Abstract
The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.
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Acknowledgements
The study was supported by grants from the State Major Research Program of China (2007CB947100, 2010CB945603, and 2011CB965301), National Science and Technology Major Project (2009ZX09503-024), Knowledge Innovation Program of CAS (KSCX2-YW-R-233, KSCX1-YW-R-46), Strategic Priority Research Program of CAS (XDA01030000), and the Ministry of Agriculture (2009ZX08010-016B). We thank Zhongyan Chen for performing fetal rat heart isolation, Lan Wu for technique assistance in the measurement of Ca2+ transients, Lei Qian and Jihui Zhang for performing rESCs culture, and Dr Ying Jin for kindly providing the Oct4 antibody.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Figure S1
Identification of optimal conditions for in vitro differentiation protocol of rESCs. (PDF 215 kb)
Supplementary information, Figure S2
Analysis of the significance of rEFM for cardiac differentiation of rESCs. (PDF 72 kb)
Supplementary information, Figure S3
Comparisons of EB generation methods. (PDF 250 kb)
Supplementary information, Table S1
Primer Sequences for RT- PCR (PDF 6 kb)
Supplementary information, Table S2
Primer Sequences for Q-PCR (PDF 6 kb)
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Cao, N., Liao, J., Liu, Z. et al. In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes. Cell Res 21, 1316–1331 (2011). https://doi.org/10.1038/cr.2011.48
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DOI: https://doi.org/10.1038/cr.2011.48
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