期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 117, 期 7, 页码 2177-2186出版社
WILEY
DOI: 10.1002/bit.27352
关键词
cell cycle; CRISPR; Cas9; FUCCI; gene editing; heart regeneration; human pluripotent stem cells
资金
- Davidson School of Chemical Engineering at Purdue University
- College of Engineering at Purdue University
- NIH Trailblazer Award [R21EB026035]
- NSF CAREER Award [1943696]
- Div Of Chem, Bioeng, Env, & Transp Sys
- Directorate For Engineering [1943696] Funding Source: National Science Foundation
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 G(1) and S/G(2)/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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