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.

Keywords: cardiac fibroblasts; cardiomyocytes; digital droplet PCR (ddPCR); direct reprogramming; microRNAs.

FIGURE 1

DharmaFECT1 mediated delivery of miR-1 and TWF-1 target downregulation in AHCFs. (A) Fold change of miR-1 expression (relative to untransfected control) in AHCFs transfected with negmiR or miR-1 using DharmaFECT1 at 24 h (i.e., immediately post-transfection) and 48 h (i.e., 24 h post-transfection) culture times, analyzed by ddPCR. Data are representative of three independent experiments. Stated p-value ^∗∗∗p < 0,001 is reported versus negmiR control. (B) Fold change of TWF-1 mRNA target expression (relative to untransfected control) in AHCFs transfected with negmiR or miR-1 using DharmaFECT1 at 24 h (i.e., immediately post-transfection) and 48 h (i.e., 24 h post-transfection) culture times. Data are representative of three independent experiments. Stated p-value ^∗∗∗p < 0,001 is reported versus negmiR control. CTRL, untransfected control.

FIGURE 2

MiRcombo-transfected AHCFs show increased cardiac transcription factor expression. (A) Representative scheme of experimental design. AHCFs were transfected with miRcombo or negative control (negmiR). The acquisition of cardiomyocyte-associated features was evaluated after 7 days (cardiac transcription factors), 15 days (cardiomyocyte and fibroblast markers) and 30 days (calcium transients). (B–F) Gene expression of cardiac transcription factors. The expression of GATA4, MEF2C, TBX5, HAND2, and NKX2.5 was evaluated by ddPCR 7 days post transfection in AHCFs transfected with miRcombo (red) or negmiR (blue). Data are representative of three independent experiments, each performed in triplicate. Stated P-value is versus negmiR controls.

FIGURE 3

Induction of cardiomyocyte markers in miRcombo-transfected AHCFs. (A,B) Gene expression of cardiomyocyte markers TNNT2 and TNNI3 in AHCFs transfected with miRcombo (red) or negmiR (blue) evaluated by ddPCR. Data are representative of three independent experiments, each performed in triplicate. (C) Representative flow plots (left panel) and percentage (right panel) of cTnT positive cells in AHCFs transfected with miRcombo (n = 3) and negmiR (n = 3) 15 days after transfection. (D–F) Gene expression of fibroblastic markers VIM, DDR2, and FSP-1 in AHCFs transfected with miRcombo (red) or negmiR (blue) by ddPCR. Data are representative of three independent experiments, each performed in triplicate. Stated P-value is versus negmiR.

FIGURE 4

miRcombo-transfected cells exhibit calcium transients. (A,B) Representative image, recorded with fluorescence microscopy (A) and trace (B) of Ca⁺² oscillations in AHCFs transfected with miRcombo (n = 3) and negmiR (n = 3) after 30 days from transfection. Scale bar: 25 μm. (C) Bar graph reports the percentage of cells showing Ca⁺² transients after 30 days from miRcombo and negmiR transfection. Stated P-value is versus negmiR.