Transcriptome analysis in cardiomyocyte-specific differentiation of murine embryonic stem cells reveals transcriptional regulation network.

The differentiation to cardiomyocytes is a prerequisite and an important part of heart development. A good understanding of the complicated cardiomyocyte differentiation process benefits cardiogenesis study. Embryonic stem cells (ESCs), cell lines with infinite ability to proliferate and to be differentiated into all cell types of the adult body, are important research tools for investigation of differentiation and meanwhile good models for developmental research. In the current study, genome-wide gene expression of ESCs is profiled through high throughput platform during cardiomyocyte-specific differentiation and maturation. Gene expression patterns of undifferentiated ESCs and ESC-derived cardiomyocytes provide a global overview of genes involved in cardiomyocyte-specific differentiation, whereas marker gene expression profiles of both ESC-related genes and cardiac-specific genes presented the expression pattern shift during differentiation in a pure ESC-derived cardiomyocyte cell culture system. The differentiation and maturation process was completed at day 19 after initiation of differentiation, according to our gene expression profile results. Functional analysis of regulated genes reveals over-represented biological processes, molecular functions and pathways during the differentiation and maturation process. Finally, transcription factor regulation networks were engineered based on gene expression data. Within these networks, the number of identified important regulators (Trim28, E2f4, Foxm1, Myc, Hdac1, Rara, Mef2c, Nkx2-5, Gata4) and possible key co-regulation modules (Nkx2-5 - Gata4 - Tbx5, Myc - E2F4) could be expanded. We demonstrate that a more comprehensive picture of cardiomyocyte differentiation and its regulation can be achieved solely by studying gene expression patterns. The results from our study contribute to a better and more accurate understanding of the regulation mechanisms during cardiomyocyte differentiation.

MicroRNA profiling during cardiomyocyte-specific differentiation of murine embryonic stem cells based on two different miRNA array platforms.

MicroRNA (miRNA) plays a critical role in a wide variety of biological processes. Profiling miRNA expression during differentiation of embryonic stem cells will help to understand the regulation pathway of differentiation, which in turn may elucidate disease mechanisms. The identified miRNAs could then serve as a new group of possible therapeutic targets. In the present paper, miRNA expression profiles were determined during cardiomyocyte-specific differentiation and maturation of murine embryonic stem (ES) cells. For this purpose a homogeneous cardiomyocyte population was generated from a transgenic murine ES cell line. Two high throughput array platforms (Affymetrix and Febit) were used for miRNA profiling in order to compare the effect of the platforms on miRNA profiling as well as to increase the validity of target miRNA identification. Four time points (i.e. day 0, day 12, day 19 and day 26) were chosen for the miRNA profiling study, which corresponded to different stages during cardiomyocyte-specific differentiation and maturation. Fifty platform and pre-processing method-independent miRNAs were identified as being regulated during the differentiation and maturation processes. The identification of these miRNAs is an important step for characterizing and understanding the events involved in cardiomyocyte-specific differentiation of ES cells and may also highlight candidate target molecules for therapeutic purposes.

Chemically induced cardiomyogenesis of mouse embryonic stem cells.

A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of alpha-myosine heavy chain (alpha-MHC) promoter (palpha-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80 % increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets.

Identification of small signalling molecules promoting cardiac-specific differentiation of mouse embryonic stem cells.

Identification of signalling cascades involved in cardiomyogenesis is crucial for optimising the generation of cardiomyocytes from embryonic stem cells (ES cells) (in vitro). We used a transgenic ES cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-myosin heavy chain (alpha-MHC) promoter (palphaMHC-EGFP) to investigate the effects of 33 small molecules interfering with several signalling cascades on cardiomyogenesis. Interestingly, the L-Type Ca2+ channel blocker Verapamil as well as Cyclosporin, an inhibitor of the protein phosphatase 2B, exerted the most striking pro-cardiomyogenic effect. Forskolin (adenylate cyclase stimulator) exerted the most striking anti-cardiomyogenic effect. The cardiomyogenic effect of Cyclosporin and Verapamil correlated with an expression of early cardiac markers Nkx2.5 and GATA4. Compared to the effects on late developmental stage embryoid bodies (EBs) stimulation of early developmental stage EBs (1-day old) with Verapamil or Cyclosporin for 48 h resulted in a potent cardiomyogenic effect. Accordingly, enhanced expression of alpha-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of alpha-smooth muscle actin or platelet endothelial cell adhesion molecule-1 (PECM-1) as well as of neuronal genes (Nestin, Neurofilament H) has been observed demonstrating a preferentially pro-cardiomyogenic effect by both molecules.