Publication Date

2017-01-04

Availability

Embargoed

Embargo Period

2019-01-04

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2016-12-05

First Committee Member

Joshua M. Hare

Second Committee Member

Michael Kapiloff

Third Committee Member

Ivonne H. Schulman

Fourth Committee Member

Danuta Szczesna-Cordary

Fifth Committee Member

Christian Faul

Sixth Committee Member

Nevis Fregien

Abstract

Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells’ regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O2). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiologic (5%) or hypoxic (0.5%) O2 concentrations. Physiologic O2 levels increased proliferation (P<0.05), but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of beta-galactosidase activity (-4203 fluorescent units, P<0.05), p16 protein expression (0.58-fold, P<0.001), mitochondrial content (0.18-fold, P<0.001), and activation of a pro-survival phenotype in hypoxia suggests that transition from high (21%) to low (0.5%) O2 reduces senescence and promotes quiescence. Additionally, physiologic O2 levels increased migration (P<0.05) compared to room air and hypoxia, and treatment with mesenchymal stem cell conditioned media rescued CSC migration under hypoxia to levels comparable to physiologic O2 migration (2-fold, P<0.05 relative to CSC media control). Furthermore, we confirmed a hypoxic induction of a quiescent phenotype in mCSCs and identified c-Myc as a regulatory stress sensor downregulated upon hypoxic stress. Induction of quiescence was associated with a decrease in the expression of c-Myc through mechanisms involved in protein degradation and subsequent downregulation of Sirt1 and upregulation of the cell cycle blocker p21. Our finding that physiologic O2 concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential and that hypoxic stress induces quiescence through modulation of c-Myc. Therefore, modulating pathways downstream of c-Myc may re-activate their regenerative potential under ischemic conditions. This study provides novel insights into the modulatory effects of O2 concentration on CSC biology and has important implications for refining stem cell therapies.

Keywords

Cardiac Stem Cells; Oxygen; Hypoxia

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