Fluorescent indicators for continuous and lineage-specific reporting of cell-cycle phases in human pluripotent stem cells

Yun Chang, Peter B. Hellwarth, Lauren N. Randolph, Yufei Sun, Yuxian Xing, Wuqiang Zhu, Xiaojun Lance Lian, Xiaoping Bao

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)2177-2186
Number of pages10
JournalBiotechnology and Bioengineering
Volume117
Issue number7
DOIs
StatePublished - Jul 1 2020

Keywords

  • cell cycle
  • CRISPR/Cas9
  • FUCCI
  • gene editing
  • heart regeneration
  • human pluripotent stem cells

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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