Abstract 1118: Novel Lentiviral Vectors Bearing the Cardiac-Specific Promoter of the Sodium-Calcium Exchanger (NCX1) Report Cardiogenic Differentiation in Stem Cells
Background: Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have differentiated down the cardiomyocyte lineage.
Methods and Results: Lentiviral vectors achieved significantly higher transduction efficiency (>90%) and longer-term transgene expression in human CDCs than any of the nine adeno-associated viral serotypes tested (AAV 1– 6, 2.5, 8, 9). To define the best reporter of cardiogenic differentiation, five cardiac-specific promoters (sodium-calcium exchanger [NCX1], L-type calcium channel α-subunit, α-myosin heavy chain, troponin I and myosin ventricular light chain) were subcloned upstream of GFP in lentiviral vectors. Cardiac specificity was assessed by transducing neonatal rat ventricular myocytes (NRVMs), HEK293, cardiac fibroblasts, skeletal myoblasts, endothelial and vascular smooth muscle cells with each vector and then measuring GFP fluorescence by flow cytometry. The cardiac NCX1-promoter conveyed the highest degree of cardiac specificity, with expression limited to NRVMs. At a multiplicity of infection of 35, NCX1-GFP vectors did not affect cell death or apoptosis of CDCs. Expression persisted for up to 6 months in vitro. NCX1-GFP positive CDCs subpopulations, were FACS-sorted and expanded in vitro, demonstrating enhanced expression of a variety of cardiac markers by real-time PCR. The utility of lentiviral vectors bearing the cardiac NCX1-promoter was further exemplified by treating CDCs with cardiogenic sulfonylhydrazones (SHZ). SHZ potently activated a NCX1-firefly luciferase reporter (211 ± 21% greater than control cells) and upregulated cardiac transcripts (including BNP, troponin I, α-tropomyosin, GATA-4).
Conclusion: Lentiviral vectors carrying the NCX1 promoter represent a useful tool for genetically marking stem cells and their progeny that have differentiated down the cardiomyocyte lineage. The ability to selectively enrich for cardiomyocytes and their precursors has potential relevance for the development of cell-based therapies.