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PLoS One. 2018 Mar 7;13(3):e0192652. doi: 10.1371/journal.pone.0192652. eCollection 2018.

Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

Author information

1
Clinic of Cardiothoracic Surgery, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
2
Clinic of Cardiology and Angiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
3
Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
4
Institute of Physiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
5
Institute of Human Genetics, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.
6
Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.
7
Chair of Livestock Biotechnology, Technical University Munich, Freising-Weihenstephan, Germany.

Abstract

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

PMID:
29513687
PMCID:
PMC5841662
DOI:
10.1371/journal.pone.0192652
[Indexed for MEDLINE]
Free PMC Article

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