MicroRNA-Mediated Direct Reprogramming of Human Adult Fibroblasts Toward Cardiac Phenotype

Abstract

Modulation of microRNA expression holds the promise to achieve direct reprogramming of fibroblasts into cardiomyocyte-like cells as a new strategy for myocardial regeneration after ischemic heart disease. Previous reports have shown that murine fibroblasts can be directly reprogrammed into induced cardiomyocytes (iCMs) by transient transfection with four microRNA mimics (miR-1, 133, 208, and 499, termed "miRcombo"). Hence, study on the effect of miRcombo transfection on adult human cardiac fibroblasts (AHCFs) deserves attention in the perspective of a future clinical translation of the approach. In this brief report, we studied for the first time whether miRcombo transient transfection of AHCFs by non-viral vectors might trigger direct reprogramming of AHCFs into cardiomyocyte-like cells. Initially, efficient miRNA delivery to cells was demonstrated through the use of a commercially available transfection agent (DharmaFECT1). Transient transfection of AHCFs with miRcombo was found to upregulate early cardiac transcription factors after 7 days post-transfection and cardiomyocyte specific marker cTnT after 15 days post-transfection, and to downregulate the expression of fibroblast markers at 15 days post-transfection. The percentage of cTnT-positive cells after 15 days from miRcombo transfection was ∼11%, as evaluated by flow cytometry. Furthermore, a relevant percentage of miRcombo-transfected AHCFs (∼38%) displayed spontaneous calcium transients at 30 days post-transfection. Results evidenced the role of miRcombo transfection on triggering the trans differentiation of AHCFs into iCMs. Although further investigations are needed to achieve iCM maturation, early findings from this study pave the way toward new advanced therapies for human cardiac regeneration.

Advanced Technologies to Target Cardiac Cell Fate Plasticity for Heart Regeneration

“…As such, novel methods allowing the temporal over-expression of reprogramming factors would be of great importance. Preliminary results were already obtained by transient transfection of human CFs by non-viral vectors with miR combo [173].…”

Stem Cells in Cardiovascular Diseases: 30,000-Foot View

“…The feasibility of these approaches will likely be dictated by whether an analogy of “Moore’s law” in the technology field applies to the costs and methods of induced pluripotent stem cell technology development, and to what degree enhanced efficiencies in this regard might make this approach competitive with transplantation. One strategy which may offer simplicity and greater efficiency is direct fibroblast reprogramming, which has garnered significant preclinical attention [ 149 , 150 , 151 , 152 ]. In addition to more efficient generation of cardiomyocytes for direct administration, these approaches are exploring generation of multipotent but cardiac committed progenitors, as well as in vivo reprogramming of fibroblast and other cardiac cells to enact cellular regeneration without direct stem cell administration [ 153 ].…”

Large Animal Models of Cell-Free Cardiac Regeneration

“…Thus far, no in vivo large animal models of direct cardiac reprogramming have been published. However, the method has been demonstrated in in vitro cultures of pig [ 99 ], canine [ 100 ], and human cells [ 99 , 101 , 102 ]. This, combined with the scarcity of in vivo studies of in situ direct cardiac reprogramming in chronic ischemic models, where the fibrotic scar would constitute the perfect target for this method, represents a major future research opportunity in large animal models of cell-free cardiac regeneration therapies.…”