Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

Abstract

The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs). We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C), known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

Cardiac fibrosis in myocardial infarction—from repair and remodeling to regeneration

“…Human fibroblasts have proven to be more resistant to reprogramming than murine fibroblasts: the successful cardiac reprogramming of human fibroblasts into iCMs requires more transcription factors than reprogramming of murine fibroblasts, and the process is slower and less efficient with human cells compared with murine cells. The origins of the fibroblasts also affect reprogramming efficiency, and cardiac fibroblasts are more easily converted to iCMs than other fibroblasts (Ieda et al 2010 ; Addis et al 2013 ; Palazzolo et al 2016 ). This is in agreement with the report showing that cardiac fibroblasts express a number of cardiogenic genes such as transcription factors Gata4 , Tbx20 , Tbx5 , Nkx2-5 , Hand2 , and Mef2c at significantly higher levels than tail fibroblasts (Furtado et al 2014 ).…”

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.…”

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries

“…45)-47),104),105),207) Direct reprogramming technology has the potential to change the pharmaceutical industry. In direct reprogramming studies, cells from different species, e.g., rat, 208) pigs, 208) dogs, 209) goats, 210) and marmosets, 211) have been generated. Some of the animals studied were industrial animals, such as livestock or pets, since direct reprogramming technology might contribute to animal industries, such as veterinary medicine.…”