Reprogramming of primary human fetal fibroblasts towards cardiomyoctes

Abstract

The mammalian heart is primarily composed of fibroblasts, cardiomyocytes, endothelial and smooth muscle cells. Cardiomyocytes show little regenerative capabilities following damage (e.g. myocardial infarction, MI) and subsequently cardiac fibroblasts migrate to the site of injury and secrete extracellular matrix proteins to form scar tissue which supports the damaged myocardium. Thus, there is a therapeutic need for cell-based therapies for the generation of functional cardiomyocytes. One method to achieve this is to directly reprogram fibroblasts into cardiomyocytes, which has previously been achieved in mice through overexpression of the transcription factors: Gata4, Mef2c and Tbx5. The present study has hypothesised that fibroblast cells, isolated from human fetal heart and skin, can be reprogrammed into cardiomyocytes by overexpressing GATA4, MEF2C and TBX5 (GMT).

Initial experiments aimed to characterise the phenotype of cardiac cell types within the human fetal heart using protein markers. Flow cytometry data revealed that the human fetal heart is composed of approximately 75-80% cardiomyocytes and 20-25% non-myocytes. The results also showed that Thy-1 and vimentin, considered to be fibroblast markers, localise in a proportion of cells that also express sarcomeric proteins (cardiomyocyte markers), confirming that they are not specific markers of fibroblasts. Isolation of fibroblasts from primary human fetal tissue was achieved through explant migration and phenotyped by RT-PCR, immunocytochemistry and flow cytometry. These cells were transfected with two vectors that allowed bicistronic expression of GATA4 with GFP and MEF2C with TBX5. Three transfection methods were compared: JetPEI, FuGENE6 and nucleofection. Nucleofection was found to be the best method of vector delivery into primary fibroblasts and enabled the selection of G418- resistant cells that were viable for 8 weeks.

Flow cytometry analysis showed upregulation of NKX2.5, cardiac troponin I (TnI) and α-actinin in skin fibroblasts transfected with GMT and treated with G418, however, expression was not sustained beyond two weeks, implying that these cells were not stably expressing GMT. Treatment with the signalling molecule TGFβ-1 increased the percentage of cells expressing NKX2.5, TnI and α-actinin.

This study has demonstrated the viability of a non-viral system for the delivery of GMT into primary human fetal fibroblasts and has shown that overexpression of GMT in these cells appears to initiate the early stages of direct cardiomyocyte reprogramming.

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Identifiers

ORCID for David Wilson: orcid.org/0000-0002-6304-2836

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