Human amniotic fluid stem cell-extracellular vesicles as paracrine candidates to rejuvenate cardiomyocyte renewal
Authors: Costa A , Balbi C , Garbati P , De Palma A , Ceresa D , Santamaria S , Cortese K , Coviello D , De Biasio P , Paladini D , Barile L , Malatesta P , Mauri P , Bollini S 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): University of Genova

Background
Reactivation of cardiomyocyte renewal represents a milestone for cardiac regeneration. While broadly active in the early neonatal mammalian myocardium, such mechanism is lost soon after birth, with transition to defective repair. We previously showed that human amniotic fluid stem cells (hAFS) possess cardioprotective and cardioactive paracrine potential in a preclinical adult mouse model of myocardial infarction (MI)1, by sustaining cardiomyocyte cell cycle re-entry up to DNA duplication.

Purpose
Here we analyse whether hAFS secretome formulations, namely hAFS-conditioned medium (hAFS-CM) over extracellular vesicles (hAFS-EVs) separated from that, can enhance cardiomyocyte renewal in vitro and in vivo.

Methods
hAFS were obtained by leftover samples of prenatal screening amniocentesis and primed under 1% O2 preconditioning to enrich hAFS-CM and hAFS-EVs with cardioactive factors. hAFS-EVs were concentrated from hAFS-CM by serial ultracentrifugation. hAFS-EVs were profiled by electron microscopy, nanoparticle tracking analysis, proteomics and RNA sequencing (RNAseq). Neonatal mouse ventricular cardiomyocytes (mNVCM) were enzymatically isolated from R26pFUCCI2 mice with fluorescent tagging of cell-cycle by nuclear signal (G1-phase by Cdt1-mCherry; S-G2-M-phases by Geminin-mVenus1)2. mNVCM were stimulated by hAFS-CM or hAFS-EVs to identify the most promising formulation for further analysis in a R26pFUCCI2 4-day-old MI mouse model via intra-peritoneal delivery, according to principles of laboratory animal care and in compliance with authorization from the Italian Ministry of Health.

Results
hAFS-EVs sustained R26pFUCCI2 mNVCM transition from S- to M-phase by 2.1-fold (*p&lt;0.05) in vitro, while triggering cytokinesis by 4.5-fold (*p&lt;0.05) increase by Aurora B kinase expression at midbodies. Treated mNVCM showed disorganized expression of cardiac α-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with significant down-regulation (0.6-fold, ****p&lt;0.0001) of Cofilin-2 (CFL-2) an actin cytoskeleton regulator antagonizing cardiomyocyte proliferation3. RNAseq on hAFS-EVs revealed some of the mostly enriched microRNAs (miRNAs) targeting CFL-2 (miR-93-5p; miR-152-3p; miR-200b-3p; miR-429; miR-199a-3p; miR-20a-5p; miR-425-5p), while proteomics reported enrichment of a secreted form of Agrin, an important mediator for neonatal heart regeneration4. hAFS-EVs increased cardiomyocyte S- to M phase transition by 1.8-fold (*p&lt;0.05) in the mouse neonatal left ventricle 3 days post-MI.

Conclusions
Our current findings suggest that EVs secreted by developmentally juvenile hAFS may be endowed with relevant potential to support cardiomyocyte renewal for future paracrine therapy in regenerative medicine with putative molecular candidate(s) of action in their cargo content including Agrin and non-coding miRNAs.