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: 4.66). 04/2013; 60(1). DOI: 10.1016/j.yjmcc.2013.04.004
Source: PubMed


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.

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    • "Subsequent reports have described improved conversion to iCMs with inclusion of different transcription factor combinations, including Hand2 (H) [13] as well as combinations of microRNAs [8]. Our group recently employed a genetically-encoded calcium indicator (GECI, GCaMP) driven by the cardiac Troponin T promoter to identify the transcription factor combination of HGMT plus Nkx2.5 (N), which led to enhanced iCM generation from mouse embryonic fibroblasts (MEFs) compared to the aforementioned transcription factor combinations [5]. This Troponin T-GCaMP reporter system is advantageous compared to traditional genetic reporters in that the intensity of GFP is responsive to intracellular calcium levels [15]. "
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    ABSTRACT: Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.
    PLoS ONE 02/2014; 9(2):e89678. DOI:10.1371/journal.pone.0089678 · 3.23 Impact Factor
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    • "It was found that the most efficient combination for generating cardiomyocyte-like cells with cardiomyocyte marker expression, consisted of Hand2, NK2 homeobox 5 (Nkx2.5), GATA4, Mef2c, and Tbx5 (HNGMT) and was >50-fold more efficient than GMT alone.75 Epigenetics are also of importance when it comes to reprogramming. "
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    ABSTRACT: The procedure of using mature, fully differentiated cells and inducing them toward other cell types while bypassing an intermediate pluripotent state is termed direct reprogramming. Avoiding the pluripotent stage during cellular conversions can be achieved either through ectopic expression of lineage-specific factors (transdifferentiation) or a direct reprogramming process that involves partial reprogramming toward the pluripotent stage. Latest advances in the field seek to alleviate concerns that include teratoma formation or retroviral usage when it comes to delivering reprogramming factors to cells. They also seek to improve efficacy and efficiency of cellular conversion, both in vitro and in vivo. The final products of this reprogramming approach could be then directly implemented in regenerative and personalized medicine.
    Stem Cells and Cloning: Advances and Applications 02/2014; 7(1):19-29. DOI:10.2147/SCCAA.S38006
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    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. · 3.63 Impact Factor
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