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Hescheler: Pluripotent stem cells – from physiology to cell therapy, disease models and test systems

Owing to their ability to reproduce the embryonic, fetal and neonatal differentiation of all different organotypic cellular phenotypes, pluripotent stem cells represent an ideal tool to study physiological processes of embryogenesis under in vitro conditions, as well as provide the basis of cellular therapeutics to develop novel disease models and to build up test assay systems for drug discovery and toxicology. In particular, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can reproduce all organotypic electrophysiology, signalling cascades and genes involved in development (functional genomics). This spontaneously occurs within three-dimensional cell aggregates – embryoid bodies – which were developed 25 years ago. To select only one lineage (e.g. the cardiac lineage) and to allow the identification of the transplanted cells, transgenic ES and iPS cells were used. They contained a vector with two cloning sites for enhanced green fluorescent protein and a puromycin resistance gene for selection under the α-MHC (myosin heavy chain) promoter. We aimed to generate iPS cell-derived cardiomyocytes and their molecular and functional characterization in comparison with cardiomyocytes derived from established ES cells on a transcriptomic and electrophysiological level. To demonstrate the ability of ES cells for regenerative medicine and tissue repair, cardiomyocytes differentiated from ES cells were injected into the cryoinfarcted left ventricular wall of adult wild-type mice. Translation from the laboratory into the clinic is one of the remaining key issues for applied stem cell research.

Reprogramming of fibroblasts from patients with long QT syndrome type 3 or catecholaminergic polymorphic ventricular tachycardia syndrome by ectopic expression of the Yamanaka’s transcription factors resulted in the generation of iPS cells for disease modelling. This novel approach may also enable patient-specific cell replacement therapies, which appear to be an indispensable prerequisite for later use in clinics.

Within two European consortia, embryonic stem cell-based novel alternative testing strategies (ESNATS) and detection of end points and biomarkers for repeated dose toxicity using in vitro systems (DETECTIVE), and under my coordination, we developed a battery of toxicity tests using human ES or human iPS cell lines subjected to different standardized culture protocols. Tests will cover embryoid bodies in different developmental stages and differentiated derivatives including gamete and neuronal lineages, complemented by test systems for hepatic metabolism. Predictive toxicogenomics and proteomics markers will be identified. The individual tests will be integrated into an ‘all-in-one’ test system. To enable future industrial use, we will prepare automating and scaling-up of human ES cell culture, and the predictability, quality and reproducibility of these will be evaluated in a proof-of-concept study. Benefits include increasing safety as a result of better predictivity of human test systems; reducing, refining and replacing animal tests; lowering testing cost; and supporting medium- and high-throughput testing.

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