Fluorescent indicators for continuous and lineage-specific reporting of cell-cycle phases in human pluripotent stem cells |
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Authors: | Yun Chang Peter B Hellwarth Lauren N Randolph Yufei Sun Yuxian Xing Wuqiang Zhu Xiaojun Lance Lian Xiaoping Bao |
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Institution: | 1. Davidson School of Chemical Engineering, Purdue University, West Lafayette, Indiana;2. Department of Biomedical Engineering, Huck institutes of the Life Sciences, Department of Biology, Pennsylvania State University, University Park, Pennsylvania;3. Department of Cardiovascular Medicine, Physiology and Biomedical Engineering, Mayo Clinic, Scottsdale, Arizona |
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Abstract: | Proper cell-cycle progression is essential for the self-renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination-based cell-cycle indicator (FUCCI) has allowed the dual-color visualization of the G1 and S/G2/M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage-specific FUCCI reporters have not yet been developed to analyze complex tissue-specific cell-cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell-cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell-cycle-dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac-specific TNNT2-FUCCI reporter for lineage-specific cell-cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell-cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration. |
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Keywords: | cell cycle CRISPR/Cas9 FUCCI gene editing heart regeneration human pluripotent stem cells |
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