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Genetic modification of adipose derived stem cells to enhance wound healing
Upasana Niyogi1, Priyanka Sharma1, Gregory C. Gould2, Sunishka M. Wimalawansa2, R. Michael Johnson2, Katherine J.D.A. Excoffon1,2
Department of Biological Sciences Wright State University
2016-02-15
Presenter: Upasana Niyogi
Affidavit:
Katherine J.D.A. Excoffon
Director Name: Katherine J.D.A. Excoffon
Author Category: Medical Student
Presentation Category: Basic Science Research
Abstract Category: General Reconstruction
Chronic wounds have become a major clinical and economic burden in our society. New treatments are desperately needed. Angiogenesis and vascularization play a critical role in healing. An essential angiogenic factor that promotes 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. Our preliminary work has shown that nonpathogenic adeno-associated virus 5 (AAV5) is able to transiently transduce ASC with higher efficiency than other AAV serotypes. We hypothesize that administration of AAV-VEGF genetically-modified ASC directly into the wound site will accelerate rejuvenation of ischemic tissue. To test our hypothesis, the VEGF-165 gene was synthesized (IDT) and cloned (Clontech) into two AAV5 plasmids, pFBAAV5CMVmcswtIRESeGFP and pFBAAV5CAGmcswtIRESeGFP. The AAV5-VEGF-165 and control plasmids were then transfected into HEK-293, COS-7, and primary ASC cells. The transfection efficiency was determined by number of GFP positive cells observed by fluorescence microscopy. The amount of VEGF-165 produced was quantified by VEGF ELISA. Greater transfection and VEGF expression was observed in the cells transfected with pFBAAVCAGmcswtIRESeGFP than pFBAAVCMVmcswtIRESeGFP plasmid. Therefore, AAV-CAG-VEGF-wtIRES-eGFP virus produced from pFBAAVCAGmcswtIRESeGFP using the Baculovirus system was used to transduce the ASC. Wound healing, as measured by scratch assay, was performed on monolayers of ASC and healing was observed. Future work will be to test the wound healing efficacy of transduced ASC expressing VEGF relative to control transduced ASC in a novel multidimensional wound model culture system.
