Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02302-4

CRISPR/dCas9VPR-mediated gene activation of cyclin promotes cell cycle re-entry in human induced pluripotent stem cell-derived cardiomyocytes

R. Kim1, S. Nagel1, W.-H. Zimmermann1, L. Cyganek2, L. Zelarayán1, E. Schoger1

1Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; 2Herzzentrum Göttingen - Stem Cell Unit, Universitätsmedizin Göttingen, Göttingen;

The proliferative capacity of adult cardiomyocytes is limited, resulting in an inability of endogenous mechanisms to replenish their loss caused by cardiovascular diseases. The fetal heart has the intrinsic capacity to regenerate after myocardial damage but the cardiomyocyte proliferation rate declines after birth and remains at a low level in adulthood. In line, most cell cycle regulators are significantly downregulated in adult hearts, however the exact mechanisms leading to the loss of regenerative capacity are not well understood. In this study, I aimed to induce the cell cycle re-entry of cardiomyocytes by modulating the expression of cell cycle regulators (cyclins) with CRISPR/dCas9VPR mediated endogenous gene activation (CRISPRa) using an enzymatically inactive Cas9-based programmable transcription factor targeted to gene regulatory regions via guide RNAs (gRNA). To monitor cell cycle activity, the fluorescence ubiquitination dependent cell cycle indicator (FUCCI) was used and delivered into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) by lentiviral transduction. The system employed fluorescently tagged cell cycle regulatory proteins specific to different cell cycle phases (mCherry-tagged Chromatin licensing and DNA replication factor 1 (CDT1) to detect G1 phase and a fusion of Geminin and ECFP for G2 phase). Ubiquitination and degradation of CDT1 or Geminin throughout the cell cycle therefore allow for identifying bona fide cell cycle progression. To validate FUCCI in hiPSC-CM, Wnt signaling activator CHIR99021 (3 µM) was added to 1 week old hiPSC-CM. The cell cycle re-entry was observed by mCherry and ECFP positive nuclei in TNNT2 expressing cells while hiPSC-CMs cultured over 1 month were incapable of cell-cycle re-entry highlighting a temporally limited cell cycle activation potential. To examine the effects of cyclins in hiPSC-CM, the CRISPRa was employed to endogenously activate cyclins (CCNA2, CCNB1, CCND1, and CCND2). Two to six gRNAs were designed and validated by qPCR in HEK293T for the CRISPR activation of each cyclin. 10 days after lentiviral delivery of gRNAs, significantly increased mRNA expression of cyclins was observed compared to control (non-targeted gRNAs, NT) in both 1 month old and 6 months old dCas9VPR expressing hiPSC-CMs (CRISPRa CM) (n ≥ 3, CCNA2 in 1-month CRISPRa CM: 5.1-fold to NT, in 6-months CRISPRa CM: 15.8-fold to NT). Cell cycle activity and karyokinesis were detected with the FUCCI system when CCNA2 and CCNB1 were activated. The immunofluorescence results suggested that elevated expression of CCNA2 and CCNB1 could induce repetitive karyokinesis (> 10 nuclei in a single TNNT2 positive cell) and cell division in hiPSC-CM. To ensure homogenous expression of FUCCI, heterozygous integration of the FUCCI construct was achieved by CRISPR/Cas9 homology-directed repair into the ROSA26 safe harbor locus on human chromosome 3 of endogenously citrine-tagged Alpha-actinin-2 (ACTN2) reporter expressing hiPSC, currently used for hiPSC-CM differentiation and 3D engineered human myocardium (EHM) generation to evaluate cell cycle activity in a cardiomyocyte-specific manner. In conclusion, we demonstrated cell cycle re-entry of FUCCI labeled hiPSC-CM via endogenous CCNA2 and CCNB1 activation. The tools established in this work are valuable assets to the field of heart regeneration and can be further employed to decipher mechanisms of cardiomyocyte proliferation in hiPSC-CM based models.

https://dgk.org/kongress_programme/ht2023/aBS137.html