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In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.

Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D - Nature (2012)

Bottom Line: Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling.Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function.These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

View Article: PubMed Central - PubMed

Affiliation: 1Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA.

ABSTRACT
The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

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Genetic lineage tracing demonstrates in vivo reprogramming of cardiac fibroblasts to cardiomyocyte-like cellsa, Quantification of FACS analyses for Thy1+ cells from sham-operated mouse hearts or hearts 2 days after myocardial infarction (MI) (n=3, *p<0.05). b, FACS analyses of Thy1+ dsRed+ cells from sham-operated or post-MI hearts injected with dsRed-expressing retrovirus, with quantification (left) and representative FACS plots (right) (n=3, *p<0.05). c, qPCR analysis of Gata4, Mef2c and Tbx5 in Thy1+ dsRed+ cells or endogenous cardiomyocytes (CMs) compared toThy1+ dsRed− cells sorted two days after post-MI intramyocardial GMTR (Gata4, Mef2c, Tbx5, and dsRed) injection. n=3 with technical quadruplicates. d, Schematic diagram showing the genetic fate mapping method to trace the lineage of CMs reprogrammed from Periostin-Cre:R26R-lacZ or Fsp1-Cre:R26R-lacZ cells. e, Immunofluorescent staining for α-Actinin (green), βGalactosidase (βGal, red) and DAPI (blue) on sham-operated or post-MI Periostin-Cre:R26R-lacZ mouse hearts 4 weeks post-surgery. Images are from distant or border zones where endogenous CMs were labeled by α-Actinin, but were never co-localized with βGal (n=5 hearts/condition, 8 sections/heart). Scale bar, 50 μm. f–g, Immunofluorescent staining for α-Actinin, βGal and DAPI in infarct areas of dsRed- or GMT-injected Periostin-Cre:R26R-lacZ (f) or Fsp1-Cre:R26R-lacZ (g) mouse hearts 4 weeks post-MI. Boxed areas indicate regions of magnification. Scale bar, 50 μm. Error bars indicate standard error of the mean (SEM).
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Figure 1: Genetic lineage tracing demonstrates in vivo reprogramming of cardiac fibroblasts to cardiomyocyte-like cellsa, Quantification of FACS analyses for Thy1+ cells from sham-operated mouse hearts or hearts 2 days after myocardial infarction (MI) (n=3, *p<0.05). b, FACS analyses of Thy1+ dsRed+ cells from sham-operated or post-MI hearts injected with dsRed-expressing retrovirus, with quantification (left) and representative FACS plots (right) (n=3, *p<0.05). c, qPCR analysis of Gata4, Mef2c and Tbx5 in Thy1+ dsRed+ cells or endogenous cardiomyocytes (CMs) compared toThy1+ dsRed− cells sorted two days after post-MI intramyocardial GMTR (Gata4, Mef2c, Tbx5, and dsRed) injection. n=3 with technical quadruplicates. d, Schematic diagram showing the genetic fate mapping method to trace the lineage of CMs reprogrammed from Periostin-Cre:R26R-lacZ or Fsp1-Cre:R26R-lacZ cells. e, Immunofluorescent staining for α-Actinin (green), βGalactosidase (βGal, red) and DAPI (blue) on sham-operated or post-MI Periostin-Cre:R26R-lacZ mouse hearts 4 weeks post-surgery. Images are from distant or border zones where endogenous CMs were labeled by α-Actinin, but were never co-localized with βGal (n=5 hearts/condition, 8 sections/heart). Scale bar, 50 μm. f–g, Immunofluorescent staining for α-Actinin, βGal and DAPI in infarct areas of dsRed- or GMT-injected Periostin-Cre:R26R-lacZ (f) or Fsp1-Cre:R26R-lacZ (g) mouse hearts 4 weeks post-MI. Boxed areas indicate regions of magnification. Scale bar, 50 μm. Error bars indicate standard error of the mean (SEM).

Mentions: Fibroblasts are embryo logically distinct from CMs in their origin15, and following myocardial infarction (MI) become activated, migrate to the injury site, and proliferate16,17. We induced cardiac injury by coronary artery ligation and injected dsRed retrovirus into the myocardium bordering the infarct zone. While cells co-expressing dsRed and α-Actinin were still undetectable, many Vimentin-positive cells were also positive for dsRed (Suppl. Fig. 1). By fluorescence-activated cell sorting (FACS), over 4% of cells (98,238 ± 5523) from the left ventricle of injected hearts were dsRed+Thy1+ 2 days after injury, suggesting successful delivery of virus into cardiac fibroblasts and possibly other non-myocytes upon injury (Fig. 1a,b). By quantitative (q)PCR, dsRed+Thy1+-sorted cells expressed 60-fold moreGata4, Mef2c, and Tbx5 than dsRed−Thy1+ cells, and 6–8-fold more than endogenous CMs (Fig. 1c). A similar number of dsRed+Thy1− cells represented other non-myocyte cell types. Endothelial cells (PECAM+) and some peri-vascular cells (NG2+) were also transduced by the retrovirus, but hematopoietic (CD34+) and pericardial (WT1+) cells were not (Suppl. Fig. 1).


In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.

Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, Fu JD, Srivastava D - Nature (2012)

Genetic lineage tracing demonstrates in vivo reprogramming of cardiac fibroblasts to cardiomyocyte-like cellsa, Quantification of FACS analyses for Thy1+ cells from sham-operated mouse hearts or hearts 2 days after myocardial infarction (MI) (n=3, *p<0.05). b, FACS analyses of Thy1+ dsRed+ cells from sham-operated or post-MI hearts injected with dsRed-expressing retrovirus, with quantification (left) and representative FACS plots (right) (n=3, *p<0.05). c, qPCR analysis of Gata4, Mef2c and Tbx5 in Thy1+ dsRed+ cells or endogenous cardiomyocytes (CMs) compared toThy1+ dsRed− cells sorted two days after post-MI intramyocardial GMTR (Gata4, Mef2c, Tbx5, and dsRed) injection. n=3 with technical quadruplicates. d, Schematic diagram showing the genetic fate mapping method to trace the lineage of CMs reprogrammed from Periostin-Cre:R26R-lacZ or Fsp1-Cre:R26R-lacZ cells. e, Immunofluorescent staining for α-Actinin (green), βGalactosidase (βGal, red) and DAPI (blue) on sham-operated or post-MI Periostin-Cre:R26R-lacZ mouse hearts 4 weeks post-surgery. Images are from distant or border zones where endogenous CMs were labeled by α-Actinin, but were never co-localized with βGal (n=5 hearts/condition, 8 sections/heart). Scale bar, 50 μm. f–g, Immunofluorescent staining for α-Actinin, βGal and DAPI in infarct areas of dsRed- or GMT-injected Periostin-Cre:R26R-lacZ (f) or Fsp1-Cre:R26R-lacZ (g) mouse hearts 4 weeks post-MI. Boxed areas indicate regions of magnification. Scale bar, 50 μm. Error bars indicate standard error of the mean (SEM).
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Related In: Results  -  Collection

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Figure 1: Genetic lineage tracing demonstrates in vivo reprogramming of cardiac fibroblasts to cardiomyocyte-like cellsa, Quantification of FACS analyses for Thy1+ cells from sham-operated mouse hearts or hearts 2 days after myocardial infarction (MI) (n=3, *p<0.05). b, FACS analyses of Thy1+ dsRed+ cells from sham-operated or post-MI hearts injected with dsRed-expressing retrovirus, with quantification (left) and representative FACS plots (right) (n=3, *p<0.05). c, qPCR analysis of Gata4, Mef2c and Tbx5 in Thy1+ dsRed+ cells or endogenous cardiomyocytes (CMs) compared toThy1+ dsRed− cells sorted two days after post-MI intramyocardial GMTR (Gata4, Mef2c, Tbx5, and dsRed) injection. n=3 with technical quadruplicates. d, Schematic diagram showing the genetic fate mapping method to trace the lineage of CMs reprogrammed from Periostin-Cre:R26R-lacZ or Fsp1-Cre:R26R-lacZ cells. e, Immunofluorescent staining for α-Actinin (green), βGalactosidase (βGal, red) and DAPI (blue) on sham-operated or post-MI Periostin-Cre:R26R-lacZ mouse hearts 4 weeks post-surgery. Images are from distant or border zones where endogenous CMs were labeled by α-Actinin, but were never co-localized with βGal (n=5 hearts/condition, 8 sections/heart). Scale bar, 50 μm. f–g, Immunofluorescent staining for α-Actinin, βGal and DAPI in infarct areas of dsRed- or GMT-injected Periostin-Cre:R26R-lacZ (f) or Fsp1-Cre:R26R-lacZ (g) mouse hearts 4 weeks post-MI. Boxed areas indicate regions of magnification. Scale bar, 50 μm. Error bars indicate standard error of the mean (SEM).
Mentions: Fibroblasts are embryo logically distinct from CMs in their origin15, and following myocardial infarction (MI) become activated, migrate to the injury site, and proliferate16,17. We induced cardiac injury by coronary artery ligation and injected dsRed retrovirus into the myocardium bordering the infarct zone. While cells co-expressing dsRed and α-Actinin were still undetectable, many Vimentin-positive cells were also positive for dsRed (Suppl. Fig. 1). By fluorescence-activated cell sorting (FACS), over 4% of cells (98,238 ± 5523) from the left ventricle of injected hearts were dsRed+Thy1+ 2 days after injury, suggesting successful delivery of virus into cardiac fibroblasts and possibly other non-myocytes upon injury (Fig. 1a,b). By quantitative (q)PCR, dsRed+Thy1+-sorted cells expressed 60-fold moreGata4, Mef2c, and Tbx5 than dsRed−Thy1+ cells, and 6–8-fold more than endogenous CMs (Fig. 1c). A similar number of dsRed+Thy1− cells represented other non-myocyte cell types. Endothelial cells (PECAM+) and some peri-vascular cells (NG2+) were also transduced by the retrovirus, but hematopoietic (CD34+) and pericardial (WT1+) cells were not (Suppl. Fig. 1).

Bottom Line: Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling.Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function.These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

View Article: PubMed Central - PubMed

Affiliation: 1Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA.

ABSTRACT
The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.

Show MeSH
Related in: MedlinePlus