞的组织工程设备，以改善患者护理。

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.