Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success

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.

Pioneer transcription factors in cell reprogramming

“…Later studies revealed that MyoD induces myogenin through cooperation with the Pbx factor, which is constitutively bound to MyoD target sites in fibroblasts prior to MyoD expression (Berkes et al 2004). There are many other examples of direct cell conversion within the same germ layer: the combination of PU.1 and C/EBPa into macrophage-like cells from fibroblasts (Feng et al 2008) and GATA4, MEF2C, TBX5, Hand2, and NKX2.5 for cardiomyocytelike cells from fibroblasts (Ieda et al 2010;Addis et al 2013). …”

Inhibitors of suppressive histone modification promote direct reprogramming of fibroblasts to cardiomyocyte-like cells

“…This wave-like pattern is typical of cardiomyocytes created by direct reprogramming, whereas cardiomyocytes prepared from embryonic heart show a simultaneous increase of the fluorescence intensity in the entire cytoplasm. 4 The wave-like pattern appears to represent partially reprogrammed cardiomyocytelike cells. Indeed, only 30% of the cells expressed cTnT, a marker for terminally differentiated cardiomyocytes (see Supplementary material online, Figure S4A) on the same day that the wave-like pattern was observed.…”

“…Ca 2+ oscillations in the cells were monitored using a transgene encoding the Ca 2+ indicator protein GCaMP5, which emits stronger green fluorescence in the presence of a higher intracellular Ca 2+ concentration. 4 When GSK126 was used from Day 1 to 4, numerous cells exhibited asynchronous oscillation of the fluorescence intensity, and thus the intracellular Ca 2+ concentration, on Day 14 (see Supplementary material online, Figure S1C and Videos 1A and 2A). Some cells were spontaneously beating (Phase and Hoechst panels in Supplementary material online, Figure S1C and Videos 1B and 2B).…”

Reprogramming Approaches to Cardiovascular Disease: From Developmental Biology to Regenerative Medicine

“…Since then, other labs around the world have reported success in reprogramming mouse fibroblasts into iCMs with similar cocktails of reprogramming factors [6][7][8][9][10]. We and others have also directly converted human cardiac and dermal fibroblasts into cardiac cells [11][12][13].…”