Date on Master's Thesis/Doctoral Dissertation

8-2015

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Hong, Kyung

Committee Co-Chair (if applicable)

Jones, Steven

Committee Member

Jones, Steven

Committee Member

Bhatnagar, Aruni

Committee Member

Clark, Geoffrey

Committee Member

Liu, Yongqing

Subject

Heart cells; Tissue engineering

Abstract

c-kit+ cardiac progenitor cells (CPCs) have recently gained much attention due to the therapeutic effects they exert on cardiac function following their administration into the infarcted heart as evidenced by animal studies and by a recent clinical trial (SCIPIO). However, injecting these cells in the heart is associated with poor differentiation into specialized cardiac cell types and with rapid death of the engrafted cells. With the ultimate goal of advancing cardiac stem cell therapy, we sought to facilitate the differentiation of human CPCs into cardiac cell types (e.g. cardiomyocytes, smooth muscle cells, endothelial cells and cardiac fibroblasts) by overexpressing selected cardiac transcription factors in vitro. To achieve that, Gata4, MEF2C, NKX2.5 and TBX5, were overexpressed in CPCs via lentivirus. When individually overexpressed, Gata4 upregulated some cardiomyocyte, smooth muscle cell, and fibroblast markers. TBX5, however, induced only few cardiomyocyte markers, indicating partial differentiation. In addition, these changes in CPC cardiac gene expression observed with Gata4 overexpression were accompanied by marked morphological changes, manifested by the cells becoming wider and largely polygonal. However, introducing the aforementioned transcription factors in various combinations largely failed to further enhance the cardiac differentiation of CPCs induced by Gata4 or TBX5, underscoring the complexity of the interaction between the cardiac transcription factors. Likewise, addition of the chromatin remodeling transcription factor BAF60C to Gata4 and/or TBX5 did not further potentiate their pro-differentiation effects in CPCs. In addition to inducing differentiation, we also endeavored to promote CPC survival by overexpressing a pro-survival gene. To that end, a constitutively active mutant form of Nrf2 (caNrf2) was overexpressed in CPCs. caNrf2 overexpression protected CPCs against hydrogen peroxide- and 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ)-induced oxidative stress in vitro without altering the overall growth characteristics of the cells. Taken together, our results highlight the potential of Gata4 in facilitating differentiation and the protective role of caNrf2 in CPCs. These effects of Gata4 and caNrf2 may enhance the regenerative capabilities of CPCs and could thus be utilized to advance cell-based heart therapies.

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