Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5Novelty and Significance by Jenny X. Chen, Markus Krane, Marcus-Andre.

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Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5Novelty and Significance by Jenny X. Chen, Markus Krane, Marcus-Andre Deutsch, Li Wang, Moshe Rav-Acha, Serge Gregoire, Marc C. Engels, Kuppusamy Rajarajan, Ravi Karra, E. Dale Abel, Joe C. Wu, David Milan, and Sean M. Wu Circulation Research Volume 111(1):50-55 June 22, 2012 Copyright © American Heart Association, Inc. All rights reserved.

Lentiviral-mediated overexpression of Gata4/MEF2C/Tbx5 factors in CFs and TTFs. A, A diagram of CF and TTF isolation from αMHC-Cre/ROSA26mTmG and Nkx2.5-Cre/ROSA26mTmG mice. Lentiviral-mediated overexpression of Gata4/MEF2C/Tbx5 factors in CFs and TTFs. A, A diagram of CF and TTF isolation from αMHC-Cre/ROSA26mTmG and Nkx2.5-Cre/ROSA26mTmG mice. B, Schematic representation of the effect of Cre recombinase excision on the expression of dTomato and membrane tethered eGFP. C, Whole-mount fluorescence imaging of heart of 3-week-old αMHC-Cre/ROSA26mTmG mouse (left) and Nkx2.5-Cre/ROSA26mTmG day 9.5 embryo (right). Note the specific expression of eGFP in the heart of embryonic mice. D, Fluorescence microscopy (top) and flow cytometric analysis (bottom) of αMHC-Cre/ROSA26mTmG TTFs with and without infection with GMT lentiviruses and treatment with doxycycline for 3 weeks. E, Nkx2.5-Cre/ROSA26mTmG TTFs were assayed as in panel D. F, αMHC-Cre/ROSA26mTmG CFs were assayed as in panel D. G, Nkx2.5-Cre/ROSA26mTmG CFs were assayed as in panel D. H, Cardiac genes expression in uninfected (−) and GMT infected (+) TTFs at 3 weeks postinfection in comparison with cells from E10.5 hearts (H). Mean±SEM from 3 independent experiments is shown for each gene. Symbols above brackets denote statistically significant differences in gene expression (*P<0.05, #P<0.01, ns=not significant). I, CFs were assayed as in panel H. All micrographs were acquired at 10× magnification. CF, cardiac fibroblasts; TTF, tail tip fibroblasts. Jenny X. Chen et al. Circ Res. 2012;111:50-55 Copyright © American Heart Association, Inc. All rights reserved.

Gene expression and functional analysis of GMT infected fibroblasts. Gene expression and functional analysis of GMT infected fibroblasts. A, Flow cytometric analysis of infected and uninfected cTnT-Cre/ROSA26mTmG TTFs. B, Fluorescence microscopy of infected and uninfected cTnT-Cre/ROSA26mTmG TTFs. Images obtained at 10× magnification. C, Pacing-evoked potentials from cardiomyocytes derived from in vitro differentiated ES cells (left), uninfected TTFs (middle), and GMT overexpressing TTFs (right). Note the passive exponential decay (τ=112 ms) of uninfected TTFs and the transient depolarization in GMT-infected fibroblasts. D, Graded voltage response of GMT overexpressing TTFs to increasing pacing amplitudes (0.5, 1, and 2 nA of 5-ms duration from resting membrane potential). E, Pacing-evoked potentials of GMT-infected TTFs in the presence (red) or absence (black) of 5 μmol/L nifedipine. In all traces the dotted line represents resting membrane potential. Jenny X. Chen et al. Circ Res. 2012;111:50-55 Copyright © American Heart Association, Inc. All rights reserved.

Survival and reprogramming of GMT-infected fibroblasts in an experimental model of MI. A, Schematic diagram of experimental procedure to transplant GMT reprogrammed luciferase+eGFP+ CFs into the injured hearts of SCID female mice. Survival and reprogramming of GMT-infected fibroblasts in an experimental model of MI. A, Schematic diagram of experimental procedure to transplant GMT reprogrammed luciferase+eGFP+ CFs into the injured hearts of SCID female mice. Transplanted mice were subjected to bioluminescent imaging for 8 days before eGFP+ cells were recovered for single-cell PCR array analysis. B, Representative bioluminescent imaging of mice injected with GMT overexpressing luciferase+eGFP+ CFs. C, Quantitative analysis of cell survival (ie, luciferase activity) in transplanted hearts. D, Single-cell PCR array analysis of FACS-purified eGFP+ cells after in vivo engraftment for 8 days. Jenny X. Chen et al. Circ Res. 2012;111:50-55 Copyright © American Heart Association, Inc. All rights reserved.