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Upasana niyogi MS thesis.pdf (2.78 MB)

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Adeno-associated virus-VEGF-165 Mediated Modification of Adipose Derived Stem Cells for Cell Therapy

Niyogi, Upasana
http://rave.ohiolink.edu/etdc/view?acc_num=wright1472043948

Abstract Details

2016, Master of Science (MS), Wright State University, Microbiology and Immunology.
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.
Katherine J.D.A. Excoffon, Ph.D. (Advisor)
R. Michael Johnson, M.D., FACS (Committee Member)
Barbara Hull, Ph.D. (Committee Member)
122 p.
Biology
Chronic wounds, adipose derived stem cells, vascular endothelial growth factor, ASC- VEGF therapy, red light therapy

Recommended Citations

Citations

  • Niyogi, U. (2016). Adeno-associated virus-VEGF-165 Mediated Modification of Adipose Derived Stem Cells for Cell Therapy [Master's thesis, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472043948

    APA Style (7th edition)

  • Niyogi, Upasana. Adeno-associated virus-VEGF-165 Mediated Modification of Adipose Derived Stem Cells for Cell Therapy . 2016. Wright State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wright1472043948.

    MLA Style (8th edition)

  • Niyogi, Upasana. "Adeno-associated virus-VEGF-165 Mediated Modification of Adipose Derived Stem Cells for Cell Therapy ." Master's thesis, Wright State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472043948

    Chicago Manual of Style (17th edition)

wright1472043948
171
© 2016, some rights reserved.Creative Commons License Adeno-associated virus-VEGF-165 Mediated Modification of Adipose Derived Stem Cells for Cell Therapy by Upasana Niyogi is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Wright State University and OhioLINK.