Cardiac Tissue-like 3D Microenvironment Enhances Route towards Human Fibroblast Direct Reprogramming into Induced Cardiomyocytes by microRNAs.

Camilla Paoletti,Daria Nurzynska,Carla Divieto,Elena Marcello,Maria Luna Melis,Valeria Chiono

doi:10.3390/cells11050800

Camilla Paoletti, Daria Nurzynska + Show 4 more

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https://doi.org/10.3390/cells11050800

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Abstract

The restoration of cardiac functionality after myocardial infarction represents a major clinical challenge. Recently, we found that transient transfection with microRNA combination (miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocytes (iCMs) in vitro. However, achieving efficient direct reprogramming still remains a challenge. The aim of this study was to investigate the influence of cardiac tissue-like biochemical and biophysical stimuli on direct reprogramming efficiency. Biomatrix (BM), a cardiac-like extracellular matrix (ECM), was produced by in vitro culture of AHCFs for 21 days, followed by decellularization. In a set of experiments, AHCFs were transfected with miRcombo and then cultured for 2 weeks on the surface of uncoated and BM-coated polystyrene (PS) dishes and fibrin hydrogels (2D hydrogel) or embedded into 3D fibrin hydrogels (3D hydrogel). Cell culturing on BM-coated PS dishes and in 3D hydrogels significantly improved direct reprogramming outcomes. Biochemical and biophysical cues were then combined in 3D fibrin hydrogels containing BM (3D BM hydrogel), resulting in a synergistic effect, triggering increased CM gene and cardiac troponin T expression in miRcombo-transfected AHCFs. Hence, biomimetic 3D culture environments may improve direct reprogramming of miRcombo-transfected AHCFs into iCMs, deserving further study.

Highlights

Ischemic heart disease is the main cause of death worldwide [1]
The results demonstrate that a biomimetic 3D culture microenvironment can enhance the direct reprogramming efficiency of miRcombo-transfected human adult cardiac fibroblasts into induced cardiomyocytes (iCMs)
We investigated whether the long-term culturing of adult human cardiac fibroblasts (AHCFs) on PS plates culture plates could induce their transition into a myofibroblast phenotype

Summary

Introduction

Ischemic heart disease is the main cause of death worldwide [1]. During myocardial infarction (MI), billions of cardiomyocytes are irreversibly lost, leading to scar tissue formation, followed by left ventricle remodeling, which may result in progressive heart failure.Over the past few decades, several therapeutic approaches have been investigated with the aim to recover cardiac functionality, encompassing tissue engineering strategies as well as cell-based and gene therapies [2,3]. Different direct reprogramming strategies have been investigated, including the expression of cardiac lineage specific TFs by viral vectors [4], the use of small molecules [6], the suppression of fibroblast signatures [7], the expression of cardiac microRNAs (miRs-1, 133, 208 and 499, called “miRcombo”) [8], or combinations of these methods [9]. We demonstrated that a single transient transfection with miRcombo, previously validated in vitro [8] and in vivo [10] in a mouse model, was able to trigger direct reprogramming of adult human cardiac fibroblasts into iCMs, as demonstrated by the expression of cardiac lineage specific TFs and cardiomyocyte markers and spontaneous calcium flux in reprogrammed cells [11]. Direct reprogramming of human adult cardiac fibroblasts into iCMs showed limited efficiency, with ~11%.cardiac troponin T (cTnT)-positive cells after 15 days post-transfection [11]

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