Exploring in vitro markers of potency of extracellular vesicles from cardiosphere-derived cells in the treatment of cardiac ageing
Authors: Gomez-Cid L , Cervera-Negueruela M , Campo-Fonseca A , Suarez-Sancho S , Pinto A , Gil Jaurena J M , Fernandez-Santos M E , Fernandez-Aviles F , Grigorian-Shamagian L 
Published: October 2022

Abstract

Background
Cardiac ageing is associated to heart failure with preserved ejection fraction (HF-pEF) and increased senescence, hypertrophy and diastolic dysfunction. No treatments have yet proved to reduce HF-pEF morbidity and mortality. Cardiosphere-derived cells (CDCs) and their secreted extracellular vesicles (CDC-EVs) have demonstrated efficacy in old animals with cardiac dysfunction, but variability of effect and lack of adequate potency tests remain as challenges.

Purpose
To explore in vitro predictors of cardiac protective potency of CDC-EVs, focusing on the chronological age of the CDC-donors, CDC-senescence, and their in vitro anti-senescent and pro-angiogenic effect.

Methods
CDCs derived from 34 patients (age range 0–81 years old, both sexes) were characterized in terms of senescence, proliferative and migration capacities, VEGF secretion, expression of specific surface markers and cardiosphere size. CDC-EVs were purified and their in vitro anti-senescent potential (at genetic, secretory and cellular level over cardiac stromal cells) and their pro-angiogenic potential (ability to induce tube formation over endothelial cells) quantified. According to the performance in each of these tests, potency was scored and CDC-EVs were classified as potent (P-EVs) and non-potent (NP-EVs). The effect of P-EVs and NP-EVs were then tested in vivo in rats with induced cardiac aging. SD-rats received 3-months of daily intraperitoneal injections (IP) of saline (healthy control) or D-galactose. Rats in the D-Gal group were randomly allocated to receive IP saline (sham control, n=12), P-EVs (n=7) or NP-EVs (n=6) and followed-up for one month.

Results
Chronological age of the donor or expression of surface markers did not relate to most CDC properties nor to their in vitro potency. CDC senescence did relate to other CDC bioactive properties, but this was insufficient to predict CDC-EV anti-senescence and pro-angiogenic in vitro potency. In vivo, EV classified as P-EVs, but not NP-EVs, prevented D-gal induced hypertrophy (2.4 vs. 2.9 mg/gr, p=0.05). This finding was in parallel to the levels of galactosidase-beta 1 expression in cardiac tissue, which were increased in sham vs. P-EVs (1.22 vs. 0.73, p=0.03) but levels in NP-EVs were not significantly different to the sham group (0.86, p=0.1). P-EVs tended to reduce TGFB1 expression, while NP-EVs significantly increased cardiac fibrosis and reduced cardiac perfusion. At systemic level, while P-EVs significantly improved glucose metabolism and tended to drive total antioxidant capacity and hair growth to a healthier profile, NP-EVs did not significantly improve any of the explored parameters and significantly increased total antioxidant capacity.

Conclusions
After further validation, the matrix potency assay proposed here, scoring the anti-senescent and pro-angiogenic in vitro effect of CDC-EVs could be used to predict EV suitability as an allogenic product in the treatment of cardiac ageing.

Funding Acknowledgement
Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaciόn, Spain: PI16/01123; PI19/00161; Red de Terapia Celular, Tercel, (RD16.0011.0029) and CIBERCV (CB16.11.00292)