Reprogramming fibroblasts into cardiomyocytes.

A deficiency of cardiomyocytes underlies most cases of heart failure, and scientists have long sought to repopulate the heart with new cardiomyocytes. Most attempts at remuscularization have been based on the transplantation of stem cells or their derivatives, and success has been modest to date. A recent study by Ieda and colleagues1 points to a new therapeutic strategy: reprogramming fibroblasts to become cardiomyocytes. The paradigm of cellular reprogramming originated with the discovery, in 1987, that expression of the skeletal muscle gene, MYOD1, could induce multiple cell types to differentiate into skeletal muscle.2 MYOD1 encodes a transcription factor that regulates RNA transcription from target genes. This finding fueled the hypothesis that the activation of master transcription factors drives the differentiation of specific cell types. Unfortunately, most transcription factors appear to regulate only a subgroup of the genes necessary for the differentiation of different cell types, and a vigorous search did not identify other master regulators of equivalent potency to MYOD1. Interest in reprogramming waned until Takahashi and Yamanaka3 made the landmark discovery in 2006 that differentiated fibroblasts could be transcriptionally reprogrammed into induced pluripotent stem cells.3 Although no single transcription factor that was tested produced such cells, a combination of 25 transcription factors that are normally present in embryonic stem cells reprogrammed fibroblasts to pluripotency. A careful winnowing identified four transcription factors that were required for this reprogramming. Impressively, entire mice can now be generated from induced pluripotent stem cells, proving their bona fide stem-cell status. Buoyed by these results, Ieda and colleagues expressed in mouse cardiac fibroblasts 14 cardiac regulatory genes, most of which encode transcription factors. To accelerate the screening process, the fibroblasts were engineered to express green fluorescent protein (GFP) on cardiac differentiation. Transduction with all 14 factors induced GFP expression in about 1% of cells. The 14 factors were carefully winnowed to 3 transcription factors — Mef2C, Gata4, and Tbx5 — that were necessary and sufficient to activate GFP expression in approximately 20% of cells (Fig. 1). Fibroblasts from the tail tip also could be reprogrammed in this way, albeit less efficiently, which effectively ruled out contamination by mature cardiomyocytes or a cardiac progenitor cell. Maintenance of the induced-cardiomyocyte state did not require continued expression of the reprogramming factors. When the fibroblasts were transplanted into the heart 1 day after transduction (but before activation of GFP), some of the cells underwent cardiac differentiation in vivo as well. Activation of a cardiac-restricted reporter gene, of course, is not the same as full cardiac differentiation. One must look for loss of the fibroblast phenotype, expression of endogenous cardiac proteins, and most important, evidence that the cells function as cardiomyocytes. Using microarrays to simultaneously profile thousands of RNA transcripts, Ieda et al. found that the GFPpositive cells down-regulated fibroblast markers, such as collagens, and expressed many cardiac genes. A minority of GFP-positive cells expressed other cardiac sarcomere genes and contained organized sarcomeres, and a small fraction displayed complex cardiac phenotypes, such as spontaneous calcium transients and spontaneous beating activity (Fig. 1). Thus, a subgroup of reprogrammed cells had physiological properties of cardiomyocytes, but the majority of cells were only partially reprogrammed. Reprogramming of fibroblasts to cardiomyocytes holds tremendous potential for the treat-