Article

Optimization of Direct Fibroblast Reprogramming to Cardiomyocytes Using Calcium Activity as a Functional Measure of Success.

Institute for Regenerative Medicine and Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address: .
Journal of Molecular and Cellular Cardiology (Impact Factor: 5.22). 04/2013; 60. DOI: 10.1016/j.yjmcc.2013.04.004
Source: PubMed

ABSTRACT Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.

0 Followers
 · 
241 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Damage in cardiac tissues from ischemia or other pathological conditions leads to heart failure; and cell loss or dysfunction in pacemaker tissues due to congenital heart defects, aging, and acquired diseases can cause severe arrhythmias. The promise of successful therapies with stem cells to treat these conditions has remained elusive to the scientific community. However, recent advances in this field have opened new opportunities for regenerative cardiac therapy. Transplantation of cardiomyocytes derived from human pluripotent stem cells has the potential to alleviate heart disease. Since the initial derivation of human embryonic stem cells, significant progress has been made in the generation and characterization of enriched cardiomyocytes and the demonstration of the ability of these cardiomyocytes to survive, integrate, and function in animal models. The scope of therapeutic potential from pluripotent stem cell-derived cardiomyocytes has been further expanded with the invention of induced pluripotent stem cells, which can be induced to generate functional cardiomyocytes for regenerative cardiac therapy in a patient specific manner. The reprogramming technology has also inspired the recent discovery of direct conversion of fibroblasts into cardiomyocyte-like cells, which may allow endogenous cardiac repair. Regenerative cardiac therapy with human pluripotent stem cells is now moving closer to clinic testing.
    Discovery medicine 06/2013; 15(85):349-356. · 3.50 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Regeneration of cardiac tissue has the potential to transform cardiovascular medicine. Recent advances in stem cell biology and direct reprogramming, or transdifferentiation, have produced powerful new tools to advance this goal. In this Review we examine key developments in the generation of new cardiomyocytes in vitro as well as the exciting progress that has been made toward in vivo reprogramming of cardiac tissue. We also address controversies and hurdles that challenge the field.
    Nature medicine 07/2013; 19(7):829-36. DOI:10.1038/nm.3225 · 28.05 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: As the adult mammalian heart has limited potential for regeneration and repair, the loss of cardiomyocytes during injury and disease can result in heart failure and death. The cellular processes and regulatory mechanisms involved in heart growth and development can be exploited to repair the injured adult heart through 'reawakening' pathways that are active during embryogenesis. Heart function has been restored in rodents by reprogramming non-myocytes into cardiomyocytes, by expressing transcription factors (GATA4, HAND2, myocyte-specific enhancer factor 2C (MEF2C) and T-box 5 (TBX5)) and microRNAs (miR-1, miR-133, miR-208 and miR-499) that control cardiomyocyte identity. Stimulating cardiomyocyte dedifferentiation and proliferation by activating mitotic signalling pathways involved in embryonic heart growth represents a complementary approach for heart regeneration and repair. Recent advances in understanding the mechanistic basis of heart development offer exciting opportunities for effective therapies for heart failure.
    Nature Reviews Molecular Cell Biology 07/2013; DOI:10.1038/nrm3619 · 36.46 Impact Factor