Abstract 1573: Genomics of Developing Cardiomyocytes from Recombinant Murine Embryonic Stem Cells
Introduction Embryonic stem cells (ESC) have the potential to develop differentiated tissues. However, little is known on the regulation of the transcriptome during differentiation of ESC. We analysed a recombinant ESC line with a neomycine-resistance gene under the control of the alpha myosin heavy chain promotor, which allows selection of pure ESC-derived murine cardiomyocytes. We analysed by microarrays the transcriptome during differentiation and compared the profiles to adult murine myocardium.
Methods Withdrawal of LIF from culture medium induced differentiation of ESC-line CM7/1 and embroid body (EB) formation. Selection with geniticine leads to formation of beating cardiobodies (CB), from which isolated cardiomyocytes can be derived. Total RNA of 7 timepoints plus adult murine heart was hybridized on Affymetrix whole genome MOE430A arrays. Analysis of expression data was performed using Genedata Analyst software. Expression of selected genes was confirmed by Real-time PCR (TaqMan).
Results One dimensional clustering of transcription factors (TF) revealed 3 characteristic groups of TF, which appear transiently in the early and late phase of the cell culture and are different from isolated cardiomyocytes. The TF-pattern of the ESC-derived cardiomyocytes showed only few differences to ventricular myocardium. Dissociation of cardiobodies leads to downregulation of TF regulating heart development (hand2) or from cardiac precursor cells (Isl1), respectively. Early transcription factors, which regulate pluripotency like Oct4 and Nanog are downregulated early after induction of differentiation. During formation of EBs hand2 and GATA6 are expressed throughout differentiation to cardiomyocytes, whereas SNAI2 and HMGA2 are transiently expressed.
Discussion Little is known on the regulation of transcription factors needed for differentiation from ESC to cardiomyocytes. From our data we conclude, that in the early undifferentiated state of the ESC a set of transcription factors is transiently transcribed, which is distinct from those needed for pattern formation in embroid bodies and differentiated cardiomyocytes. The expression pattern of isolated cardiomyocytes and the ventricular myocardium was found to be extremely similar.