Adeno-associated virus-VEGF-165 Mediated Modification of Adipose Derived Stem Cells for Cell Therapy
Publication Date
2016
Document Type
Thesis
Committee Members
Katherine J.D.A. Excoffon (Advisor), Barbara Hull (Committee Member), R. Michael Johnson (Committee Member)
Degree Name
Master of Science (MS)
Abstract
Chronic wounds have become a major clinical and economic burden in our society. New approaches that accelerate wound healing are desperately needed. Angiogenesis and vascularization play a critical role in healing. One of the essential angiogenic factors that promote the formation of vascular beds is vascular endothelial growth factor (VEGF). Moreover, adipose-derived stem cells (ASC), through their regeneration and differentiation properties, may promote healing when transplanted into a wound bed. I have proposed to develop a novel organotypic wound model to facilitate the study of wound healing process. I hypothesize that administration of genetically-modified ASC secreting VEGF via an adeno-associated viral vector serotype (AAV5) directly into the wound site will accelerate rejuvenation of ischemic tissue. To test this hypothesis, the VEGF-165 gene was synthesized (IDT) and cloned (Clontech) into two AAV5 plasmids, pAAV5-CMV-GFP and pAAV5-CAG-GFP and transfected in HEK293, Cos-7 and primary ASC cells for gene expression. Based on the higher transfection efficiency and VEGF secretion in pAAV5-CAG-VEGF-GFP, AAV5-CAG-VEGF-GFP virus was produced by the University of Iowa using the Baculovirus system. I used AAV5-CAG-VEGF-GFP and AAV5-CMV-GFP (control virus) to transduce the ASC. Wound healing assays in vitro measured by scratch assay were performed on monolayers of ASC and were observed for healing. In vivo wound healing assay was performed on 2mm or 3mm organotypic wound models by treating them with ASC-AAV5-CAG-VEGF-GFP, ASC-AAV5-CMV-GFP, non-transduced ASC or ASC media. In vitro wound healing assays showed complete closure of non-transduced ASC in three days whereas the ASC-AAV5-CAG-VEGF-GFP healed within two days indicating the efficiency of VEGF in accelerating closure. In vivo wound healing assays on organotypic wounds showed some closure in wounds treated with ASC compared to control wounds treated with media alone. However wounds treated with ASC-AAV5-CAG-VEGF-GFP facilitated enhanced in complete closure compared to non-transduced ASC-treated wounds. No significant difference was observed between ASC-AAV5-CMV-GFP or non-transduced ASC treated wounds. We observed similar results in both 2 mm or 3 mm wounded models and with autologous or allogenic stem cells. Moreover, red light therapy (670 nm) also caused partial closure of wounds. Future studies should involve personalization of the wound model system depending on patient's disease condition and developing ASC therapy with a cocktail of growth factors that can facilitate complete wound closure.
Page Count
122
Department or Program
Microbiology and Immunology
Year Degree Awarded
2016
Copyright
Copyright 2016, some rights reserved. My ETD may be copied and distributed only for non-commercial purposes and may be modified only if the modified version is distributed with these same permissions. All use must give me credit as the original author.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.
