Enhanced efficiency of genetic programming toward cardiomyocyte creation through topographical cues.

Generation of de novo cardiomyocytes through viral over-expression of key transcription factors represents a highly promising strategy for cardiac muscle tissue regeneration. Although the feasibility of cell reprogramming has been proven possible both in vitro and in vivo, the efficiency of the process remains extremely low. Here, we report a chemical-free technique in which topographical cues, more specifically parallel microgrooves, enhance the directed differentiation of cardiac progenitors into cardiomyocyte-like cells. Using a lentivirus-mediated direct reprogramming strategy for expression of Myocardin, Tbx5, and Mef2c, we showed that the microgrooved substrate provokes an increase in histone H3 acetylation (AcH3), known to be a permissive environment for reprogramming by "stemness" factors, as well as stimulation of myocardin sumoylation, a post-translational modification essential to the transcriptional function of this key co-activator. These biochemical effects mimicked those of a pharmacological histone deacetylase inhibitor, valproic acid (VPA), and like VPA markedly augmented the expression of cardiomyocyte-specific proteins by the genetically engineered cells. No instructive effect was seen in cells unresponsive to VPA. In addition, the anisotropy resulting from parallel microgrooves induced cellular alignment, mimicking the native ventricular myocardium and augmenting sarcomere organization.

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

UNLABELLED: 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.

PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium.

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT-PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα(+) cells. Clonal progeny of single Sca1(+) SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα(-) cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα(+)/CD31(-) cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα(-)/CD31(+) cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1(+) stem/progenitor cell.

Effect of YB-1 on the regulation of micro RNA expression in drug-sensitive and drug-resistant gastric carcinoma cells.

The multifunctional Y-Box protein 1 (YB-1) exerts positive and negative regulatory effects on gene expression by different mechanisms. Since transcription can be controlled by micro RNAs (miRNAs), YB-1 could also cause effects on gene expression by regulation of cellular miRNAs. To test this hypothesis, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells, in which the expression of YB-1 can be inhibited by tetracycline-dependent triggering of the RNA interference (RNAi) pathway, was investigated concerning their miRNA expression profiles in the presence and absence of YB-1. Microarray hybridizations demonstrated that six miRNAs (miR-96*, miR-210, miR-503, miR-623, miR-1275, miR-1290) were up-regulated more than 1.5-fold in drug-sensitive cells following YB-1 inhibition, but no differences in miRNA expression could be detected in multidrug-resistant cells. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction did not confirm these effects. Likewise, an in silico analysis of potential regulatory effects of the miRNAs on their target genes did not support the potential miRNA regulatory effects of YB-1. In conclusion, the data provide evidence that YB-1 has no direct influence on global miRNA expression pattern in different variants of gastric carcinoma cells and, therewith, does not control gene expression by regulation of miRNAs.

Regulation of mRNA expression in drug-sensitive and drug-resistant gastric carcinoma cells is independent of YB-1 expression.

Y-Box protein 1 (YB-1) is a multifunctional cellular protein expressed in a range of mammalian cells, including human cancer cells. It is involved in the regulation of various genes including cancer-associated genes, but the full range of target genes and regulatory mechanisms have not been fully elucidated. To identify global mRNA expression patterns that are potentially regulated by YB-1, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells in which the expression of YB-1 can be inhibited by tetracycline-dependent activation of the RNA interference (RNAi) pathway, was analyzed by microarray technology. By this approach, various potentially regulated genes encoding members of important cellular pathways such as the Jak/STAT, VEGF and the MAP-kinase signaling pathways were identified. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction and Western blot did not confirm these regulatory effects. In conclusion, the findings suggest that YB-1 is not directly involved in the regulation of mRNA expression in drug-sensitive or drug-resistant gastric carcinoma cells.