Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5

Abstract

Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)—a co-activator not only for serum response factor, but also for Gata4 and Tbx5—is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. In summary: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

Enhanced efficiency of genetic programming toward cardiomyocyte creation through topographical cues

“…Clones were generated as previously described [46] , [47] and maintained in clonal growth medium (CGM) comprising 65% (v/v) Dulbecco's Modified Eagle's Medium/Ham F-12 (Life Technologies), 35% (v/v) Iscove's Modified Dulbecco's Medium (IMDM; Life Technologies), 3.5% (v/v) bovine growth serum (Thermo Scientific), 100 U/mL Antibiotics-Antimycotics (Life Technologies), 2 mM l -glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma), 1.3% (v/v) B27 media supplement (Life Technologies), 6.5 ng/mL EGF (Petrotech), 13 ng/mL FGF (Petrotech), 0.0005 U/mL thrombin (Roche), 0.345 ng/mL cardiotrophin-1 (Cell Science). For reprogramming experiments we used a previously described protocol [46] . Briefly cells were first transduced with a lentiviral rtTA-IRES-Puro vector and subsequently selected with puromycin (Life Technologies) for 14 days.…”

“…Briefly cells were first transduced with a lentiviral rtTA-IRES-Puro vector and subsequently selected with puromycin (Life Technologies) for 14 days. Cells were then transduced with the transcription factor-encoding lentiviral vectors: TRE-Myocd-ING, TRE-Tbx5-INR (kindly obtained from Lei Zhou, Texas Heart Institute, Houston, Texas, USA) [48] and TRE-Mef2c-ING (cDNA from BioScience LifeSciences cloned onto the Myocardin lentiviral backbone [46] ). One day following transfection, CGM was changed for IMDM supplemented with 10% (v/v) bovine growth serum, 100 U/mL Antibiotics-Antimycotics, 2 mM l -glutamine, 0.1 mM β-mercaptoethanol and 1 μg/mL doxycycline (Sigma) and medium changed every 48 h for the duration of the reprogramming experiment.…”

The Role of Cardiac Side Population Cells in Cardiac Regeneration

“…They have the ability to differentiate into four major cell types in the heart: cardiomyocytes, endothelial cells, fibroblasts, and smooth muscle cells (Table 1 ). Several different in vitro approaches have been used to differentiate cSPCs into cardiomyocytes (Pfister et al, 2005 ; Oyama et al, 2007 ; Yamahara et al, 2008 ; Lushaj et al, 2012 ; Belian et al, 2015 ). When cSPCs are co-cultured with adult rat ventricular cardiomyocytes, they differentiate into cardiomyocytes that are structurally and functionally comparable to adult cardiomyocytes.…”

microRNA Perspective on Cardiomyocyte Development and Cardiovascular Diseases

“…Direct differentiation of fibroblasts to cardiomyocytes was initially achieved using transcription factors: Hand2, GATA4, MEF2c and TBX5 [35], and later enhanced by the addition of MYOCD, SRF, SMARCD3 and MESP1, never addressed the role of miR's [33,36,37].…”