Abstract
We have recently shown that a combination of microRNAs, miR combo, can directly reprogram cardiac fibroblasts into functional cardiomyocytes in vitro and in vivo. Reprogramming of cardiac fibroblasts by miR combo in vivo is associated with improved cardiac function following myocardial infarction. However, the efficiency of direct reprogramming in vitro is relatively modest and new strategies beyond the traditional two-dimensional (2D) culture should be identified to improve reprogramming process. Here, we report that a tissue-engineered three-dimensional (3D) hydrogel environment enhanced miR combo reprogramming of neonatal cardiac and tail-tip fibroblasts. This was associated with significantly increased MMPs expression in 3D vs. 2D cultured cells, while pharmacological inhibition of MMPs blocked the effect of the 3D culture on enhanced miR combo mediated reprogramming. We conclude that 3D tissue-engineered environment can enhance the direct reprogramming of fibroblasts to cardiomyocytes via a MMP-dependent mechanism.
Highlights
Cardiac injury in humans results in irreversible loss of cardiomyocytes followed by expansion of cardiac fibroblasts, collagen deposition, and formation of scar tissue[1,2]
We have previously shown that a combination of microRNAs that we termed miR combo, directly reprograms cardiac fibroblasts into cardiomyocytes both in vitro and in vivo[5,7]
We showed that when seeded into 3D fibrin-based hydrogel tissues, cardiomyocytes derived from embryonic stem cells (ESCs), induced pluripotent stem cells or neonatal heart tissues displayed enhanced structural and functional maturation[11,12,14]
Summary
Cardiac injury in humans results in irreversible loss of cardiomyocytes followed by expansion of cardiac fibroblasts, collagen deposition, and formation of scar tissue[1,2]. Functional cardiac tissue patches would be typically seeded with cardiomyocytes derived from human pluripotent stem cells[12]. These cell sources are associated with safety and ethical concerns, which may limit their human applications. We sought to explore the effects of a fibrin-based 3D culture environment[11,12] on the direct miR combo reprogramming of cardiac fibroblasts into a cardiomyocyte fate. We show that culturing fibroblasts within a 3D fibrin-based hydrogel environment (“tissue bundle”) significantly improves the efficiency of direct cardiac reprogramming by miR combo as assessed by gene and protein expression of early and later cardiac differentiation markers. We further demonstrate that the improved cardiac reprogramming is mediated by the enhanced expression of MMPs in the 3D culture environment
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