Aggregation of Child Cardiac Progenitor Cells Into Spheres Activates Notch Signaling and Improves Treatment of Right Ventricular Heart Failure

Abstract

Rationale: Congenital heart disease can lead to life-threatening right ventricular heart failure (RVHF). Results from clinical trials support expanding cardiac progenitor cell (CPC) based therapies. However, our recent data show that CPCs lose function as they age, starting as early as 1 year. Objective: To determine whether the aggregation of child (1 to 5-year-old) CPCs into scaffoldfree spheres can improve differentiation by enhancing Notch signaling, a known regulator of CPC fate. We hypothesized that aggregated (3D) CPCs will repair RVHF better than monolayer (2D) CPCs. Methods and Results: Spheres were produced with 1500 CPCs each using a microwell array. CPC aggregation significantly increased gene expression of Notch1 compared to 2D CPCs, accompanied by significant upregulation of cardiogenic transcription factors (GATA4, HAND1, MEF2C, NKX2.5, and TBX5) and endothelial markers (CD31, FLK1, FLT1, vWF). Blocking Notch receptor activation with the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) diminished these effects. To evaluate the therapeutic improvements of CPC aggregation, RVHF was induced in athymic rats by pulmonary artery banding and cells were implanted into the RV free wall. Echocardiographic measurements 28 days post-implantation showed significantly improved RV function with 3D compared to 2D CPCs. Tracking implanted CPCs via DiR-labeling showed improved retention of 3D CPCs. Transducing 3D CPCs with Notch1-shRNA did not reduce retention, but significantly reduced RV functional

Predicting Functional Responses of Progenitor Cell Exosome Potential with Computational Modeling

“…1C). Exosomes from 3D 7-day CPCs significantly reduced CTGF, COL1A1, COL1A2, and VIM transcript expression compared with TGF-β only control (+), as previously published [5]. RCFs had greater uptake of exosomes than CECs, with no significant differences in uptake between exosome treatment groups (Supporting Information Fig.…”

“…CTGF, COL1A1, and COL1A2 had positive loadings values in principal components 1 and 2. This suggests that these responses are negatively correlated with 3D 7-day exosome cues, as affirmed by our previously published data showing reduced expression of CTGF, COL1A1, and COL1A2 with 3D 7-day exosome treatment in vitro [5]. Similarly, VIM and COL3A1 were negatively correlated with 3D 3-day exosome cues.…”

“…*, p ≤ .05 versus TGF‐β only with one‐way ANOVA with Tukey's post hoc analysis. Fibrotic gene expression in TGF‐β stimulated RCFs after treatment with exosomes from 2D or 3D CPCs at day 7 were recently published in reference . 1048, 1063, and 1092 represent different child CPC patients.…”

“…Notch signaling plays an important role in regulating CPC differentiation . Moreover, we have recently shown that 3D CPCs improve RV vessel density in a RVHF rat model in a Notch‐dependent manner . However, the effect of Notch1 inhibition on 3D CPC exosome‐mediated angiogenic function was not explored.…”

“…But older CPCs, starting as early as 1 year old, have been shown to reduce reparative ability [3,4]. In a recent publication, we show that aggregating CPCs into spheres can improve their ability to repair RVHF, likely due to exosome-mediated effects [5].…”