This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Embryonic stem cell-derived cardiomyocytes

Microarray analysis reveals that the specific pattern of gene expression in cardiomyocytes derived from embryonic stem cells reflects the biological, physiological and functional processes occurring in mature cardiomyocytes.

Background/Aims: Embryonic stem (ES) cells may represent an alternative source of functionally mature cardiomyocytes for the treatment of heart diseases. ES cells spontaneously differentiate into spheroidal aggregates, also referred to as embryoid bodies (EBs). The identification of growth factors playing a decisive role in cardiogenesis is a crucial issue for the generation of mature cardiomyocytes. Methods: In order to identify growth factors promoting cardiac development, we established a new differentiation protocol using a defined serum-replacement medium (SRM) containing 5µg/ml insulin and 5µg/ml transferrin in combination with Dulbecco’s Modified Eagle Medium (DMEM). Furthermore, we added platelet-derived growth factor-BB (PDGF-BB) or sphingosine-1-phosphate (SPP) to promote cardiac differentiation. Results: Using SRM/DMEM, we obtained a 6-fold increase of cardiac specific myosin heavy chain α and β (cMHCα/β) in relation to 0,2% foetal calf serum (FCS)/DMEM (= 100%). Stimulation of EBs with PDGF-BB in the presence of SRM/DMEM resulted in a further 2,6-fold enhancement in comparison with the SRM/DMEM-induced increase of cMHCα/β (= 100%). A parallel increase in the number of beating EBs was observed. Similar results were obtained after stimulation of EBs with 5µg/ml SPP. Conclusion: We established a serum-free protocol and identify PDGF-BB and SPP as potent factors promoting cardiogenesis in ES 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 α-myosin heavy chain (α-MHC) promoter (pαMHC-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 α-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of ?-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.

Transcriptome and phenotype of BMP2 + cells

Transcriptome analysis of BMP2⁺ cells in comparison to the undifferentiated BMP2 ES cells and the control population from 7-day old embryoid bodies led to the identification of 479 specifically upregulated and 193 downregulated transcripts.

No abstract