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

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.

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy

“…Donor plasmid construction. The donor plasmids targeting AAVS1 locus were constructed as previously described (Chang et al, 2020). Briefly, to generate the CAG-IL13 T-CAR plasmid, the TQM-IL13 CAR fragment (Kim et al, 2020b) Maintenance and differentiation of hPSCs.…”

“…The donor plasmids targeting AAVS1 locus were constructed as previously described (Chang, et al, 2020). Briefly, to generate the CAG-IL13 T-CAR plasmid, the TQM-IL13 CAR fragment (Kim et al, 2020b) was amplified from Addgene plasmid #154054 and then cloned into the AAVS1-Puro CAG-FUCCI donor plasmid (Addgene; #136934), replacing the FUCCI.…”

Optogenetic‐mediated cardiovascular differentiation and patterning of human pluripotent stem cells

“…H9 wildtype hPSCs were obtained from WiCell and maintained on Matrigel‐coated 6‐well plate in mTeSR1 or mTeSR plus medium. To make OptoWnt H9 cells, Cry2‐LRP6c‐2A‐mCherry construct was cloned into AAVS1 donor plasmid (Addgene #80945) and nucleofected into H9 hPSCs along with Cas9/AAVS1 gRNA plasmid 24 . Puromycin‐resistant mCherry+ hPSC colonies were picked and genotyped for homozygosity.…”

“…To make OptoWnt H9 cells, Cry2-LRP6c-2A-mCherry construct was cloned into AAVS1 donor plasmid (Addgene #80945) and nucleofected into H9 hPSCs along with Cas9/AAVS1 gRNA plasmid. 24 Puromycinresistant mCherry+ hPSC colonies were picked and genotyped for homozygosity. The resulting OptoWnt hPSCs were maintained in dark (foil cover) and used for the downstream applications.…”

Induced Cardiomyocyte Proliferation: A Promising Approach to Cure Heart Failure

“…Interestingly, the use of the FUCCI cell cycle reporter revealed that small molecule regulators of PPARd signaling could modulate the cardiomyocyte cell cycle [ 125 ]. Furthermore, in vivo models [ 126 , 127 ] and hiPS-derived cardiomyocytes [ 128 ] have been generated to study cell cycle progression in the heart using FUCCI indicators. Interestingly, a recent study generated a Ki67-based lineage and traced mice to track the cell cycle activation in cardiomyocytes [ 129 ].…”