Abstract 16720: Induction of Notch Activity Promotes a Cardiac Differentiated Phenotype in Adult Cardiac Progenitor Cells

Abstract

Introduction: Notch is a critical mediator of heart development, stem cell self-renewal and specification; appropriate timing and levels of Notch signaling are essential for proper cell fate determination. However, constitutive activation of Notch does not recapitulate the physiological signal driving cardiac differentiation. Hypothesis: Controlled pulses of Notch activation enhance cardiac differentiation in CPCs. Methods: Ligand-regulated or tetracycline inducible versions of intracellular Notch designed to control timing and activation level were engineered into cultured adult cardiac progenitor cells (CPCs). Stable CPC lines were created carrying constructs for GFP (CPCe), ligand-regulated intracellular Notch (CPCeK), or tetracycline inducible intracellular Notch (CPCtetKmER). Induction was performed using 1 µM tamoxifen and/or 1 µM doxycycline with proliferation measured over a period of 6 days. Markers of Notch signaling and cardiac differentiation were assessed by quantitative PCR, immunoblotting, or immunostaining up to 6 days after induction. Results: Increased levels of hey1, jagged1, cardiac troponin T (cTnT) and smooth muscle actin (SMA) transcripts were observed following one day of Notch induction in CPCeK and CPCtetKmER. Changes indicative of differentiation were present in CPCeKs and CPCtetKmERs following one week after Notch induction including flattened and enlarged morphology, elevated levels of smooth muscle actin (SMA) and jagged1 mRNA, and expression of SMA protein. Proliferation decreased after three days of sustained Notch signaling. Conclusions: Notch signaling promotes CPCs to acquire phenotypic characteristics of smooth muscle or myocyte differentiation. Engineering CPCs allows for appropriate “physiologic” temporal control to regulate the potent cardiogenic activity of Notch signaling. Regulation of Notch activity in CPCs utilizing temporal and reversible expression will enhance commitment and differentiation when utilized in conjunction with cell based therapy for heart disease, thereby enhancing myocardial regeneration and repair.