© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4267-4278 doi:10.1242/dev.114025
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors
ABSTRACT Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtypespecific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5+ progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols. KEY WORDS: Direct reprogramming, Pacemaker, Cardiomyocyte, Mouse
INTRODUCTION
The canonical view of development depicts lineage specification as a unidirectional process, beginning with pluripotent progenitor cells that progressively differentiate into specialized cell types with restricted potential. Recent studies, however, have directly refuted a simple linear model of lineage specification. Yamanaka and colleagues demonstrated that overexpression of four transcription factors (Oct4/Sox2/Klf4/Myc; OSKM) was sufficient to unlock the restricted potential of committed cells by directly reprogramming differentiated fibroblasts into induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006). 1
Department of Molecular Biology, UT Southwestern Medical Center, Dallas, 2 TX 75390, USA. Department of Medicine, Division of Cardiovascular Medicine, 3 Vanderbilt University, Nashville, TN 37232, USA. Department of Internal Medicine, Division of Cardiology, UT Southwestern Medical Center, Dallas, TX 75390, USA. 4 Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical 5 Center, Dallas, TX 75390, USA. McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, TX 75390, USA. *Authors for correspondence ([email protected]; [email protected]) Received 6 June 2014; Accepted 6 September 2014
Building on this reprogramming paradigm, Ieda and colleagues showed that the combination of Gata4, Mef2c and Tbx5 (GMT) can generate cardiomyocyte-like cells, albeit with low efficiency (Ieda et al., 2010). These cells display many of the key features of bona fide cardiomyocytes (CMs), including expression of sarcomeric genes, spontaneous beating activity, calcium oscillations and characteristic action potentials. Interestingly, follow-up studies have revealed that many different combinations of transcription factors, including GHMT (H; Hand2), can function to reprogram fibroblasts into CM-like cells in vitro (Addis et al., 2013; Christoforou et al., 2013; Fu et al., 2013; Nam et al., 2013; Protze et al., 2012; Song et al., 2012; Wada et al., 2013). Based on these results, several groups subsequently demonstrated direct reprogramming of activated fibroblasts toward a cardiac phenotype in vivo (Inagawa et al., 2012; Jayawardena et al., 2012; Mathison et al., 2012; Qian et al., 2012; Song et al., 2012). Taken together, these studies provide a basis for converting fibroblasts into CMs in vivo to treat a variety of cardiovascular disorders. In addition to potential therapeutic applications, cardiac reprogramming represents a platform to dissect the molecular details of cardiomyogenesis. Although direct cardiac reprogramming is intriguing as a model system, several important hurdles remain to be addressed. Currently, the estimated efficiency of generating reprogrammed CMs is
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors
ABSTRACT Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtypespecific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5+ progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols. KEY WORDS: Direct reprogramming, Pacemaker, Cardiomyocyte, Mouse
INTRODUCTION
The canonical view of development depicts lineage specification as a unidirectional process, beginning with pluripotent progenitor cells that progressively differentiate into specialized cell types with restricted potential. Recent studies, however, have directly refuted a simple linear model of lineage specification. Yamanaka and colleagues demonstrated that overexpression of four transcription factors (Oct4/Sox2/Klf4/Myc; OSKM) was sufficient to unlock the restricted potential of committed cells by directly reprogramming differentiated fibroblasts into induced pluripotent stem (iPS) cells (Takahashi and Yamanaka, 2006). 1
Department of Molecular Biology, UT Southwestern Medical Center, Dallas, 2 TX 75390, USA. Department of Medicine, Division of Cardiovascular Medicine, 3 Vanderbilt University, Nashville, TN 37232, USA. Department of Internal Medicine, Division of Cardiology, UT Southwestern Medical Center, Dallas, TX 75390, USA. 4 Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical 5 Center, Dallas, TX 75390, USA. McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, TX 75390, USA. *Authors for correspondence ([email protected]; [email protected]) Received 6 June 2014; Accepted 6 September 2014
Building on this reprogramming paradigm, Ieda and colleagues showed that the combination of Gata4, Mef2c and Tbx5 (GMT) can generate cardiomyocyte-like cells, albeit with low efficiency (Ieda et al., 2010). These cells display many of the key features of bona fide cardiomyocytes (CMs), including expression of sarcomeric genes, spontaneous beating activity, calcium oscillations and characteristic action potentials. Interestingly, follow-up studies have revealed that many different combinations of transcription factors, including GHMT (H; Hand2), can function to reprogram fibroblasts into CM-like cells in vitro (Addis et al., 2013; Christoforou et al., 2013; Fu et al., 2013; Nam et al., 2013; Protze et al., 2012; Song et al., 2012; Wada et al., 2013). Based on these results, several groups subsequently demonstrated direct reprogramming of activated fibroblasts toward a cardiac phenotype in vivo (Inagawa et al., 2012; Jayawardena et al., 2012; Mathison et al., 2012; Qian et al., 2012; Song et al., 2012). Taken together, these studies provide a basis for converting fibroblasts into CMs in vivo to treat a variety of cardiovascular disorders. In addition to potential therapeutic applications, cardiac reprogramming represents a platform to dissect the molecular details of cardiomyogenesis. Although direct cardiac reprogramming is intriguing as a model system, several important hurdles remain to be addressed. Currently, the estimated efficiency of generating reprogrammed CMs is
