Analysis of G protein-coupled receptors on human embryonic stem cells
Zusammenfassung der Projektergebnisse
The aim of this project was to determine the profile of G protein-coupled receptors (GPCR) during cardiac differentiation of human embryonic stem cells. GPCR could serve as surface markers to identify and isolate subpopulations during differentiation as well as tools to modulate differentiation by modifying receptor activity with agonists or antagonists. High differentiation efficiency and/or a suitable selection marker are prerequisites for using differentiated cell populations to generate engineered cardiac constructs. Major progress of this study was the establishment of a cardiac differentiation protocol from human embryonic stem cells and the determination a total of 26 candidate receptors for undifferentiated embryonic stem cells (4), cardiac progenitors (9) and early cardiomyocytes (13), determined by SOLiD™ technology and verified by individual quantitative PCR. Current effort focuses on independent verification of these GPCR on cardiac differentiation of human induced pluripotent stem cells and testing the effect of agonists and antagonists or molecular means of knocking down the receptors. The establishment of robust cardiac differentiation protocols turned out to be difficult, but finally resulted in the differentiation of cell populations with more than 40% cardiomyocytes. These preparations were used to generate the first engineered heart tissues (EHT) from hESCs applicable to automated evaluation of contractility. Key findings with human EHTs were stable and regular beating over weeks, partial maturation (excellent cellular alignment and sarcomeric organisation, relatively high ß-myosin heavy chain expression), but electrophysiological immaturity, and high sensitivity to detect proarrhythmic toxicity of hERG channel blockers. This part of the study attracted substantial attention by academic and commercial collaborators, was the basis of a recently approved DFG grant to determine disease phenotypes of iPS cells from patients with genetic cardiac diseases and was awarded by the DFG Ursula M. Händel-Animal welfare price in 2011.
Projektbezogene Publikationen (Auswahl)
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Künstliche Herzgewebe aus humanen embryonalen Stammzellen als Testsystem für Arzneistoffe. Dissertation, FBCh 3019
Sebastian Schaaf
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(2010). Development of a Drug Screening Platform Based on Engineered Heart Tissue. Circulation Research, 107(1), 35-44
Hansen, A., Eder, A., Bönstrup, M., Flato, M., Mewe, M., Schaaf, S., Aksehirlioglu, B., et al.
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(2011). Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. (L. J. de Windt, Ed.)PloS one, 6(10), e26397
Schaaf, S., Shibamiya, A., Mewe, M., Eder, A., Stöhr, A., Hirt, M. N., Rau, T., et al.
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Bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (EHT). J Vis Exp. 2011 Jun 1;(52). pii: 2605
Conradi L, Pahrmann C, Schmidt S, Deuse T, Hansen A, Eder A, Reichenspurner H, Robbins RC, Eschenhagen T, Schrepfer S
