Abstract 345: Aggregation of Child Cardiac Progenitor Cells into Spheres Activates Notch Signaling and Improves Treatment of Right Ventricular Heart Failure

Abstract

Congenital heart disease is rarely cured by surgery and can lead to life-threatening, intractable right ventricular heart failure (HF). Recent stem cell-based clinical trials have been limited by poor differentiation rates and low cell retention. We propose the aggregation of child cardiac progenitor cells (CPCs) into scaffold-free spheres to improve the differentiation of CPCs into mature cardiac phenotypes by enhancing intercellular Notch signaling, a key regulator of CPC fate decisions. Child CPC spheres were produced at a size of 1500 cells per sphere using a microwell array and cultured in suspension for up to 14 days with 70-80% cell viability. By 7 days in culture, aggregation of CPCs increased protein expression of Notch1 intracellular domain (3.8-fold) and transcript expression of Notch1 signaling pathway members, including ADAM10 (3.6-fold), DLL1 (25.0-fold), and JAG1 (3.2-fold), compared to parallel monolayer cultures. Increased Notch signaling activity was accompanied by high transcript levels of cardiac differentiation markers, including MEF2C, MYH7, and cTNNI. In particular, Nkx2.5, an important transcription factor for cardiomyocyte development, was robustly expressed within the nuclei of aggregated CPCs. Inhibition of Notch signaling activity using the small molecule γ-secretase inhibitor DAPT abrogated these effects, including the nuclear translocation of Nkx2.5. To evaluate the therapeutic effects of CPC spheres compared to single CPCs, right ventricular HF was induced in rats by pulmonary artery banding and cells were transplanted into the right ventricular free wall. By 28 days, echocardiographic measurements showed significantly improved right ventricular function with CPC sphere transplantation compared to single CPC transplantation (tricuspid annular plane systolic excursion: 2.03±0.2 vs. 1.61±0.22 mm, P<0.001). Additionally, CPC aggregation improved cell retention at 28 days compared to single CPCs as measured using DiR-labeled CPCs (29.2±4.0 vs. 11.1±7.3% of day 0 fluorescence, P<0.005). The results of our project will facilitate the development of autologous stem-cell based therapies for pediatric HF.